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Experimental arthritis : in vitro and in vivo models
Wang, Xuanhui
DOI:10.6100/IR634844
Published: 01/01/2008
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Citation for published version (APA):Wang, X. (2008). Experimental arthritis : in vitro and in vivo models Eindhoven: Technische UniversiteitEindhoven DOI: 10.6100/IR634844
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Experimental Arthritis: in vitro and in vivo Models
A catalogue record is available from the Eindhoven University of Technology Library
ISBN: 978‐90‐386‐1270‐6
Printed in the United States of America
Wang, Susanne X.
Experimental Arthritis: in vitro and in vivo Models/ Susanne X. Wang
Cover design: Ryan H. Tam and Susanne X. Wang
Copyright ©2008 by Susanne Xuanhui Wang
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Experimental Arthritis: in vitro and in vivo Models
PROEFSCHRIFT
ter verkrijging van de graad van doctor aan de
Technische Universiteit Eindhoven, op gezag van de
Rector Magnificus, prof.dr.ir. C.J. van Duijn, voor een
commissie aangewezen door het College voor
Promoties in het openbaar te verdedigen
op donderdag 3 juni 2008 om 16.00 uur
door
Xuanhui Wang
geboren te Liaoning, China
Dit proefschrift is goedgekeurd door de promotor:
prof.dr.ir. H.W.J. Huiskes
Copromotor:
dr. C.C. van Donkelaar
To my daughters Lauren and Martina
Thank you for being the most wonderful
children in the world.
For since the creation of the world God's invisible
qualities—his eternal power and divine nature—have
been clearly seen, being understood from what has been
made, so that men are without excuse.
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Table of Contents
Table of Contents ..............................................................................................................1
Summary ............................................................................................................................9
Chapter 1 ‐ Introduction ................................................................................................. 13
1. Introduction to Arthritis ......................................................................................... 14
1.1. Overview of OA ................................................................................................ 14
1.2. Overview of RA ................................................................................................ 14
2. Introduction of Articular Joints ............................................................................... 15
3. Introduction to Articular Cartilage and Function ................................................... 16
3.1. Chondrocytes in Articular Cartilage ................................................................. 16
4. Problems of Articular Cartilage Repair ................................................................... 18
5. Current OA Treatments .......................................................................................... 19
5.1. Medications ..................................................................................................... 19
5.2. Surgery ............................................................................................................. 19
5.3. Alternatives ..................................................................................................... 20
6. Development and Progression of OA ..................................................................... 21
6.1. Typical OA Progression .................................................................................... 21
6.2. OA as a Whole Joint Disease ........................................................................... 22
6.3. Relationship between Articular Cartilage and Subchondral Bone .................. 23
6.4. Bone Structure and Metabolism ..................................................................... 23
6.5. Bone Structure, Material, and Mechanical Properties .................................... 24
6.6. Bone Remodeling ............................................................................................ 25
6.7. Possible Initiation of OA as a Bone Disease ..................................................... 25
6.8. Anti‐resorptive Drugs as a Potential Treatment for OA .................................. 26
6.9 Development of Osteophytes .......................................................................... 27
6.10. Complexity of OA Development and Progression Pathways ......................... 27
7. Need for Animal Models ......................................................................................... 28
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8. Objectives and outlines .......................................................................................... 29
References .................................................................................................................. 30
Chapter 2 ‐ Subchondral Bone Microarchitecture Changes in Animal Models of Arthritis Quantified by Micro‐CT .................................................................................................. 35
1. Introduction ............................................................................................................ 37
2. Bone changes in human arthritis ............................................................................ 38
3. Bone changes in OA animal models ....................................................................... 38
4. Micro‐CT imaging in arthritis ................................................................................. 39
5. Methodology of using micro‐CT in arthritis research ............................................. 40
5.1 Sample preparation: ......................................................................................... 40
5.2 Scanning procedures: ....................................................................................... 41
5.3 Reconstruction: ................................................................................................ 41
5.4 Three‐dimensional analysis .............................................................................. 42
5.5 Three‐dimensional isosurfaces ......................................................................... 42
6. Cartilage imaging using micro‐CT and micro MRI .................................................. 46
7. The future of using micro‐CT in arthritis research ................................................. 46
References .................................................................................................................. 48
Chapter 3 ‐ Tissue Engineering of Biphasic Cartilage Constructs Using Various Biodegradable Scaffolds: an in vitro Study ..................................................................... 51
1. Introduction ............................................................................................................ 53
2. Materials and methods .......................................................................................... 54
2.1. Articular chondrocyte isolation ....................................................................... 54
2.2. Chamber seeding and construct formation .................................................... 54
2.3. Histological evaluation .................................................................................... 56
2.4. Confocal laser scanning microscopy (CLSM) cell viability (live/dead assay) ... 56
2.5. Immunohistochemical staining for collagen types I, II and X .......................... 56
2.6. Biochemical analyses (SDS‐PAGE) ................................................................... 56
2.7. Transmission electron microscopy (TEM) ....................................................... 57
2.8. Scanning electron microscopy (SEM) .............................................................. 57
3. Results .................................................................................................................... 57
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3.1. Macroscopic and histological observations..................................................... 57
3.2. Cell viability ...................................................................................................... 59
3.3. Biochemistry and immunohistochemistry ...................................................... 60
3.4. Transmission electron microscopy .................................................................. 60
3.5. Scanning electron microscopy (SEM) .............................................................. 61
4. Discussion ............................................................................................................... 63
5. Conclusions ............................................................................................................. 65
Acknowledgements .................................................................................................... 65
References .................................................................................................................. 66
Chapter 4 ‐ Disease‐Modifying Effects of N‐butyryl Glucosamine in a Streptococcal Cell Wall (SCW) Induced Arthritis Model in Rats ................................................................... 69
1. Introduction ............................................................................................................ 71
2. Materials and Methods .......................................................................................... 73
2.1 Animal model and experiment design. ............................................................ 73
2.2 Bone mineral density (BMD) measurement. .................................................... 74
2.3 Micro‐CT imaging protocol. .............................................................................. 74
2.4 Three‐dimensional trabecular bone measurements. ....................................... 74
2.5 Three‐dimensional isosurface. ......................................................................... 75
2.6 Two‐dimensional subchondral bone analysis. ................................................. 75
2.7 Statistical analysis. ............................................................................................ 76
3. Results .................................................................................................................... 76
3.1 Effects of GlcNBu on the SCW‐induced inflammation. .................................... 76
3.2 Decline of BMD by the arthritis and reversal by GlcNBu.................................. 77
3.3 Micro‐architecture of the trabecular bone. ..................................................... 80
3.4 Cancellous bone connectivity. .......................................................................... 82
3.5 Site‐specific structure change. ......................................................................... 82
3.6 Joint integrity. ................................................................................................... 86
3.7 Structure model index (SMI). ........................................................................... 87
4. Discussion ............................................................................................................... 87
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Acknowledgements .................................................................................................... 89
References .................................................................................................................. 90
Chapter 5 ‐ The Effect of Glucosamine Hydrochloride on Subchondral Bone Changes in an Animal Model of OA .................................................................................................. 93
1. Introduction ............................................................................................................ 95
2. Materials and Methods .......................................................................................... 97
2.1 Animals and OA model. .................................................................................... 97
2.2 Macroscopic examination of cartilage. ............................................................ 98
2.4 Processing of undecalcified tissue. ................................................................. 100
2.5 Histomorphometric analysis. .......................................................................... 100
2.6 Bone structure. ............................................................................................... 101
2.7 Bone connectivity. .......................................................................................... 101
2.8 Assessment of subchondral bone plate thickness.......................................... 101
2.9 Bone mineralization assessment. ................................................................... 101
2.10 Statistical analysis. ........................................................................................ 102
3. Results .................................................................................................................. 102
3.1 Macroscopic features of cartilage. ................................................................. 102
3.2 DXA findings........................................................................................................ 103
3.3 Bone static histomorphometric findings. ....................................................... 105
3.4 Subchondral bone thickness. .......................................................................... 106
3.5 Bone architecture and connectivity. .............................................................. 107
3.6 Mineralization (BSE microscopy) .................................................................... 108
4. Discussion ............................................................................................................. 109
Reference ................................................................................................................. 112
Chapter 6 ‐ Stereological Study of Cartilage and Subchondral Bone Changes in Spontaneous Knee Osteoarthritis: Results from Two Strains of Guinea Pig ................ 117
1. Introduction .......................................................................................................... 119
2. Materials and Methods ........................................................................................ 120
2.1 In vivo study .................................................................................................... 120
2.2 Biomarker measurement ................................................................................ 121
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2.3 Tissue processing ............................................................................................ 121
2.4 3‐D and 2‐D structure analysis on histology slides ......................................... 122
2.5 3‐D histological analysis: ................................................................................ 123
2.6 2‐D histological analysis: ................................................................................ 123
2.7 Micro‐CT imaging analysis .............................................................................. 124
2.8 Three‐dimensional trabecular bone measurements ...................................... 125
2.9 Three‐dimensional isosurface ........................................................................ 125
2.10 Statistical analysis. ........................................................................................ 126
3. Results: ................................................................................................................. 126
3.1 Stereology Results: ......................................................................................... 126
3.2 MicroCT data .................................................................................................. 127
3.3 Biomarker Results ........................................................................................... 129
4. Discussion: ............................................................................................................ 129
References ................................................................................................................ 133
Chapter 7 ‐ Progressive Change of Cartilage and Subchondral Bone in Partial Medial Menisectomy and Accelerated Spontaneous OA Models ‐ A 6 Month Study with Dunkin Hartley Guinea Pigs ...................................................................................................... 135
1. Introduction: ......................................................................................................... 137
2. Materials and methods ........................................................................................ 139
2.1. Animal model................................................................................................. 139
2.2. Samples collection ......................................................................................... 140
2.3. Dual Energy X‐ray Absotiometry (DEXA): ...................................................... 140
2.4 Cartilage Biochemistry: ................................................................................... 140
2.5 Histological Grading of Cartilage: ................................................................... 140
2.6 Subchondral bone plate mineralization and thickness change ...................... 141
2.7 Histomorphometry analysis using Bioquant Nova Pro System ...................... 141
2.8 Femoral subchondral bone structure and connectivity analysis using BSE images ................................................................................................................... 142
2.9 Bone connectivity ........................................................................................... 142
2.10 Microhardness .............................................................................................. 142
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2.11 Data analysis ................................................................................................. 143
3. Results: ................................................................................................................. 143
3.1 Animal Weight Change ................................................................................. 143
3.2 Histology ......................................................................................................... 143
3.3 Cartilage Biochemistry: ................................................................................... 144
3.4 Bone Mineral density: .................................................................................... 147
3.5 Bone histomorphometric analysis on Goldner’s trichrome stained femur slides .............................................................................................................................. 148
3.6 Bone structure analysis on femoral BSE images ............................................ 149
3.7 Bone connectivity analysis on femoral BSE images ........................................ 150
3.8 Subchondral bone plate change: .................................................................... 151
3.9 Osteophyte formation in the osteoarthritic joint: ......................................... 153
4. Discussion: ............................................................................................................ 155
4.1 Histology ......................................................................................................... 155
4.2 Biochemistry‐ DNA ......................................................................................... 156
4.3 Biochemistry‐ GAG ......................................................................................... 156
4.4 Biochemistry‐ Hydroxproline .......................................................................... 157
4.5 Bone Mineral Changes: ................................................................................... 157
4.6 Bone formation: ............................................................................................. 158
4.7 Bone Structure Analysis .................................................................................. 158
4.8 Bone connectivity analysis ............................................................................. 159
4.9 Subchondral bone plate change ..................................................................... 159
4.10 Osteophyte formation .................................................................................. 159
References ................................................................................................................ 161
Chapter 8 ‐ The Effects of Alendronate on the Progression of OA in Cartilage and Subchondral Bone Tissues using a Partial Medial Menisectomy Guinea Pig Model .... 165
1. Introduction .......................................................................................................... 167
2. Material and method ............................................................................................ 168
2.1 OA Model and Experiment Design ................................................................. 168
2.2 Sample Collection ........................................................................................... 169
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2.3 Bone Mineral Density Measurement ............................................................. 170
2.4 Histology, Mankin grading .............................................................................. 170
2.5 Cartilage Biochemistry .................................................................................... 171
2.6 Undecalcified Sample Processing ................................................................... 172
2.7 Bone Static and Dynamic Histomorphometry ................................................ 172
2.8 Backscattered Electron Microscopy ............................................................... 172
2.9 Femoral Subchondral Bone Structure and Connectivity Analysis .................. 173
2.10 Subchondral Bone Plate Thickness ............................................................... 173
2.11 Subchondral Bone Plate Mineralization Assessments ................................. 173
2.12 Osteophyte Incidence and Area ................................................................... 174
2.13 Statistical Analysis ........................................................................................ 174
3. Results .................................................................................................................. 174
3.1 Gross and Microscopic Morphology ............................................................... 174
3.2 Cartilage Biochemistry: ................................................................................... 177
3.3 Bone Mineral density ..................................................................................... 178
3.4 Bone structure and connectivity analysis ....................................................... 179
3.5 Subchondral Bone Plate thickness ................................................................. 181
3.6 Static and dynamic histomophometry ........................................................... 182
3.7 Inhibition of osteophyte formation by alendronate ...................................... 184
4. Discussions ............................................................................................................ 184
References ................................................................................................................ 187
Chapter 9 ‐ Discussion and Conclusions ....................................................................... 191
1. General Discussion ............................................................................................... 192
2. Concluding Remarks: ............................................................................................ 196
References ................................................................................................................ 197
Samenvatting ................................................................................................................ 199
Acknowledgement ........................................................................................................ 203
Curriculum Vitae ........................................................................................................... 205
Bibliography .................................................................................................................. 207
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Book chapter: ........................................................................................................... 207
Peer reviewed articles: ............................................................................................. 207
Conference proceedings: .......................................................................................... 208
Summary
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Summary
Experimental Arthritis: in vivo and in vitro Models.
As the primary cause of disability for people over the age of 45, arthritis actually consists of more than hundred different conditions. Osteoarthritis (OA) is the most common form of arthritis followed by rheumatoid arthritis (RA). OA is characterized by progressive articular cartilage loss and destruction, osteophyte formation, subchondral bone changes and synovial inflammation. The pathophysiology of OA is not yet completely understood, but mechanical influences, effects of aging, and genetic factors play a vital role in OA initiation and progression.
Arthritis is a complex disease for two major reasons: the large number of contributing factors both in disease initiation and propagation; unknown mechanisms behind the disease development involving unknown interactions between the aforementioned factors. Studies to elucidate the pathogenesis of OA are further deterred by the relatively long dormant period where critical changes develop in both the bone and cartilage tissue with little to no outward symptoms. In order to properly address the problem of OA with effective therapeutic and preventative interventions, the mechanisms for its pathogenesis must be more clearly understood. However, as a disease not usually detected in patients until its last stages, OA proved to be a difficult subject of study. As such, both in vivo and in vitro models are employed as powerful tools for the research of OA, each with different strengths and limitations. The in vivo models address the complex and interacting mechanisms and factors for disease initiation and propagation, allowing for the study of natural disease progression over time. On the other hand, in vitro studies are better suited for the isolation of specific factors and the analysis of their contribution to the overall disease progression. By isolating a particular factor in vitro, these models have the advantage over their in vivo counterparts as a cost-effective and high throughput solution without the problem of variability between animals. The selection of an appropriate study model is important; each model introduces unique experimental conditions affects the results and provides unique insights in understanding the disease, and the results from different studies are therefore often complementary. The aim of this thesis is to combine a number of in vivo and in vitro models to gain better insights in the progression of OA, specifically focusing on the interactions between bone adaptation and cartilage degradation.
Experimental Arthritis: in vitro and in vivo Models
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Chapter 1 reviews the current status of arthritis research and the various models currently employed in the study of OA and RA. Chapter 2 explores the subchondral bone microarchitecture changes in animal models of OA and RA using high resolution micro-computed tomography (micro CT) technique. The author had developed several in vitro arthritis models over the years, namely monolayer, multi-layered, and pellet culture using primary chondrocytes. In addition, the author also employed a co-culture model of chondrocytes, osteoblasts, and synovial cells. The best in vitro model was found to be the tissue engineered cartilage that resulted from a closed-chamber bioreactor. The resultant tissue engineered cartilage can be either non-scaffold or scaffold. Chapter 3 presents a study on the development of biphasic implants that consist of the aforementioned tissue engineered cartilage with or without various underlying biodegradable osteoconductive support materials.
RA is a systemic autoimmune disease characterized by chronic joint inflammation and various degrees of bone and cartilage erosion. Study of RA animal models provides an understanding of the bone damage and its treatment. Chapter 4 presents a study utilizing a cell wall antigen induced arthritis model in rats. The aim of the study is to 1. Evaluate subchondral bone micro architecture change and 2. Investigate the efficacy of N-butyryl glucosamine (GlcNBu). The results show that GlcNBu inhibits inflammatory ankle swelling and preserves bone mineral density and bone connectivity, thus preventing further bone loss in this rat model of chronic arthritis.
Subchondral bone change is hypothesized to play a significant role in the initiation and/or development of OA. Chapter 5 examines the periarticular subchondral bone changes, including bone mineral density, subchondral trabecular bone turnover, architecture, and connectivity, as well as subchondral plate thickness and mineralization using a rabbit anterior cruciate ligament transection model of osteoarthritis. Results show that orally administered Glucosamine HCl presents protective effects in subchondral bone changes in the abovementioned experimental OA model.
The complexity in the development and progression of OA can be attributed to the close relationship between cartilage, subchondral bone, and neighboring tissues. Due to the complicated nature of OA progression, it is difficult to predict exactly when and how it is initiated. Numerous animal models were developed and their use has become indispensable in this field of study. To bring further clarity to the many unanswered questions concerning the role and importance of the subchondral bone in OA development, this thesis approaches the problem from two primary directions. First, we examine the minute changes of subchondral bone and cartilage to elucidate their relationship and impact on OA progression. Chapter 6 presents a study using three dimensional micro CT analyses combined with stereological histology assessment of cartilage changes in spontaneous knee osteoarthritis of two strains of guinea pig. A connection between bone remodeling and cartilage destruction is established by
Summary
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correlating three dimensional cartilage changes with bone remodeling. The second direction taken by this thesis is to study the OA development in a time course experiment using a slow progressive OA model. Chapter 7 examines OA progression in detail over time on both surgical induced OA (mimic secondary OA) and spontaneous OA (mimic primary OA) in guinea pigs, with special emphasis on the early stage of disease development. The progressive changes of subchondral bone over a 6 month time period is described in details for this experimental guinea pig OA model. It is now clear that increased subchondral bone turnover is a crucial step in the progression of OA and that the presence of cartilage lesion is always matched with significant bone remodeling directly below. This discovery has significant implications in both the understanding and treatment of OA.
Having recognized the role of the subchondral bone in the OA progression, we hypothesize that the reduction of cartilage degeneration by suppressing subchondral bone turnover is highly achievable. Chapter 8 investigates the effect of Alendronate, a drug that prohibits bone resorption, in the aforementioned guinea pig OA model. This study demonstrates that by suppressing bone turnover, Alendronate exhibits positive effects on articular surface erosion, cartilage degradation and subchondral bone structure and mineralization; it also protected collagen and proteoglycan content of the articular cartilage. We conclude that anti-resorptive treatments have positive effects on both cartilage and bone degradation.
Taken together, the thesis shows that cartilage and bone are tightly coupled together as a whole organ system. The two tissues cannot be considered separately in the study of arthritis pathogenesis; the interaction between subchondral bone and cartilage is one of the most important factors in OA progression. By suppressing subchondral bone turnover we have achieved cartilage protection in the guinea pig model of OA. This proves that increased subchondral bone turnover is a causal factor in OA progression. The combination of in vitro and in vivo models in this thesis has contributed to a better understanding of the etiology. In particular, in vitro models based on tissue engineered cartilage have been important for studying changes to the cartilage surface, and for screening of potential medication. For the study of progression of OA in the long term, the guinea pig model is very useful. This model simulates many aspects of normal development of OA in humans and can be used to evaluate treatments of OA in vivo.
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Chapter 1
Chapter 1 - Introduction
Experimental Arthritis: in vitro and in vivo Models
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1. Introduction to Arthritis
Arthritis is one of the leading causes of disability in aging humans. Arthritis (from Greek arthro-, joint + -itis, inflammation), as its name indicates, is a group of diseases that targets and attacks the articular joints of the body.
There are many different types of arthritis, with highly diverse causes. Rheumatoid arthritis (RA) and psoriatic arthritis are autoimmune diseases where the body is under the attack of its own immune system. Septic arthritis is initiated by joint infection. The deposition of uric acid crystals in the joint and the resulting subsequent inflammation is responsible for gouty arthritis. The most common form of arthritis is osteoarthritis (OA), which can occur following trauma to or infection of the joint, or simply as a result of aging. This thesis will limit the scope of its study primarily to the development and progression of OA and RA.
1.1. Overview of OA
Osteoarthritis (OA) is a slowly progressive disease that primarily strikes load-bearing joints such as hands, knees, hips, and shoulders. Although its exact pathophysiology not completely understood, this debilitating disease generally affects the aging population. It is estimated that 68% of Americans over the age of 55 have radiographic evidence of OA, with almost 85% of the population affected by age 75. The prevalence of OA is expected to increase. Clinically, OA is characterized by pain, inflammation, deformity, osteophyte formation, and limited range of motion. Pathological changes include progressive articular cartilage loss and destruction, osteophyte formation, subchondral bone sclerosis and synovial inflammation.
Joints affected by OA often exhibit symptoms in neighboring tissues. The joint capsule is usually thickened and may adhere to the deformed bony structures, which may contribute to the limited joint movement. Histological evaluation of the capsule often shows, especially in cases of advanced OA, areas of inflammatory infiltrate, neovascularity, hyalinization, amyloid deposition, and cell loss.
1.2. Overview of RA
Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disorder where the immune system targets and attacks the joints. It is a disabling and painful inflammatory condition, which can lead to substantial loss of mobility due to pain and joint destruction. RA is a systemic disease, often affecting extra-articular tissues throughout the body. As a joint disease, it is commonly polyarticular [1] and its involvement of many joints distinguishes rheumatoid and other inflammatory arthritis from non-inflammatory arthritis such as OA [2, 3].
Chapter 1: Introduction
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In RA, damage to articular cartilage begins at the cartilage-pannus interface, with progressive erosions also developing into the subchondral bone. The bone damage in the joint includes focal erosions and juxtaarticular osteopenia. The effect of RA on bone, however, is also observed systemically in the axial and appendicular skeleton, with reductions in bone mineral density (BMD) causing osteoporosis and, as a consequence, increased risk of fracture. Bone loss is a typical pathological feature of RA and many patients with RA have radiographic evidence of substantial joint damage within the first 2 years of disease with evidence of bone erosion detected with magnetic resonance imaging as early as in the first few months [4, 5]. Most vulnerable to inflammatory damage is the subchondral bone adjacent to inflamed synovial tissue, and, early after disease onset, this particular area faces rapid destruction, which results in local bone erosion and periarticular demineralization.
As the pathology progresses the inflammatory activity leads to erosion and total destruction of the joint surface, and impairs their range of movement and finally leads to deformity of the joint. About 60% of RA patients are unable to work 10 years after the onset of their disease.
2. Introduction of Articular Joints
Human articular joints are composed of several different tissues (cartilage, calcified cartilage, bone, synovium, ligament) that function interdependently to allow the joint to function for many years under normal conditions. These tissues are all important to the health of the whole joint organ, and when one tissue is compromised, it inevitably has an impact on the others. When this delicate balance between the tissues is upset, it often triggers a cascade of abnormal physiological reactions, ultimately leading to the total failure of the joint.
Normal joint anatomy: The primary bearing surface in a synovial joint is the articular cartilage (Fig. 1). The collagen and proteoglycans in the articular cartilage are arranged to withstand primarily tensile and shear stresses at the surface, and compressive stresses in the deeper cartilage layers6. Collagen tends to be oriented parallel to the surface in its superficial layers, and gradually is re-oriented to be perpendicular to the surface as one moves into the deeper radial zone just above the tidemark (i.e., the junction between the articular cartilage and the calcified cartilage)7,8. At the same time, the proteoglycan content increases in the matrix from the articular surface to the tidemark6. Deep to the articular cartilage, and separated from it by the tidemark, is a layer of calcified cartilage. The calcified cartilage is not very vascular normally, if it is vascular at all, and so the remodeling process is not going to be very effective here. But there is a process of ongoing endochondral ossification at the tidemark that can cause the calcified cartilage to thicken, and may contribute to subchondral sclerosis, as observed in radiographs (Fig. 2).
Experimental Arthritis: in vitro and in vivo Models
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3. Introduction to Articular Cartilage and Function
Articular cartilage uniquely designed to provide diarthrodial joints with excellent wear, friction, and lubrication properties required to sustain continuous gliding movements. Its resilient structure also serves to absorb mechanical shock and to evenly spread the applied load unto the bony supportive tissue below.
Articular cartilage consists of a highly organized extracellular matrix (ECM) sparsely populated with specialized cells called chondrocytes. The ECM primarily consists of a combination of proteoglycan, collagen, and water, along with trace amounts of other proteins. These components all combine to give articular cartilage its complex structure and unique mechanical properties.
One unique characteristic of articular cartilage is how its structure and composition vary according to its depth from the articular surface to the subchondral bone. These differences include cell shape and volume, collagen fibril size and orientation, and proteoglycan density. From its structural characteristics, the cartilage can be divided into four distinct zones: the superficial zone, the transitional zone, and the deep zone, and the zone of calcified cartilage.
The superficial zone is the uppermost zone of the cartilage and forms the gliding surface for the articular joint. Its thin collagen fibrils are arranged parallel to the surface to lend strength against the shear stress and it contains the lowest density of proteoglycan. It can readily be identified by the presence of chondrocytes elongated with the long axis parallel to the surface. The transitional zone contains collagen fiber with larger diameters and the chondrocytes take on a more rounded appearance. In the deep zone, collagen fibers of large diameters are aligned vertical to the joint surface; this layer contains the highest density of proteoglycan and the lowest concentration of water. The chondrocytes there are spherical and are often arranged in columns. The final layer, the zone of calcified cartilage (ZCC), separates the cartilage tissue from the underlying subchondral bone and is characterized by small pyknotic cells distributed in a highly mineralized cartilaginous matrix.
3.1. Chondrocytes in Articular Cartilage
Chondrocytes are responsible for the synthesis and maintenance of articular cartilage. Chondrocytes are originally derived from mesenchymal cells, which differentiate during development to the chondrocytes phenotype. During development, densely populated chondrocytes generate the large amount of ECM, but by maturity, they occupy less than 10% of the total tissue volume. Although the low cell density limits the tissues ability to heal and respond to trauma, the metabolic properties of chondrocytes are found to be essential for the maintenance of the ECM against normal wear and tear. Studies have shown chondrocytes to respond to a variety of stimuli, including soluble mediators such
Chapter 1: Introduction
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as growth factors, interleukins; matrix composition; mechanical loads; and hydrostatic pressure changes.
3.1.1. Biochemistry of Articular Cartilage
Collagen in Articular Cartilage Articular cartilage is composed of 68-85 % water. The second most abundant component of cartilage is collagen, which constitute approximate 10% to 20% of the tissue’s wet weight. Collagens are structural macromolecules within the ECM and there are at least 15 distinct collagen types composed of over 29 genetically distinct chains. All members of the collagen family contain a characteristic triple helical structure made up of three polypeptide strands, which is interrupted by one or more nonhelical domains. Over 50% of the dry weight of articular cartilage consists of collagen. The most common form of collagen in hyaline cartilage is type II (90%-95%), with trace amounts of collagen type V, VI, IX, X, and XI also in the found in the matrix. In general, the primary function of collagen in articular cartilage is to provide the tissue’s tensile properties and furnish the matrix infrastructure that immobilizes the proteoglycans within the ECM. Collagen fibers in cartilage are generally thinner than those seen in tendon or bone, and vary from 10 to 100 nm, although their width may increase with age and disease.
Proteoglycan in Articular Cartilage The third most abundant component of articular cartilage is proteoglycan molecules and it constitutes 4% to 7% of the cartilage’s wet weight. Proteoglycan are complex macromolecules that, by definition, consisted of a protein core to which are linked extended polysaccharide (glycoaminoglycan) chains. Aggregate size can vary with age and disease state, but an average aggregate can contain up to 200 aggrecan molecules. By assembling the small precursor molecules produced by the chondrocytes, these large proteoglycans are embedded within the collagenous network of the cartilage.
Once packed into the collagen network, the charged proteoglycans molecules create positive Donnan osmotic pressure in their physiological environment, giving rise to swelling pressure of cartilage. The size of the proteoglycan molecules prevents significant diffusion or hydrodynamic convective transport of these molecules through the ECM. Trapped within the ECM, the size, structural rigidity, and molecular conformation of the charged proteoglycans has a strong influence on the mechanical behavior of the cartilage matrix. In addition, the capacity of proteoglycans to network amongst themselves further enhances the ability of cartilage to maintain structural rigidity and contributes to the stiffness and strength of the ECM. Also of note is the lack of covalent bonds between the proteoglycans and collagen, which may allow the collagen fibers to slide easily through the proteoglycan gel and better resist the tensile stresses developed within the matrix during variable loading conditions.
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3.1.2. Biomechanics of Articular Cartilage The articular cartilage of diarthrodial joints is subject to high loads applied statically, cyclically, and repetitively for many decades. Thus, the structural molecules, that is collagen, proteoglycan, and other quantitatively minor molecules, must be organized into a strong, fatigue-resistant solid matrix capable of sustaining the high stresses and strains that develop within the tissue from these loads. As a material, this solid matrix can be described as being porous and permeable, and very soft. Water, 65% to 80% of the total weight of the tissue, resides in the microscopic pores, and its hydraulic pressure effect that is responsible for much of the load absorbing properties of articular cartilage. The water embedded in the matrix may be caused to flow through the porous-permeable solid matrix by a pressure gradient or by matrix compaction. Thus, the biomechanical properties of articular cartilage are mainly defined by the swelling properties of proteoglycans and the reinforcement by collagen.
4. Problems of Articular Cartilage Repair
Under normal physiological condition, articular cartilage can perform these essential biomechanical functions over the entire human lifespan. However, in the instances of trauma, this delicate balance of matrix metabolism and maintenance may be disrupted. The same structural properties of articular cartilage that lends it its mechanical toughness and function now retards its ability to heal and recover from injuries.
Many mechanical tissues, such as skin and bones, undergo a typical healing response to injury by filling the injury site with scar tissue, consisted usually of densely packed collagen fibrils and scatter fibroblasts. Scar tissue is then remodeled over an extended period of time where cells and ECM content is replaced and reorganized. In many tissues, the dense scar tissue, composed primarily of collagen type I molecules, serves to restore structural integrity and limited function. Because the mechanical properties in normal cartilage arise from a combination of both its material constitution as well as its complex architecture, newly formed scar tissues cannot effectively replace normal cartilage functions in the high stress environment.
One major obstacle to cartilage repair is the lack of a blood supply. The repair of musculoskeletal tissue defects normally begins with inflammatory response and requires a population of cells that migrate into the injury site, proliferate, differentiate, and synthesize new matrix. The lack of blood vessels in cartilage tissue prevents the migration of undifferentiated mesenchymal cells from entering the injury site. The only cell type to be found in the cartilage tissue is the highly differentiated chondrocyte, which has a limited capacity for migration and proliferation due to its encasement within the dense ECM.
The native population density of chondrocytes in mature cartilage is very low compared to that of cartilage tissue in development. While chondrocytes in mature cartilage do
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synthesize sufficient quantities of matrix macromolecules to maintain the matrix under normal wear and tear conditions, the level of matrix production is insufficient for the repair of defects. The number of chondrocytes present in the cartilage tissue also declines with age, further reducing the tissue’s capacity to repair itself.
Another major obstacle to cartilage repair is its location at the ends of diarthrodial joints, where it is usually subjected repetitive stress throughout the healing process. Mature cartilage tissue is able to sustain these relatively high loads due to its unique matrix composition and complex organization of structural molecules into a tough, biphasic composite. However, the fibrous scar tissues that usually form exhibit lower elastic modulus and higher permeability than normal tissue. Because the original tissue matrix is so highly organized, newly formed scar tissues also do not effectively integrate into native matrix, creating high stress area that negatively affect the fluid-pressure load-carrying capacity of the original tissue.
5. Current OA Treatments
Due to complexity of the disorder and the large number of obstacles to tissue repair, the current treatment of OA is usually limited to relieving pain, improving the range of motion, and promoting partial regeneration and/or slowing the degeneration of the cartilage tissue.
5.1. Medications
Most drug treatments of OA primarily focus on pain relieve, although some are target at other symptoms and slowing disease progression. Analgesics are drugs designed to relieve pain without dealing with the issues of inflammation or swelling. Although these drugs tend to have fewer side effects, they play no part in slowing disease progression. NSAIDs play the dual role of pain relief as well as reduction inflammation and swelling associated with OA. Cox-2 drugs are targeted NSAIDs that do not cause the stomach irritation associated with traditional NSAIDs. Injectable glucocorticoids are steroids that are injected intra-articularly for fast, targeted pain relief. Viscosupplementation involves a series of hylauronic acid (HA) intra-articular injections over a period of weeks, aiming to restore the lubricating properties of the synovial fluid.
5.2. Surgery
Surgical intervention is also available for the treatment of OA, with most procedures aiming to relieve pain and delay disease progression, without the potential for true tissue restoration or reverse of disease progression. Arthroscopic surgery is often used to remove loose cartilage tissues, repair tears, smooth a rough surface or remove diseased synovial tissues. Osteotomy can used to increase joint stability by redistributing the
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weight and altering the loading mechanics to protect the injured joint tissues. Osteotomy is most useful for patients with unilateral hip or knee osteoarthritis, who are usually too young for a total joint replacement.
Arthroplasty allows for the total replacement of a joint with engineered materials and is usually recommended for patients over 50 years old or have severe disease progression. Although this procedure usually yields pain-free, limited joint functions, the new joint typically only last between 20 and 30 years and do not serve as a permanent treatment solution.
5.3. Alternatives
Osteoarthritis (OA) may also respond to some alternative or complementary therapies, such as:
Glucosamine and Chondroitin Sulfate. Glucosamine is a naturally occurring aminomonosaccharide in the human body and is one of the principal substrates used in the biosynthesis of glycosaminoglycans, proteoglycans, and hyaluronan, all of which are fundamental components of articular cartilage. In several European double-blind studies in the early 1980s, investigators reported that oral glucosamine decreases pain and improves mobility in human OA with no significant side effects (ref). Chondroitin sulfate is part of a protein that gives cartilage elasticity. Taken together, these two dietary supplements have shown significant relieve of OA symptoms and similar pain relief as achieved with NSAIDs, although the supplements may take longer to begin working.
Vitamins. Some research has shown that certain antioxidants in vitamins may help relieve symptoms of osteoarthritis. In general, vitamins from whole foods are believed to be more readily absorbed by the body than supplements. Vitamin C has been shown to counteract the wearing away of cartilage in animals with OA and is associated with decreased OA progression and pain in humans. Vitamin E provides some pain relief to people with OA and Vitamin D may also have preventative qualities for OA.
Tissue Engineering Due to the lack of inherent healing capacity, recent attempts to repair cartilage lesions and defects include the construct of cell-seeded scaffolds to facilitate the formation and ingrowth of cartilage matrix [6] [7]. Although chondrocyte seeding were met with some success in the repair of cartilage defect, no cell transplantation was used for the treatment of OA. The complex diseased conditions in the OA joint poses many obstacles to successful treatments with the transplantation with bare cells, which are very sensitive to changes in their environment.
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Recent tissue engineering approaches focus on the development of whole tissue implants. One of the problems with cartilage repair is the tendency of the repair tissue form fibrocartilage instead of hyaline cartilage, which has markedly inferior mechanical and physiological function under loading conditions. By developing 3D tissue cultures, researchers have reduced the tendency of chondrocyte to de-differentiate into fibroblasts, thus preserving the matrix content of the engineering tissue.
However, even with the availability of engineered hyaline cartilage, repair of cartilage defect is still far from a complete success. One of the major obstacles with cartilage tissue implantation is the lack of integration with the native tissue [6, 8, 9]. This creates high stress boundaries at the implant edges and ultimately leads to the destruction of the implant. One of the study focuses in this thesis is to overcome the issues of tissue integration. The study examined the feasibility and development of biphasic cartilage constructs for implantation [7].
6. Development and Progression of OA
Despite its prevalence in the elderly population, the exact etiology of OA is unknown, although age, obesity[10], degree of physical activity [11, 12], coexisting metabolic diseases [13], abnormal joint loading [14] [15, 16], trauma, muscular disorders, and genetic factors may be contributory factors. Epidemiologic studies demonstrated a consistent correlation of obesity with the development of OA [17-19]. On the whole, the onset of OA is insidious, usually without a history of trauma. Pain, usually associated with motion, is the first noticeable symptom and progressively become more severe with the development of OA, although little or no other evidence may be found in early physical examination.
6.1. Typical OA Progression
Although OA is a disorder primarily associated with aging, cartilage tissue can remain perfectly healthy for many older individuals. Studies have established that there is a higher water content and decreased proteoglycan density in aging cartilage tissue, which can compromise the tissue performance under normal physiological loads. As the mechanical integrity of the tissue degenerates, the native cells and surrounding tissues are placed under higher than normal stress, often eliciting an inflammation response from the tissues. Inflammation of the joint in turn triggers a series of biological events that further degenerates the cartilage matrix. Due to the weakening of the cartilage matrix, previously acceptable loads now becomes damaging to the embedded chondrocytes. Many studies have shown high compressive loads to trigger the apoptosis of the chondrocytes in the cartilage tissue. With the loss of the already sparse population of chondrocytes, the tissue’s ability to synthesize and maintain the matrix is even further reduced. This cycle of cell death and matrix degeneration is one of many
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mechanisms in OA that is responsible for its progression and ultimately the total destruction of the cartilage tissue.
Another factor attributed to aging is the steady decline in the quality and quantity of proteoglycans in the cartilage matrix. Studies have shown that the levels of proteoglycan decline with age [20]. The keratan sulphate content of extracted proteoglycans increased while the chondroitin sulphate content decreased [21]. Due to a decrease in the chondroitin sulphate-rich region of the proteoglycan monomers, the extracted proteoglycans molecules were smaller in the aging cartilage. At the onset of OA, changes in proteoglycan content in cartilage tissue become even more evident. The degenerated cartilage demonstrates increased levels of proteoglycans, but proteoglycan monomers extracted from the degenerated cartilage are smaller than those from normal cartilage of the same age[21]. The proteoglycan molecules from OA tissue also exhibit a smaller chondroitin sulphate-rich region and some of the molecules also appeared to lack the hyaluronic acid-binding region. These molecular changes significantly impact the aggrecan’s ability to aggregate and form large, shock absorbing complexes found in normal tissue. This degradation only become more severe with the onset of OA and contributes to the disorder’s accelerating progression.
The collagen network that is responsible for much of the tissue’s tensile strength is also significantly affected by the onset of OA [22]. While there is no evidence for collage loss relative to the total mass of the tissue, there are marked variations in the size and arrangement of the fibers. Generally, this remodeling yields a much less orderly network and may be responsible for the swelling of the surface and increased water content of the tissue. Proteoglycan aggregates cartilage tissue is also shown to be more easily extracted from OA with aging. This may be due to a combination effect of both a decrease in the size of the aggregates as well as a degradation of the quality of the collagen network that usually holds these molecules in place.
Although thought to originate in the cartilage, OA affects remarkable changes in the bony structures of the joint. Late stage OA is associated with significant remodeling of the underlying bone structure, including the thickening of the cortices and changes in trabecular stress lines.
6.2. OA as a Whole Joint Disease
Although progression of advanced OA is often characterized by the severe changes of cartilage and bony tissues, early studies focused primarily on cartilage changes in their investigations. One reason why cartilage was in the early research spotlight may be due to its lack of intrinsic healing properties, while bone is known to have a high capacity for regeneration. It was probably thought that the bottleneck for successful OA treatment lies in cartilage repair. However, because of the complex and intimate
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relationship between bone and cartilage, many treatment efforts that focus on cartilage alone invariably fail.
As OA progresses, it recruits the involvement of additional tissues, further increasing the complexity of the disorder. Due to this self-feeding nature of OA development, preventative or early stage treatments are more likely to be successful. As such, knowledge of key interactions between different tissues in the initiation of OA is critical in treatment development. Concentrating only on bone and cartilage separately provides an overly simplistic view of the OA joint. One of the scopes of this thesis is to examine the complex interactions of different tissue in OA progression, especially focusing on early stages of disease development.
More recently, OA was recognized as a disease that involves the entire joint as an organ, not just the cartilage tissue [23-26]. Although loss of cartilage is a definitive characteristic of OA progression, all tissues in the joint organ are ultimately affected by the disease. OA development and progression, as its name indicates, is especially mated to the structural, biochemical, and physiological changes in the bone tissue.
6.3. Relationship between Articular Cartilage and Subchondral Bone
The role of subchondral bone in the pathogenesis of cartilage damage has likely been underestimated. Subchondral bone is not only an important shock absorber, but it may also be important for cartilage metabolism [27].
In the normal diarthrodial joint, articular cartilage and subchondral bone act together in transmitting load pressure through joints. Therefore, the integrity of both tissues is necessary for adequate joint function [28, 29]. Also, the condition of the articular cartilage depends on the mechanical properties of the adjacent subchondral bone [30] [31]. The changes in the density and the architecture of the underlying subchondral bone have a profound effect on both the initiation and progression of cartilage degeneration [32]. The thickening of the subchondral plate influences the increase of the internal cartilage stress which leads to increased hardening of the subchondral bone or progressive thinning of the cartilage layer [33-35].
OA progression can be understood only if the relationship between bone and cartilage is fully appreciated [36]. Although limitations in current technology do not allow early detection of cartilage breakdown, changes in bone metabolism is shown to be linked to the formation of lesions, providing a potential route of detection for OA development.
6.4. Bone Structure and Metabolism
Although collectively termed as subchondral bone, this tissue is actually composed of two phases, each with very different structural and biological properties. The layer adjacent to the cartilage layer is the subchondral bone plate, analogous in structure and
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composition to the cortical layer in long bones. This layer provides a smooth, continuous supporting surface for cartilage tissue, and forms the osteochondral juncture with the zone of calcified cartilage.
The trabecular bone lattice structure beneath the subchondral plate is also classified as part of the ‘subchondral bone’. This portion of the subchondral bone differs from the bone plate morphologically, physiologically, and mechanically. The biological mechanisms for growth and remodeling are also distinct and the two portions may respond to stress and OA progression differently. Unlike the dense subchondral bone plate, the cancellous trabecular bone structure consists of a complex network of trabeculae, and is shown to exhibit a higher rate of turnover and remodeling, and is especially sensitive to changes in the loading environment.
Subchondral bone is highly vascular, although many of the vessels terminate at or before the osteochondral juncture, and do not penetrate into the articular cartilage except in cases of disease. The vascular pores average 89 µm in diameter, and while they may have served as pathways for nutrition of developing cartilage in the young, a recent study demonstrated that they lack the diffusive permeability to support that function in adult tissues. Instead, the primarily function of these vascular space is likely to be reserved for the nutrition of the subchondral bone alone and to support its remodeling in response to joint loading. The vascularity of the plate begins to decline by the third decade and continues to diminish until age 50-70 when perfusion normally associated with OA occurs. This network of vascular pores in the subchondral bone is suggested to play a critical role in the rapid densification of subchondral bone after joint overload and the formation of bony sclerosis in OA joints.
6.5. Bone Structure, Material, and Mechanical Properties
To understand the involvement of bone changes and their effects on OA progression, a deeper examination of bone properties must be considered. First, it must be clear that bone’s mechanical properties are combined products of both its structural and material properties. Structural properties of bone include its matrix deposition, the number, thickness, and orientations of its trabeculae, as well as their angle and level of the connectivity. Bone material properties, on the other hand, concern itself primarily with the material density, degree of mineralization, and molecular composition of the tissue.
Many radiographic assessments associated with the diagnosis of OA, such as the measurement of the joint space gap, are a direct reflection of the bone’s material properties. The true volume of bone, if defined as both neo and old bone tissue, cannot readily be measured by normal radiographic means. This is critical in the understanding of OA development because it is the mechanical properties of bone, rather than its material properties, that determine its true impact on the adjacent cartilage tissue. The course of bone remodeling is a combination of resorption and formation processes (see
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section on bone remodeling) and new bone matrix, while contributing to the structural properties of bone cannot be captured in radiographic imaging. Conversely, newly mineralized bone, which is usually counted towards bone density in radiographic measurements, has yet to achieve its normal levels of stiffness. This discrepancy between measured ‘apparent’ bone density, and ‘true’ bone density makes characterization of bone properties difficult, especially in case of OA, where bone remodeling is extremely active.
6.6. Bone Remodeling
Bone remodeling is a process in which damaged bone attempts to repair itself. The damage may occur from either an acute injury or as the result of chronic “wear and tear” such as that found in osteoarthritis.
Subchondral bone has long been known to be thickened in joints involved by OA; this has been observed on plain radiographs as well as detected by orthopaedic surgeons at time of total joint replacement. Approximately 20 years ago, Radin and Rose [31] proposed that alterations in underlying subchondral bone stiffness may be an important etiologic factor in the subsequent overlying cartilage damage. Dieppe and colleagues [37] studied 94 OA patients for 5 years. Their results strongly support a close relationship between progressive cartilage damage and destruction and underlying bone remodeling activity.
More recent studies have documented acceleration of subchondral bone turnover accompanied by specific architectural changes in the subchondral trabecular bone of OA joints [38-40] [41].
6.7. Possible Initiation of OA as a Bone Disease
Initially believed to be a cartilage disease, recent evidences point toward subchondral bone’s active involvement in the initiation and progression of OA, as first suggested by Radin and Rose in 1970 [42]. Many studies since then have reported dynamic morphologic changes in subchondral bone during the evolution of OA, now recognized as a hallmark of disease progression. Several reports pointed out that the subchondral bone remodeling in OA involves a delicate balance of both bone resorption and formation. In the early stage of OA, bone resorption was described as the primary feature of the bone remodeling process. In contrast, bone formation was predominant in the more advanced stage of the disease. It is generally agreed that bone remodeling and metabolism do steadily decline with age and, especially in regions of high stress and strain, turnover rates are depressed, resulting in older and more mineralized tissue. However, at late stage OA, the situation is reversed, where studies showed that the weight-bearing areas of the femoral head exhibit greater remodeling activity than their unloaded counterparts.
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Moreover, evidence is now accumulating that certain types of primary OA might initially be a bone disease rather than a cartilage disease [43, 44], although other studies concluded that subchondral bone changes may be secondary to cartilage damage and then proceeds deeper into subchondral bone with increasing cartilage degeneration. Recent studies addressed the role of bone changes during the early stage of experimental OA in animal models. By suppressing bone turnover, calcitonin and bisphosphonates have been shown to reduce the severity of cartilage lesions in animal models [45] [46] [47] of OA as well as in humans [48, 49] [50]. In addition, imaging studies have shown that bone metabolism changes occur before cartilage damage.
6.8. Antiresorptive Drugs as a Potential Treatment for OA
Due to the significant correlation between bone remodeling and OA progression, anti-resorptive drugs have been proposed as potential treatment candidates for OA [48, 49, 51]. Evidences of subchondral bone change in OA were described in many clinical studies, including subchondral sclerosis, cyst and osteophyte formation. While the exact role of the bone changes in the initiation and progression of OA is still being debated [52, 53], it is clear that the mechanical properties of the subchondral bone have a direct effect on the functional integrity of the overlying cartilage. Increased subchondral bone stiffness reduces its ability to dissipate the load and evenly distribute the strain generated within the joint, resulting in higher stress in articular cartilage during impact loading, and accelerating cartilage damage over time. Accordingly, cartilage damage progresses into full-thickness cartilage loss only upon repetitive loading over an already stiffened subchondral bone plate.
Increased subchondral bone resorption and turnover was observed both in patients and in experimental OA [25, 54]. Following this correlations, studies were conduct to investigate the potential of anti-resorptive drugs to slow the progression of OA. Calcitonin, an anti-resorptive bone drug, was reported to reduce the severity of the bone, cartilage, and synovium change in the early stages of canine ACLT experimental OA. Workers have also shown that Zoledronate, a member of the bisphosphonates family, has a partial chondroprotective effect in the Carrageenan rabbit model of inflammatory arthritis by preventing bone resorption.
Bisphosphonates is a group of chemicals that bind strongly to the mineral component of bone and have been shown to be potent inhibitors of osteoclastic resorption. Alendronate (ALN), a second generation of bisphosphonates, is a potent inhibitor of osteoclast-mediated bone resorption. At present, ALN is used clinically for the treatment and prevention of osteoporosis, Paget's disease, and for reducing osteolysis around prostheses to prevent implant loosening. Patients receiving prolonged ALN therapy have shown histologically normal bone. ALN has demonstrated to improve the mechanical properties and microstructure of trabecular bone even after short-term
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treatment in a canine experiment. One of the studies included in this thesis will examine the effects of ALN treatment on the progression of OA in a spontaneous and accelerated guinea pig model.
6.9 Development of Osteophytes
Appearance of osteophytes in OA joint is so consistent that their presence is used as for radiographic diagnosis of the disease. They initially form at the margins of joints, originally as cartilage outgrowths that form bony tissue by a process of endochondral ossification. Therefore, anti-resorptive drugs, which inhibit bone formation, have no effects on osteophyte formation. On the other hand glucocorticoid, an anti-anabolic drug for connection tissues is shown to have inhibitory effects on osteophytosis.
The exact function of osteophytes is unknown, as is their specific role in OA progression, although some speculate that they may serve to stabilize the joint. Mature osteophytes, on the other hand, may serve limit joint movement and represent a potential source of pain. Whatever their function, it is clear that osteophyte formation is part of the body’s adaptation to the altered and high stress environment induced by the progression of OA. Due to their consistent formation in OA joints, investigation of osteophytes may yield useful insight in the initiation and progression of OA.
6.10. Complexity of OA Development and Progression Pathways
The complexity in both the development and the progression of OA can be attributed to the highly intimate relationship between cartilage, subchondral bone, and neighboring tissues and the complex level of organization necessary for normal joint function. Multiple biochemical pathways, both cell and non-cell mediated, and biomechanical pathways exist to potentially initiate and to fuel the progression of the disease. This problem is only compounded by the tissues’ significant remodeling in response to the increased levels of stress, especially in the subchondral bone, and completely changes both the physiology and loading dynamic of the joint with disease progression. In addition, pain and limitation in range of motion with the progression of OA may also affect the way diseased individuals move and load their joints, adding yet another level of complexity to disease development.
At the microscale, changes to the tissue is as remarkable, if not even more profound than the macro-changes. In the cartilage alone, there exist numerous highly interdependent organization and pathways that can affect the progression and development of the disease. Its two primary matrix molecules, collagen and proteoglycan, have an intimate biochemical relationship in both their organization, synthesis, and repair, as well as a interdependence in their biomechanical function. A compromise in the function of one constituent can elicit a mechanical and potentially damaging effect on the other. The quality and quantity of their content in the ECM in turn has both a direct biochemical
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and an indirect biomechanical effect on the metabolism and production of the native chondrocytes. The resulting high stress level can lead to cell death or abnormal cell metabolism, both with evident influence on the matrix maintenance and the remaining cell population.
Bone, while it normally retains a higher capacity for healing than cartilage, do not necessarily respond in a manner that benefits the entire joint. In response to the higher stress levels, bone remodeling begins with an increase in bone metabolism, which initially decrease the level of mineralization in the tissue coupled with an increase in bone mass, followed by an increase in mineralization at later stages. The physiological change in the subchondral bone has a direct biomechanical impact on the cartilage tissue layer on top, perceived as an initial drop in stiffness and then a significant decrease in shock absorption. Other changes associated with bone remodel such as increase in vascularity and biochemical changes in the microenvironment may also have a significant effect on all tissue of the joint, in particular, perhaps playing a crucial role in the formation of osteophytes.
Because of the high level of complexity in the initiation and progression of OA, understanding the mechanical and specific pathways of the disease at different stages is critical for the development of therapeutic and preventative treatments. New evidences have shed new light on the significant of bone as not only a mediator but a potential initiator of OA. A primary focus of this thesis will be to elucidate the specific roles and functions of subchondral bone in the developing stages of the disorder, especially in the early stages.
7. Need for Animal Models
Osteoarthritis (OA) is the most common form of arthritis, characterized morphologically by destruction of cartilage, sclerosis of subchondral bone, formation of cysts, and the presence of osteophytes at the joint margins. Studies of early primary osteoarthritis in humans are difficult because of the subtle progression of the disease, and by the time it is discovered, patients are mostly at the late or advanced stages of OA. Due to the unavailability of human tissue samples in the early stages of OA progression, animal models were developed and proved to be invaluable in the study of OA development.
Therefore, animal models are commonly employed in the study of OA pathogenesis and potential therapeutic option of disease. Spontaneous OA occurs in the knee joints of guinea pigs, mice, dogs and nonhuman primates. The advantage of naturally occurring OA in animal models is that they possess similar pathology and pathogenesis to that of humans. However, such models usually exhibit slow disease progressions and are susceptible to large variability between individuals. Surgically induced OA models have also been developed in various animal species, using such methods as anterior cruciate ligament transection in dogs and rabbits, total or partial meniscectomy in guinea
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pigs and rabbits, and the combination of meniscectomy and ligament transection. Most of the surgically induced models showed rapid disease progression, which resembles secondary osteoarthritis after trauma.
Dunkin Hartley guinea pig is a widely used spontaneous OA animal model, and a partial meniscectomy could make the arthritis change occurring earlier and more predictable; the cartilage histopathological change and disease progression in this model was well documented by A. Bendele in 1987. We have modified this model by dissecting out middle 1/3 of the medial meniscus to induce a chronic osteoarthritis, the histopathological change of this model is similar to that of spontaneous guinea pig OA but in an accelerated pace.
8. Objectives and outlines
By studying and developing tools and models for the investigation of early OA, this thesis aims to contribute to the understanding of the OA development and the creation of effective treatments.
A primary focus of this thesis will be to elucidate the specific roles and functions of subchondral bone in the developing stages of the disorder, especially in the early stages. The first study in this thesis is to overcome the issues of tissue integration, this study examined the feasibility and development of biphasic cartilage constructs for implantation. The continued three studies are focused on the methods and analysis of bone quality. The focus of other three studies is to characterize the development and progression of OA and RA. One of the scopes of this thesis is to examine the complex interactions of different tissue in OA progression, especially focusing on the importance of subchondral bone in the early stages of disease development. Using previously characterized animal models, one of the studies included in this thesis will examine the effects of Alendronate (a type of Bisphosphonate) treatment on the progression of OA in a spontaneous and accelerated guinea pig model, other two studies investigated the effects of two different types of glucosamine treatment on a rat RA model and a rabbit OA models.
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30. Behets C, Williams JM, Chappard D, Devogelaer JP, Manicourt DH. Effects of calcitonin on subchondral trabecular bone changes and on osteoarthritic cartilage lesions after acute anterior cruciate ligament deficiency. J Bone Miner Res 2004; 19: 1821‐1826.
31. Radin EL, Rose RM. Role of subchondral bone in the initiation and progression of cartilage damage. Clin Orthop Relat Res 1986: 34‐40.
32. Radin EL, Swann DA, Paul IL, McGrath PJ. Factors influencing articular cartilage wear in vitro. Arthritis Rheum 1982; 25: 974‐980.
33. Radin EL, Paul IL, Rose RM. Role of mechanical factors in pathogenesis of primary osteoarthritis. Lancet 1972; 1: 519‐522.
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34. Radin EL, Abernethy PJ, Townsend PM, Rose RM. The role of bone changes in the degeneration of articular cartilage in osteoarthrosis. Acta Orthop Belg 1978; 44: 55‐63.
35. Shimizu M, Tsuji H, Matsui H, Katoh Y, Sano A. Morphometric analysis of subchondral bone of the tibial condyle in osteoarthrosis. Clin Orthop Relat Res 1993: 229‐239.
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37. Dieppe P, Cushnaghan J, Young P, Kirwan J. Prediction of the progression of joint space narrowing in osteoarthritis of the knee by bone scintigraphy. Ann Rheum Dis 1993; 52: 557‐563.
38. Burr DB. The importance of subchondral bone in osteoarthrosis. Curr Opin Rheumatol 1998; 10: 256‐262.
39. Goker B, Sumner DR, Hurwitz DE, Block JA. Bone mineral density varies as a function of the rate of joint space narrowing in the hip. J Rheumatol 2000; 27: 735‐738.
40. Pastoureau P, Leduc S, Chomel A, De Ceuninck F. Quantitative assessment of articular cartilage and subchondral bone histology in the meniscectomized guinea pig model of osteoarthritis. Osteoarthritis Cartilage 2003; 11: 412‐423.
41. Pelletier JP, Boileau C, Brunet J, Boily M, Lajeunesse D, Reboul P, et al. The inhibition of subchondral bone resorption in the early phase of experimental dog osteoarthritis by licofelone is associated with a reduction in the synthesis of MMP‐13 and cathepsin K. Bone 2004; 34: 527‐538.
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43. Dequeker J, Mokassa L, Aerssens J. Bone density and osteoarthritis. J Rheumatol Suppl 1995; 43: 98‐100.
44. Naitou K, Kushida K, Takahashi M, Ohishi T, Inoue T. Bone mineral density and bone turnover in patients with knee osteoarthritis compared with generalized osteoarthritis. Calcif Tissue Int 2000; 66: 325‐329.
45. Manicourt DH, Altman RD, Williams JM, Devogelaer JP, Druetz‐Van Egeren A, Lenz ME, et al. Treatment with calcitonin suppresses the responses of bone, cartilage, and synovium in the early stages of canine experimental osteoarthritis and significantly reduces the severity of the cartilage lesions. Arthritis Rheum 1999; 42: 1159‐1167.
46. El Hajjaji H, Williams JM, Devogelaer JP, Lenz ME, Thonar EJ, Manicourt DH. Treatment with calcitonin prevents the net loss of collagen, hyaluronan and proteoglycan aggregates from cartilage in the early stages of canine experimental osteoarthritis. Osteoarthritis Cartilage 2004; 12: 904‐911.
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48. Buckland‐Wright JC, Messent EA, Bingham CO, 3rd, Ward RJ, Tonkin C. A 2 yr longitudinal radiographic study examining the effect of a bisphosphonate (risedronate) upon subchondral bone loss in osteoarthritic knee patients. Rheumatology (Oxford) 2007; 46: 257‐264.
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50. Manicourt DH, Azria M, Mindeholm L, Thonar EJ, Devogelaer JP. Oral salmon calcitonin reduces Lequesne's algofunctional index scores and decreases urinary and serum levels of biomarkers of joint metabolism in knee osteoarthritis. Arthritis Rheum 2006; 54: 3205‐3211.
51. Burr DB. Anatomy and physiology of the mineralized tissues: role in the pathogenesis of osteoarthrosis. Osteoarthritis Cartilage 2004; 12 Suppl A: S20‐30.
52. Burr DB, Schaffler MB. The involvement of subchondral mineralized tissues in osteoarthrosis: quantitative microscopic evidence. Microsc Res Tech 1997; 37: 343‐357.
53. Findlay DM. Vascular pathology and osteoarthritis. Rheumatology (Oxford) 2007; 46: 1763‐1768.
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Chapter 2
Chapter 2 - Subchondral Bone Microarchitecture Changes in
Animal Models of Arthritis Quantified by Micro-CT
Susanne X. Wang1
1MDS Pharma Services, Bothell, WA, USA
In. Qin Ling, Harry Genant ed. “Advanced Bioimaging Technologies in
Assessment of the Quality of Bone and Scaffold Materials”. Chapter 40 Springer-
Verlag Inc. (Berlin, New York, Tokyo) 2007
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Abstract:
Animal models of osteoarthritis (OA) and rheumatoid arthritis (RA) are used to study the pathogenesis of joint degeneration and evaluate potential antiarthritic drugs for clinical use. Subchondral bone change in arthritis plays an important role in the development of the disease. The study of the subchondral bone change in human arthritis is limited by the access of human tissues especially in the earlier stage. Animal models provide the possibility of studying of the pathogenesis of the disease at different stages and with easier usage of tissues. The development of high-resolution micro computed tomography (micro-CT) scanners, capable of 3D reconstruction at a resolution of 6-50μm, enables researchers to evaluate the changes in trabecular subchondral bone in animal models of arthritis. This chapter reviews the 3D microstructure changes in OA and RA animal models, as well as describing the application of micro-CT in evaluation of subchondral bone changes using 3D and 2D methods.
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1. Introduction
Arthritis actually consists of more than 100 different conditions, osteoarthritis (OA) is the most common form of arthritis; rheumatoid arthritis (RA) is the second common form. Osteoarthritis is one of the most frequent causes of physical disability among adults. More than 20 million people in the United States have OA. Some younger people suffer from OA as a result of joint injuries, but this disease most often occurs in older people. In fact, more than half of the population aged 65 and above may show x-ray evidence of OA in at least one joint.
Osteoarthritis is characterized by the progressive destruction of articular cartilage and concomitant changes in subchondral bone. Clinical research on OA has been limited because it often presents at a late stage of the disease and there is a lack of markers indicative of disease progression. The etiology of primary OA is unknown but secondary OA has multiple causes such as trauma, synovial disease, etc. The pathological mechanisms of OA are poorly understood and current therapy is not directed at the origin of the disease and is mostly symptomatic.
Rheumatoid arthritis is an autoimmune disease that causes chronic inflammation of the joints. It affects up to 1% of the world population. Rheumatoid arthritis can also cause inflammation of the tissue around the joints, as well as other organs in the body. Bone change in RA affects the periarticular and axial skeleton and is a major cause of disability. Osteoporosis of the skeleton has long been recognized in RA patients (Suzuki and Mizushima 1997). The localized osteopenia/osteoporosis seen around inflamed joints is the earliest sign of RA seen on radiographs and is used in RA diagnostics. The presence of focal marginal joint erosions have been regarded as the radiographic hallmark of RA (Sharp et al. 1991; Goldring and Polisson 1998; Goldring and Gravallese 2000). Standard radiographic techniques have demonstrated that these focal bone changes tend to progress throughout the course of the disease, and in general, the presence of extensive erosions tends to correlate with more severe disease activity ; van Zeben et al. 1993).
A variety of animal models of arthritis have been developed to study disease pathogenesis and to evaluate potential anti-arthritis drugs for clinical use. Animal models of OA include spontaneous OA in guinea pigs (Bendele and Hulman 1988; Meacock et al. 1990; Jimenez et al. 1997) and cynomolgus macaques (Carlson et al. 1994), meniscectomy and ligament transection in guinea pigs (Bendele et al. 1991), meniscectomy in rabbits (Colombo et al. 1983), and anterior cruciate ligament (ACL) transection in rabbits (Yoshioka et al. 1996) and dogs (Manicourt et al. 1999). Animal models of RA include collagen-induced arthritis (CIA) models in mouse and rat, adjuvant-induced arthritis (AIA), antigen induced arthritis, and transgenic/knockout mice.
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2. Bone changes in human arthritis
It has long been debated whether initiating OA changes occur primarily in the cartilage or first in the underlying bone, with subsequent cartilage pathology. Unfortunately, studies that address OA changes in a kinetic way in both tissues are limited. Although significant changes in the subchondral cancellous bone, such as sclerosis and cyst formation, are frequently observed in patients with OA, little attention has been paid to changes in the subchondral bone because they have always been considered to be secondary. More than 40 years ago Johnson (Johnson 1962) suggested that bone remodeling, a natural accompaniment of aging, could lead to irregularities of the articular surface, and consequently, cartilage degeneration. Hutton et al (Hutton et al. 1986) showed that in hand OA the increase in isotope concentration was identified in joints with OA prior to the onset of radiographic changes and was predictive of subsequent radiographic abnormalities of joint space loss and osteophyte formation. In human osteoarthritis increased subchondral bone activity, as judged by enhanced uptake of technetium labeled diphosphonate, was shown to predict cartilage loss (Dieppe et al. 1993). These results suggest that cartilage lesions did not progress in the absence of significant subchondral activity.
More recent studies have shown that increased subchondral bone remodeling results in thicker subchondral bone (Grynpas et al. 1991) and increased stiffness (Burr and Schaffler 1997); however, these newly formed arthritic bones are under-mineralized (Grynpas et al. 1991;, Li and Aspden 1997). The weaker bone within thickened subchondral cortical plate and trabeculae of OA joints causes the subarticular osteoporosis (Buckland-Wright 2004). These concepts gained credence from histological and histomorphometric analysis of tibial condyles that showed cartilage degeneration to be influenced by remodeling of underlying subchondral bone (Matsui et al. 1997).
Rheumatoid arthritis is a progressive illness that has the potential to cause joint destruction and functional disability. It exhibits significant bone change, including bone erosion and remodeling. The relationship between the subchondral bone changes, cartilage damage and synovium inflammation are not yet fully understood; however, the bone and joint destruction in RA have been confirmed by biochemical markers and gene expression alterations during RA progression (Goldring 2002; Anastassiades and Rees-Milton 2005; Tchetina et al. 2005).
3. Bone changes in OA animal models
It is evident that changes in the bone occur both in instability and spontaneous OA models. After ACL transection in dogs, the regions of pronounced periarticular cancellous bone mineral density (BMD) adaptation (Boyd et al. 2000a) and microstructural changes (Boyd et al. 2000b) corresponded to focal cartilage defects was
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WERE already observed at week 3 and appeared more prominent at week 12. Similar studies in rabbits identified a correlation between enhanced blood flow and bone adaptation (Shymkiw et al. 2001). In contrast, studies in meniscectomized guinea pigs demonstrated early bone loss at the subchondral level at 1 month, but increased bone density at 3 months after meniscectomy (Pastoureau et al. 1999).
The consequences of altered subchondral bone for the overlaying cartilage layer remain unclear. It has been suggested that only the bone density close to the cartilage surface is a major determinant in altered impact loading of the cartilage and more detailed analysis of focal bone changes, close to expected cartilage lesion sites, is warranted. Interestingly, an increase of ossification is noted in the medial meniscus and not in the lateral meniscus of aging Hartley guinea pigs (Kapadia et al. 2000). Since bone remodeling and cartilage degeneration in this strain is evident with aging in the medial joint compartment, it is suggested that increased ossification of the meniscus had altered joint biomechanics and underlies OA joint destruction.
Several animal experiments (Bailey and Mansell 1997, Dedrick et al. 1993) and human biopsy (Grynpas et al. 1991) studies reported thickening of the subchondral bone plate correlates to articular cartilage degradation. Simon et al. (Simon et al. 1972) promoted the notion that OA begins in the bone by demonstrating that stiffening of subchondral bone preceded cartilage damage in guinea pigs.
In other OA animal models, changes in both bone and cartilage occur as a result of mechanical or surgical alteration of joint loading. For example, impulsive loading of rabbit knees, resulting in increased bone volume, is followed by progressive changes in articular cartilage during the following 6 months (Radin et al. 1984).
Taken together, these results demonstrate that subchondral bone remodeling is linked to cartilage destruction in both human and animals. However, the mechanism by which changes in subchondral bone result in damage to articular cartilage is not readily apparent.
4. MicroCT imaging in arthritis
There are increasing demands on arthritis imaging to identify early bone change and erosive joint damage and predict future structural and functional deterioration. Unfortunately, conventional radiography has been shown to be insensitive for insignificant bone change and erosions, particularly in early stages of the disease. Computed tomography (CT) offers advantages over projectional radiography. Micro-CT is a fast, accurate, powerful method to study skeletal structures. Micro-CT provides superior signal contrast between bone and soft tissue, making it especially appropriate for applications involving measurements of bone density (Genant et al. 1999). High-resolution micro-CT allows the quality spatial view of cancellous and cortical bone. A number of studies have applied micro-CT techniques to non-destructive evaluation of
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trabecular bone (Borah et al. 2001, Gross et al. 1999, Kapadia et al. 1998) and it has been confirmed to be an accurate tool to precisely measure changes in bone stereology (Kuhn et al. 1990, Feldkamp et al. 1989, Goulet et al. 1994, Odgaard and Gundersen 1993) as well as bone volume and microarchitecture (Goulet et al. 1994, Buchman et al. 1998).
Three-dimensional imaging of human OA as yet is limited to late stages of the disease. The strength of X-ray-based techniques like micro-CT is that it offers excellent visualization of bone, and exciting features for diagnosis of disease stages and for disease monitoring. Micro-CT provides spatial resolution better than 10 microns, but the size of the objects that can be scanned is only a maximum 12-15 centimeters. Therefore, the main application of micro-CT in arthritis has been the measurements of bone density and the analysis of three-dimensional microstructure of the bone in animal models, in vivo animal total body scanning (small rodents), human peripheral scanning, and human bone biopsies.
The arthritis animal models mentioned in the previous section develop arthritic pathological changes that carry many characteristics of human arthritis. With application of micro-CT, it is possible to monitor the prominent bony alterations such as volumetric BMD (vBMD), 3D microstructure, bone erosion, osteophyte formation trabecular remodeling (BV/TV), subchondral bone plate thickness, and subchondral sclerosis (Dedrick et al. 1993, Wachsmuth and Engelke 2004, Batiste et al. 2004).
In addition to applications involving measurements of vBMD, BV/TV and joint-space, micro-CT data sets can be rendered and reconstructed into 3D isosurfaces and subsequently used for examination of bone surface irregularities such as osteophyte formation and bone erosions. Batiste et al (Batiste et al. 2004) developed a method for volumetric quantification of osteophyte. Briefly, the osteophytes were manually outlined within each contiguous coronal image section. The total volume of osteophytic tissue was then determined in mm3, based on the known voxel volume.
5. Methodology of using microCT in arthritis research
In our experiments mentioned below we used GE micro-CT scanner (Explore Locus SP micro-CT Scanner, GE Healthcare, London, ON, Canada).
5.1 Sample preparation:
For in vitro scanning, specimens (usually the hind legs) were dislocated from the animal (rabbit, guinea pig, rat and mouse) body, soft tissue could be intact to scan the whole joint, or be dissected off to scan a single bone. Specimens were prefixed with 10% natural buffered formalin or other fixatives, or freshly dissected and immersed in fixative when scanning. Freezing of the sample should be avoided since the ice crystals can alter the fine microstructure within the bone, causing inaccurate measurement.
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5.2 Scanning procedures:
Samples should be firmly fixed in a water-filled specimen tube in a position such that the main axis was kept as parallel as possible to the Z-axis of the micro-CT image coordinate system. This minimized any beam-hardening effects, since the x-ray beam crossed a minimal volume of bone tissue. It is suggested that a sufficient space needs to remain between the edges of the sample to the cylinder wall in order to avoid interference to the scanned image from the test tubes.
The eXplore CT acquisition software from General Electric Medical Systems was used in our studies to create different protocols according to the animal size. For example, mouse and rat bones were scanned at 8-µm isotropic voxel size, with 499 projections, and a total scanning time of 150 min. Rabbit and guinea pig bones were scanned at 20 µm isotropic voxel size, with 299 projections, and the total scanning time was 45min.
The scan was corrected following image acquisition using data acquired from an empty scan in which bright and dark images are gathered. Correction accounts for variations in temperature and attenuation of the x-rays (Feldkamp et al. 1989). The reconstructed tibia images were analyzed with MicroView 2.0 software (GE Healthcare, London, ON, Canada) in two and three dimensions.
5.3 Reconstruction:
A volumetric dataset of 5123 voxels was reconstructed from the 499 (or 299) projections using the reconstruction software. Three dimensional bone analyses were conducted in the reconstructed images. Using MicroView 2.0 analysis software, different regions of interest (ROIs) were selected according to the study purpose. In our guinea pig spontaneous OA model, two of the ROIs were rectangular with dimensions 1.85mm x1.85mm x 0.6mm and positioned in the middle of the lateral and medial epiphysis to measure the 3-D micro architecture of the secondary spongiosa. Figure 1(A) and 1(B) illustrate the location of the epiphyseal ROIs. A third ROI, Figure 1(C), a cylinder 2mm x 2mm x 1mm in size, was placed 1mm below the growth plate to measure the 3-D micro architecture of the primary spongiosa.
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5.4 Threedimensional analysis
Micro-CT images were viewed and analyzed using MicroView software. In 3D analysis, the tissue volume (3D-TV, mm3) and trabecular bone volume (3D-BV, mm3) were measured directly, and the fractional trabecular bone volume (3D-BV/TV, %) was calculated. The trabecular thickness (3D-Tb.Th, µm), trabecular number (3D-Tb.N, N/mm) and bone mineral density (v-BMD) were measured directly on 3D images (Hildebrand et al. 1999). Another parameter obtained is the structure model index (SMI) established by Hildebrand and Ruegsegger (Hildebrand and Ruegsegger 1997). The SMI represents the plate-rod characteristics of trabecular structure. A negative number is indicative of a plate-like structure containing holes, 0 represents an ideal plate-like structure, while 3 represents the ideal rod (cylindrical) structure. Larger SMI values indicate that trabecular structure contains a more rod-like structure in older or diseased bone.
5.5 Threedimensional isosurfaces
A 3-D isosurface image of the specimen was obtained by the rendering of image data and reconstructed into a 3D surface view. This isosurfaces function facilitated visualization of bone surface irregularities, assisting with early detection of
Figure 1:
(A) (B) (C)
Figure 1: Locations of the regions of interest analyzed using the bone analysis application of MicroView. The region highlighted in (A) has dimensions of 1.85 x1.85 x 0.6 mm and was placed in the lateral proximal epiphysis, (B) is a closer look of the same size region at medial epiphysis. The line in (C) is 1mm in length and marks the start of the cylindrical metaphyseal ROI analyzed, this ROI has dimensions of 2mm x 2mm x 1 mm.
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morphological changes related to disease progression. Osteohpyte volume can be quantified using the method described previously (Batiste et al. 2004).
Figure 2A is a 3D reconstructed isosurface of a 12 months old guinea pig femur from our research. Spontaneous OA was developed in the knee joint of this animal, typical osteophytes were formed (highlighted in color). Figure 2B is a rabbit femur at 8 weeks after ACLT, showing massive osteophyte formation. Figure 3 are horizontal (A) and vertical (B) views of rabbits distal femur segmentations. Osteophytes were also segmented. The osteophyte located at the upper left site of (B) revealed thinner cortical shell and much less trabeculae.
Figure 2:
(A) (B)
Figure 2: (A): 3D reconstructed isosurface of a 12 months old guinea pig femur to show the out growth of bonny osteophyte (highlighted in color). (B): A rabbit femur at 8 weeks after ACLT, showing massive osteophyte formation.
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We studied bone changes in a cell wall antigen-induced rat rheumatoid arthritis model. Figure 4A shows a 3D reconstructed isosurface of a rat proximal tibia. Compared to the normal control tibia (Figure 4B), the erosions of tibia plateau are clearly visualized. The method mentioned above (Batiste et al. 2004) can also be used to quantify the volume of erosion by calculating the necessary volume needed to fill the eroded space.
Figure 3:
Figure 3: segmentations of distal femur horizontal (A) and vertical (B) view. Osteophytes were also segmented, the osteophyte located at the upper left site of (B) revealed thinner cortical shell and much less trabeculae.
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5.6 Twodimensional subchondral bone analysis.
Three coronal sections of proximal tibia were obtained from each 3-D reconstructed tibia image: anterior, central, and posterior section (1mm apart for rat and guinea pig tibias). The subchondral trabecular bone structure and connectivity in the epiphyseal region of the proximal tibia were analyzed using an image processing and analysis system (Quantimet 570, Leica, Germany) on the 2-D micro-CT images. After thresholding, a binary image was obtained at the medial and lateral side of the epiphyseal region to analyze the structure parameters according to the ASBMR guidelines (Parfitt et al. 1987): fractional trabecular bone volume (2D-BV/TV, %), trabecular thickness (2D-Tb.Th) and trabecular number (2D-Tb.N, N/mm).
The connectivity parameters of trabecular bone were measured on the skeletonized binary images using an in-house program based on the techniques described by Parisien et al (Parisien et al. 1992). The connectivity parameters obtained in the analysis include: number of multiple points (1/mm2), number of end points (1/mm2), lengths of node-node struts (mm/mm2), lengths of free-free struts (mm/mm2), and total strut length (mm/mm2). Figure 5A illustrates the area of cancellous bone analyzed by strut analysis and Figure 5B defines strut connectivity parameters.
Figure 4:
(A) (B)
Figure 4: (A): Isosurfaces of proximal rat tibiae show the erosions at articular bone of the epiphysis in the arthritis‐induced rat. (B): a smooth intact surface in a healthy rat tibia.
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The subchondral bone plate thickness (Sb.Pl.Th, mm) was also measured on the central section of the 2D micro-CT proximal tibia images using individual point-to-point distance measures. The thickness was calculated by averaging 12 measurements per tibia. All measurements were made in a standardized viewing area by a single observer who was blinded to the experiment.
6. Cartilage imaging using microCT and micro MRI
Cartilage can not be seen directly in the micro-CT images. However, it can be calculated indirectly from the joint space between the two bony edges. The use of contrast agents may help in some circumstance to image cartilage and other soft tissues. Yet this still limits the application of micro-CT to evaluate cartilage in vivo.
As compared with X-ray based imaging devices, MRI provides better soft tissue contrast and the ability to directly visualize articular cartilage, synovium, menisci, and other non-osseous structures both in vivo and ex vivo. Despite its theoretical advantages, due to the inadequacy of the MRI spatial resolution (no better than 100 micron), the current commercially available High-resolution microMRI cannot provide sufficient tissue contrast in order to accurately quantify the cartilage damage in animal models (rodents), especially for smaller lesions (Hutton and Vennart 1995).
7. The future of using microCT in arthritis research
SkyScan (Aartselaar, Belgium, www.skyscan.be) is a company specialized in the development and manufacturing of systems for 3-dimensional non-destructive investigation of an object's internal microstructure. They have put the effort into developing high-resolution micro-CT. The Skyscan 2011 nano-CT scanner obtained a spot size of 0.3 microns, which could reduce the pixel size down to 150nm. This
Figure 5:
(A) (B)
Figure 5: The area of cancellous bone analyzed by strut analysis (A) and defines strut connectivity parameters (B)
Node
End PointNode-Node Strut
End Point End Point
Free-Free Strut
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instrument enables the observation of small osteocytes and microcracks in bone. This dedicated technique gives better quality to micro-CT in studying detailed bone micro architecture and material quality.
Scanco Medical AG (Bassersdorf, Switzerland, www.scanco.ch) has advanced the in vivo application of the micro-CT system. Its in vivo micro-CT measurements provide information in a rapid and non-destructive way, which also gives a good resolution up to 12.5 microns. One has to consider the negative effects of the radiation dose of in vivo Micro-CT and potential contra indication in clinical and preclinical applications. The effective radiation dose for a standard measurement with the “XtremeCT” (in vivo CT for human) is 3mSv, which is the same dose as the natural background radiation per year, and is much lower than the exposure of one time intercontinental flight (50mSv). However, the applied dose to the animals during in vivo CT is much higher due to the high resolution. Therefore, it is important to minimize the dose of radiation without sacrificing the image quality requirements prior to an animal study.
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21. Buckland-Wright C (2004) Subchondral bone changes in hand and knee osteoarthritis detected by radiography. Osteoarthritis Cartilage 12 Suppl A: S10-19
22. Goldring SR (2002) Bone and joint destruction in rheumatoid arthritis: what is really happening? J Rheumatol Suppl 65:44-48.
23. Anastassiades T, Rees-Milton K (2005) Biochemical markers for osteoarthritis: from the present to the future and back to the past. J Rheumatol 32(4):578-579.
24. Tchetina EV, Squires G, Poole AR (2005) Increased type II collagen degradation and very early focal cartilage degeneration is associated with upregulation of chondrocyte differentiation related genes in early human articular cartilage lesions. J Rheumatol 32(5):876-886.
25. Matsui H, Shimitzu M, Tsuji H (1997) Cartilage and subchondral bone interaction in osteoarthrosis of human knee joint: a histological and histomorphometric study. Micro Res Tech 37: 333-342
26. Boyd SK, Matyas JR, Wohl GR, Kantzas A, Zernicke RF (2000a) Early regional adaptation of periarticular bone mineral density after anterior cruciate ligament injury. J Appl Physiol 89: 2359-2364
27. Boyd SK, Muller R, Matyas JR, Wohl GR, Zernicke RF (2000b) Early morphometric and anisotropic change in periarticular cancellous bone in a model of experimental knee osteoarthritis quantified using microcomputed tomography. Clin Biomech (Bristol, Avon) 15: 624-631
28. Shymkiw RC, Bray RC, Boyd SK (2001) Physiological and mechanical adaptation of periarticular cancellous bone after joint ligament injury. J Appl Physiol 90: 1083-1087
29. Pastoureau PC, Chomel AC, Bonnet J (1999) Evidence of early subchondral bone changes in the meniscectomized guinea pig. A densitometric study using dual-energy X-ray absorptiometry subregional analysis. Osteoarthritis Cartilage 7: 466-473
30. Kapadia RD, Badger AM, Levin JM, Swift B, Bhattacharyya A, Dodds RA, Coatney RW, Lark MW (2000) Meniscal ossification in spontaneous osteoarthritis in the guinea-pig. Osteoarthritis Cartilage 8: 374-377
31. Bailey AJ, Mansell JP (1997) Do subchondral bone changes exacerbate or precede articular cartilage destruction in osteoarthritis of the elderly? Gerontology 43: 296-304
32. Dedrick DK, Goldstein SA, Brandt KD, O'Connor BL, Goulet RW, Albrecht M (1993) A longitudinal study of subchondral plate and trabecular bone in cruciate-deficient dogs with osteoarthritis followed up for 54 months. Arthritis Rheum 36: 1460-1467
33. Simon SR, Radin EL, Paul IL, Rose RM (1972) The response of joints to impact loading II: in vivo behaviour of subchondral bone. J Biomech 5: 267-272
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35. Genant HK, Gordon C, Jiang Y, Lang TF, Link TM, Majumdar S (1999) Advanced imaging of bone macro and micro structure. Bone 25: 149-152
36. Borah B, Gross GJ, Dufresne TE, Smith TS, Cockman MD, Chmielewski PA, Lundy MW, Hartke JR, Sod EW (2001) Three-dimensional microimaging (MRmicroI and microCT), finite element modeling, and rapid prototyping provide unique insights into bone architecture in osteoporosis. Anat Rec 265: 101-110
37. Gross GJ, Dufresne TE, Smith T, Cockman MD, Chmielewski PA, Combs KS (1999) Bone architecture and image synthesis. Morphologie 83: 21-24
38. Kapadia RD, Stroup GB, Badger AM, Koller B, Levin JM, Coatney RW, Dodds RA, Liang X, Lark MW, Gowen M (1998) Applications of micro-CT and MR microscopy to study pre-clinical models of osteoporosis and osteoarthritis. Technol Health Care 6: 361-372
39. Kuhn JL, Goldstein SA, Feldkamp LA, Goulet RW, Jesion G (1990) Evaluation of a microcomputed tomography system to study trabecular bone structure. J Orthop Res 8: 833-842
40. Feldkamp LA, Goldstein SA, Parfitt AM, Jesion G, Kleerekoper M (1989) The direct examination of three-dimensional bone architecture in vitro by computed tomography. J Bone Min Res 4: 3-11
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41. Goulet RW, Goldstein SA, Ciarelli MJ, Kuhn JL, Brown MB, Feldkamp LA (1994) The relationship between the structural and orthogonal compressive properties of trabecular bone. J Biomech 27: 375-389
42. Odgaard A, Gundersen HJ (1993) Quantification of connectivity in cancellous bone, with special emphasis on 3-D reconstructions. Bone 14: 173-182
43. Buchman SR, Sherick DG, Goulet RW, Goldstein SA (1998) Use of microcomputed tomography scanning as a new technique for the evaluation of membranous bone. J Craniomaxillofac Surg 9: 48-54
44. Wachsmuth L, Engelke K (2004) High-resolution imaging of osteoarthritis using microcomputed tomography. Methods Mol Med 101: 231-248
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46. Hildebrand T, Laib A, Muller R, Dequeker J, Ruegsegger P (1999) Direct three-dimensional morphometric analysis of human cancellous bone: microstructural data from spine, femur, iliac crest, and calcaneus. J Bone Miner Res 14: 1167-1174
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49. Parisien M, Mellish RW, Silverberg SJ, Shane E, Lindsay R, Bilezikian JP, Dempster DW (1992) Maintenance of cancellous bone connectivity in primary hyperparathyroidism: trabecular strut analysis. J Bone Miner Res 7: 913-919
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Chapter 3
Chapter 3 - Tissue Engineering of Biphasic Cartilage Constructs Using Various Biodegradable
Scaffolds: an in vitro Study Xuanhui Wanga, Shawn P. Grogan a, Franz Rieser b, Verena Winkelmann a,
Véronique Maquet c, Martine La Berge d and Pierre Mainil-Varlet a
a Osteoarticular Research, Institute of Pathology, University of Bern, Murtenstrasse 31, Bern 3010,
Switzerland b Centerpulse, PO Box 65, Winterthur 8404, Switzerland
c Center for Education and Research on Macromolecules, University of Liege, Sart-Tilman, 4000, Liège,
Belgium d Department of Bioengineering, Clemson University, Clemson SC 29634-0905, USA
Biomaterials 2004 Aug;25(17):3681-8
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Abstract
Biological restoration of osteochondral defects requires suitable subchondral support material that also allows the induction of hyaline cartilage tissue. Biphasic implants consisting of pre-fabricated neocartilage and an underlying biodegradable osteoconductive base may meet these requirements. Here we explore various candidate biodegradable support materials onto which neo-cartilage was produced in vitro. Porcine chondrocytes were seeded in a closed and static bioreactor with a base of biomaterial consisting of either poly-L-lactide [P(L)LA], poly-D,L-lactide [P(D,L)LA] or Collagen-hydroxyapatite [Col-HA] and were cultured for 15 weeks. Viable neo-cartilage was produced on each biomaterial with differing amounts of cellular colonisation. P(D,L)LA breakdown was more rapid and uneven among the three biomaterials, leading to constructs of irregular shape. Little or no breakdown or chondrocyte colonisation was evident in P(L)LA. Col-HA constructs were superior in terms of viability, implant morphology and integration between neo-cartilage and biomaterial. These results indicate that our reported system has potential for producing biphasic implants that may be adequate for the repair of osteochondral defects.
Author Keywords: Chondrocyte; Composite; Bioreactor; Biodegradation
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1. Introduction
Current models for biological repair of osteochondral defects do not offer satisfactory long-term results and clinicians need better models to account for such failings. Cell-based therapies used for cartilage repair include: autologous chondrocyte implantation (ACI) as described by Brittberg et al. [1], the combined use of biomaterials and bioreactor systems to promote chondrogenesis, and the subsequent formation of implantable neo-cartilage tissue [2, 3, 4, 5, 6, 7, 8, 9 and 10]. None of these therapies fully restore both the subchondral bone and articular cartilage, but it is evident that a multiphasic implant design must allow for adequate subchondral repair to then support regeneration of the overlying neo-cartilage. To achieve subchondral repair, two methods may be employed: in vitro formation of composites and successive implantation, or filling the lesion directly in vivo. Autogenous cortical bone for grafting the bony defect along with the implantation of cells or an autogenous osteochondral graft is one strategy, but requires a two-step procedure that is technically demanding.
The production of osteochondral composites in vitro provides the opportunity to pre-fabricate mechanically functional implants that are already integrated with a base subchondral support. One of the most advanced procedures reported was demonstrated by Schaefer et al. [11], where cartilage and bone were produced separately on biodegradable scaffolds and then combined culture as composites. Integration between cartilage and bone was observed, but was dependent upon the maturity of the neo-cartilage. Mixed approaches and results using other combinations have been reported. Frenkel et al. [12] employed a two-layered collagen matrix consisting of a lower dense collagen layer, as subchondral support, which is in contact with bone, and a porous upper matrix to support seeded chondrocytes. This combination gave promising repair by 24 weeks in a rabbit model. In another study, chondrocytes have also been seeded into fibrin glue and then placed upon hydroxyapatite cylinders [13], but this failed to produce a stable osteochondral base and resulted in overlaying fibrocartilage. Kreklau et al. [14] used composites comprised of natural coralline material (calcium carbonate and calcite) and chondrocytes seeded into an upper polymer fleece, which were bound together by a fibrin solution. In this case, the neo-cartilage formed a new matrix, which fused with the underlying biomaterial. Materials like bioactive glass, hydroxyapatite [15], unseeded biofabricated marine carbonate materials [16], polylactic/polyglycolic acids (PLA/PGA), polyglycolic acid (PGA) fibres, bioglass or calcium sulfate [6] have been used for bone and cartilage repair. Overall these materials have been somewhat suitable in restoration of subchondral bone, but generally do not adequately support the formation of repaired hyaline cartilage in relevant large animal models.
To produce biphasic constructs in vitro Schaefer et al. [11] used spinner flask cultures and Kreklau et al. [14] employed a perfusion culture system. However, it is evident that it is also possible to produce implantable neo-cartilage using scaffold-free static systems
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[10, 17 and 18]. Thus, the aims of the present work were to study whether biphasic osteochondral implants could be produced within a static system, and to evaluate the suitability of three commonly used biomaterials compatible with bone/osteochondral defect healing: poly-L-lactide [P(L)LA], poly-D,L-lactide [P(D, L)LA] or Collagen-hydroxyapatite [Col-HA] over a 15-week culture period. Besides being a static system, the reported strategy is distinct from other approaches because the chondrocytes are seeded on top of the scaffold and not inside. The aim is therefore to have neo-cartilage to form above the base biomaterial, while also permitting some cellular in-growth for biomaterial and neo-cartilage integration. Since the biomaterial characteristics differ (in pore size and breakdown rate), the consequence on tissue/biomaterial integration, implant morphological features and cell viability was also investigated.
2. Materials and methods
2.1. Articular chondrocyte isolation
Full thickness cartilage from the distal femur of 12 porcine shoulders were collected and pooled to reduce the potential influence of individual differences. The cartilage was sliced into 1 cm3 cubes and digested in 0.1% Pronase (Roche Diagnostics GmBH, Mannheim, Germany) for 2 h at 37°C, and subsequently 0.025% Collagenase P (Roche, Basel, Switzerland) over night at 37°C. After digestion the released cells were passed through a 100 μm nylon cell strainer (Falcon, Le Pont De Claix, France) to remove undigested fragments. The filtrate was centrifuged at 2000 rpm for 8 min and the cell pellet was re-suspended in culture medium at the required density for direct chamber seeding. The culture medium consisted of DMEM-F12 supplemented with 10% FCS (Life Technologies, HyClone; Utah, USA), L-Glutamine (Fluka, Switzerland), Penicillin-Streptomycin (100IE/100 μg/ml, Life Technologies), ascorbic (50 μg/ml, Fluka, Switzerland) and 1 μg/ml insulin (1 μg/ml, Life Technologies).
2.2. Chamber seeding and construct formation
Cylindrical dialysis chambers that were 8 mm in diameter and 5 mm high were used to produce neo-cartilage (Fig. 1). The bottom of each chamber consisted of a semi-permeable membrane (MWCO: 100’000 Daltons (Spectrum, California, USA) onto which the biomaterial was placed or where the control cells (without base material) were seeded. In total, 84 experimental samples were divided in four groups and three time points (i.e. 7, 11 and 15 weeks) and seven samples were studied from each group at one time point. In Group I, which served as control, the chambers were filled only with cells, at a final density of 8×106 cells per chamber. Group II, consisted of a disk of porous poly ( -lactide), (P( )LA) (Resomer® L206 from Boehringer-Ingelheim). Group III involved the use of porous poly ( -lactide), (P( )LA) (Resomer® R206 from Boehringer-Ingelheim) and group IV made use of collagen-hydroxyapatite (Col-HA).
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Porous P(L)LA and P P(D,L)LA were prepared by a thermally induced solid–liquid phase separation of polymer solution in dioxane at a concentration of 5 wt/v% [19]. In groups II–IV the chondrocytes were seeded on the top of the biomaterial base at an ostensibly identical density to that in the control group (8×106 cells per chamber) and kept in a static (i.e. non-perfusion) environment.
The chambers were subsequently closed with an upper semi-permeable membrane (same as the base membrane) to contain the cells. Each chamber was placed in an individual well of a 6 well plate (Nunclon; Nunc, Denmark) with approximately 10 ml of medium and kept in standard cell culture conditions (37°C, 5% CO2 and humidity
Figure 1:
Fig. 1. Production of biphasic constructs within a closed and static bioreactor. Cells are seeded at high density upon (not into) a base material and are cultured within the chamber, submerged in media, in a 6 well plate for 3–4 weeks. The constructs are then removed from the chamber and are ready to implant. To study the interaction between the neo‐cartilage and the base material, the constructs were removed from the chamber after 3–4 weeks and maintained in culture for the desired duration.
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90%) with medium changes twice per week. After 3 weeks in culture, the cells/cell-polymer in each chamber was removed under sterile conditions and transferred individually into 6 well plates filled with fresh medium. After another 4 weeks (at this point a total of 7 weeks of culture), seven constructs per treatment were taken out of culture, cut into five portions and processed for different analyses. The remaining constructs were maintained in individual wells for a total culture period of either 11 or 15 weeks before analyses.
2.3. Histological evaluation
Cell-scaffold composites and controls were photographed, and then fixed in 4% paraformaldehyde for 24 h, dehydrated and paraffin embedded. Samples were cut into 4 μm sections and stained with Safranin O, H&E, Alcian-blue and Masson's Trichrom for histological analysis. Composite structure was observed by using light microscopy.
2.4. Confocal laser scanning microscopy (CLSM) cell viability (live/dead assay)
The viability of each construct was assessed employing cell viability fluorescence markers (Live/dead kit, Molecular Probes, Eugene, USA). Live pieces of each composite were prepared as previously described [20] and incubated in live/dead buffer consisting of calcein-AM (1 μM) and ethidium homodimer (8 μM) in PBS for 1 h. The sections were then visualised using a laser scanning confocal microscope (LSM410, Zeiss, Germany).
2.5. Immunohistochemical staining for collagen types I, II and X
Paraffin embedded tissue sections were deparaffinised with xylene, hydrated in a graded series of ethanol (100–30%), and then rinsed three times with Tris buffered saline (TBS). Pre-treatment included incubation with 0.1% hyaluronidase in TBS (pH 6.0) for 45 min at 37°C, blocking with 3% goat serum, and incubation for 1 h with the respective primary monoclonal antibodies (1:300). Anti-collagen type I (Quartett; Freiburg, Germany), collagen type II (II-II6B3, Developmental Studies Hybridoma Bank, University of Iowa, USA), and type X (Quartett; Freiburg, Germany), for 1 h at room temperature. Universal biotinylated goat-anti-mouse secondary antibody (Biocare Medical, Walnut Creek, USA) was applied for 35 min, followed by StrABC/AP for 45 min (DAKO, Denmark) at room temperature. Bound antibodies were rendered visible by use of New Fuchsin/Naphthol AS-BI substrate (Sigma). Micrographs were taken using a microscope equipped with a digital camera (Zeiss, Germany).
2.6. Biochemical analyses (SDSPAGE)
Newly synthesised collagen types I, II, IX, X and XI in the constructs were extracted from the neo-cartilage matrix via limited pepsinisation in 0.5M acetic acid, containing 0.4 NaCl as described by Gibson et al. [21]. Protein aliquots were solubilised in 0.5M
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tris–HCl, pH 6.8, containing 0.4% SDS and fractionated on 6.5% SDS polyacrylamide gels and stained with Coomassie blue. Gels were run in parallel and used for photographic analysis as described by Reese et al. [22].
2.7. Transmission electron microscopy (TEM)
Samples were fixed with 2.5% gluteraldehyde in 0.01M sodium potassium phosphate (pH 7.3) for 24 h and postfixed in 2% osmium tetraoxide (Sigma, Switzerland). After dehydration, in a graded series of ethanol solutions, the tissue was embedded in Spurr's resin (EMS, Washington, PA, USA). Sections (0.8 μM) were stained with toludine blue for sample assessment and orientation. Subsequently, 80 nm thick sections were mounted on 200-mesh copper grids, contra-stained with 2% uranyl acetate followed by lead citrate. The ultrastructure was examined using a TEM (E10, Carl Zeiss, Germany). Electron micrographs with final magnifications from 6000 to 80,000 times were taken. Cell morphology, cell scaffold interaction and the structural composition of the extracellular matrix were assessed.
2.8. Scanning electron microscopy (SEM)
SEM was used to make observations on the spatial construction of each biphasic implant and to document cell–material interaction. Four representative pieces from each group and at each time point (7, 11 and 15 weeks) were fixed 24 h in 2.5% glutaraldehyde in PBS, washed in 0.164 potassium (pH 7.4) and dehydrated in a graded series of ethanol (30–100%). Critical point drying was made in a Balzers Union Critical Point Dryer (BU-11120 Balzers, Liechtenstein), and followed by sputter coating in gold (10 nm) using a Balzers Union Sputtering Device (BU-07120, Balzers, Liechtenstein). Each sample was then visualised with a Philips XL 30-FEG scanning electron microscope (Philips, Eindhoven, The Netherlands).
3. Results
3.1. Macroscopic and histological observations
The control group consisted 1.3±0.2 mm thick hyaline cartilage like discs and the three biphasic implant groups were structured with an upper layer of glassy neo-cartilage, 1.3±0.2 mm thick, and a lower layer of scaffold that was 1.5±0.2 mm thick (Fig. 1). P(L)LA and Co1-HA composites sustained the disc form for 7–15 weeks in culture, while P(D,L)LA composites were not regular in shape and the surface of the scaffold side was corrugated at the end of the culture. All three types of composites remained intact when removed from the chambers and were stable enough to be handled by forceps.
Histological light micrographs indicate that a 1.3–0.2 mm thick neo-cartilage tissue formed in control group by week 7, and remained in a similar pattern until the end of
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culture (15 weeks). Cells within the tissue were spherical in shape and cell density was higher than that normally observed in mature native cartilage. However, the neo-cartilage showed evidence of chondron formation and produced an extensive amount of extracellular matrix (Fig. 2). Macroscopically, the biphasic constructs produced on all three biomaterial groups were structurally similar, having an upper layer of neo-cartilage tissue, which was apparently well integrated into with the biomaterial base. Nevertheless, upon histological examination, little apparent cell in-growth and scaffold deformation were observed in P(L)LA group until the end of 15 weeks of culturing. However, the neo-cartilage matrix was attached to the P(L)LA scaffold (Fig. 2), in that it did not become separated when extracted with forceps from the chamber. Following 7 weeks in culture, cells in the Col-HA and P(D,L)LA groups showed signs of scaffold in-growth and cell clusters were observed in the immediate areas adjacent to the upper neo-cartilage side. This progress gained intensity with incubation time, when at 11 weeks cellular in-growth could be seen within all Col-HA and P(D,L)LA constructs. Concomitantly, collapse of scaffolds were observed in these two groups, especially in the case of P(D,L)LA constructs (Fig. 2). After 15 weeks in culture, more than half of the pores collapsed in P(D,L)LA scaffolds leading to the loss of scaffold integrity and construct deformation. Cell clusters can be seen through the whole scaffold with higher chondrocyte densities confined to the upper region of the constructs adjacent to the overlaying neo-cartilage. Cell clusters were distributed through the entire Col-HA scaffolds, which retained most of its original form after 15 weeks. The extent of cellular in-growth in Col-HA constructs was less but more evenly distributed compared to that observed inside P(D,L)LA scaffolds. Bubble-like structures located in the overlying neo-cartilage were first observed in some constructs after 11 weeks of culture (Fig. 2), and this phenomenon gained intensity with culturing time. Thus, by 15 weeks, bubble-like structures could be observed in most constructs independently of the scaffold type.
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3.2. Cell viability
Confocal microscopic observations of neo-cartilage tissue produced in the control group reveal viable cells after 15 weeks of culture. The upper layer of neo-cartilage from other three groups showed similar viabilities (Fig. 2). However, the viability of chondrocytes located inside or close to the surface of the scaffold was altered to a certain degree,
Figure 2:
Fig. 2. Collagen type II immunohistochemistry, Alcian blue staining and confocal viability (live/dead) images. An extensive matrix was produced on each biomaterial, which was mainly composed of collagen type II. Generally all biphasic constructs were viable, but more death (red) can be seen in both polylactide scaffolds, especially chondrocytes located inside P(D,L)LA (confocal viability images ×4).
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depending on the biomaterial used. A thin layer of cell death was detected at the P(L)LA cell–polymer interface after 7 weeks of culture and no further increase was noted until 15 weeks in culture. Extensive cell death was observed in P(D,L)LA constructs where most death was located inside P(D,L)LA polymer and more death can be seen in the upper neo-cartilage layer compared to other biomaterial groups compared. Chondrocytes in Col-HA scaffolds revealed the least cell death than in the other scaffolds.
3.3. Biochemistry and immunohistochemistry
Immunohistochemical analysis revealed the expression of collagen type II throughout the cellular component of each group at each time point studied (Fig. 2). No positive signal for type I or type X collagen protein was observed in any samples. SDS-PAGE examination confirmed the presence of collagen type II as well as the absence of collagen types I and X. Various levels of collagen type IX and XI collagen were detected in all experimental cell–polymer constructs ( Fig. 3). No notable difference in band intensities for each subtype was noted between each group.
Figure 3. SDS‐PAGE gel displaying collagen subtypes produced in each neo‐cartilage group. In each construct, collagen types II, VI and IX collagen were detected in all groups. No collagen type I and X collagen are present. Each collagen subtype is noted on the gel in the standard lanes.
3.4. Transmission electron microscopy
Ultrastructural inspection of constructs during the course of the 15-week incubation period revealed the presence of spherical or rounded cells inside neo-cartilage tissue embedded within an extracellular matrix (ECM) exhibiting a similar morphological appearance to that of maturing chondrocytes. During the 15 weeks of culture, a majority of cells within the neo-cartilage maintained their spherical morphology and generally had the appearance of healthy chondrocytes; that is, cells with a prominent nuclei,
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possessing the usual complement of cytoplasmic organelles, such as endoplasmic reticulum, Golgi apparatus and vacuoles (Fig. 4A). An abundance of randomly organised collagenous matrix fibers was observed in pericellular, inter- and intra-territorial zones. At a higher magnification, the ECM produced by cultured chondrocytes contained a homogeneous population of fibrillar collagens with a diameter of approximately 20 nm, which were faintly cross-banded and randomly oriented (Fig. 4B). After 7 weeks in culture, some cells observed within P(D,L)LA scaffold did not appear healthy. These cells were irregular in shape, slightly shrunken and with many vacuoles and lipid bodies (Fig. 4C).
3.5. Scanning electron microscopy (SEM)
Chondrocytes located in the newly formed cartilage layer in all constructs were surrounded with a dense mat of collagen fibres that extended between the cells to form a continuous network of cell and matrix. The same structure was observed in control group (Fig. 5A). Cell–scaffold interaction was different among the three groups compared. Very few cells were found inside P(L)LA scaffold, nevertheless, it is apparent that the cells and the neo-cartilage was well connected to the P( )LA polymer through collagen fibres at the interface region (Fig. 5B). Cell in-growth was observed in the case of P(D,L)LA and collagen scaffolds and the cells maintained their spherical chondrocyte phenotype morphology (Figs. 5C and D). More cell clusters were found in Col-HA scaffold lacunae, but less frequent in the P(D,L)LA composites at all times examined.
Figure 4:
Fig. 4. Transmission electron micrographs. (A) Chondrocyte (ch) surrounded by matrix (mx) in close proximity to the col‐HA scaffold (Sc). (B) Homogeneous cross‐banded collagen fibers. (C) An unhealthy cell within a P(D,L)LA construct.
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The SEM observation at 7 weeks demonstrated that all the three types of biomaterials had uniformly distributed and interconnected pore structures. P(L)LA possesses the smallest pore size, exhibited a “ladder” like structure and did not appear to change during 15 weeks of culture (Fig. 6A). Col-HA and P(D,L)LA had comparable pore size before seeding and the porosity of both polymers increased upon culturing time. In most instances, P(D,L)LA scaffold showed a degradation process that was much faster than P(L)LA and Col-HA and in some cases lead to an entire collapse of the scaffold (Fig. 5B). In the case of Col-HA, this process was relatively slow and the whole constructs kept its original shape until 15 weeks of culture ( Fig. 5C).
Figure 5:
Fig. 5. Scanning electron micrographs upon 2 months of culture. (A) Chondrocytes surrounded by collagen network in the newly formed upper neo‐cartilage. (B) Interaction of matrix and the P(L)LA scaffold. (C) Spherical chondrocytes on the dense mat of collagenous fibrils in P(D,L)LA constructs. (D) A cluster of chondrocytes located in the lacuna of a Col‐HA scaffold.
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4. Discussion
In this study we have been able to form constructs with an upper neo-cartilage above each of the three base biomaterials tested. The neo-cartilage tissue possessed cartilage-like qualities since they all displayed a prominent proportion of collagen type II and deposited an extensive extracellular matrix that largely consisted of glycosaminoglycans. Equally, each neo-cartilage showed the formation of collagen fibrils and evidence of interteritorial region formation. Although there was variation in the extent of cellular in-growth and some undesirable interactions with the subchondral base material, all constructs were largely viable, stable enough to be handled by surgical forceps and somewhat regular in form. We have previously generated monophasic neo-cartilage constructs using this closed and static system [18] and thus appears to be also suitable for in vitro pre-fabrication of biphasic constructs.
Biphasic constructs produced using Col-HA were superior in comparison to P(L)LA and P(D,L)LA constructs in relation to cell viability, construct shape and cellular integration. Cellular enzymes are able to recognise collagen, being a natural polymer, which allows for remodelling and appropriate degradation to provide space for the growing tissue [4, 9, 23 and 24]. Employing a hydroxyapatite and collagen mix may prove to be accommodating given that hydroxyapatite is naturally part of the bone mineral component and has reduced foreign-body-type reactions and toxic degradation by-products [25]. In contrast, synthetic polymers, such as P(L)LA and P(D,L)LA used here, have been shown to produce degradation products that create acidic environments leading to cell death, at least in vitro [24 and 26], and consistent with the present findings.
The aim of the present study was to examine interaction of chondrocytes and the biomaterial and to observe the consequence of biomaterial changes, or lack thereof, over
Figure 6:
Fig. 6. Scanning electron micrographs of the three different scaffolds used following 7 weeks of culture. (A) P(L)LA, (B) P(D,L)LA and (C) Col‐HA.
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time in culture. Nevertheless, it is clear that to truly evaluate the best combination, an in vivo implantation of these pre-fabricated biphasic constructs is necessary. We noted in this current study that even though there was no discernible cellular in-growth into P()LA, the overlying neo-cartilage developed upon this material did not break away once handled when removed from the chambers. Certainly, there is evidence (via SEM) of extracelluar matrix connections to the P(L)LA scaffold to maintain adhesion, but it is yet to be determined if this is adequate when implanted into the pressures experienced in the joint. It is predicted that such superficial binding may lead to implant delamination.
The consequence of biomaterial degradation on changes in cell viability and how this may alter long-term survival of an implant is not clear. An increase in death associated with the release of acidic products during P(D,L)LA degradation is well noted. Nevertheless, the effect of this breakdown may not be overly detrimental to such an implant described here since the neo-cartilage produced in this study is very cellular. Hence, during the natural remodelling in vivo a reduction of cell numbers should naturally occur and may not be detrimental within certain limits.
Degradation time and pore size are two other important characteristics for tissue engineering such composites. The smaller pore size and slower degradation rate of P(L)LA scaffolds prohibited cell in-growth. In fact, the complete remodelling of P(L)LA has been reported to be more than 2 years [27 and 28]. Conversely, Col-HA scaffolds have a more reasonable degradation time of 2 months to 1 year [29], which may be more suitable in vivo and Col-HA materials appear to have a suitable pore size that allows cell in-growth and migration. It is therefore essential that a subchondral support structure provides an adequate structural support, sustains appropriate cell viability and permits integration and maturation of the overlying neo-cartilage. Moreover, the ability of both components to integrate with all associated native tissues is a common multidimensional problem associated with such cartilage repair strategies.
Two factors observed in this study worth noting that may be an inherited artefact due to the unique in vitro environment include the formation of ‘bubble’ like structures and perhaps an over induction of cell death. The ‘bubble’ phenomenon observed in many constructs by week 15 is most probably a consequence of longer in vitro culturing times and available nutrients. Since these structures are not observed by 4–5 weeks of culture and are already suitable for implantation, then these structures may not occur once implanted into a subchondral defect. The extent of death observed in the P(D,L)LA constructs may be more extensive compared to an in vivo environment, since the in vitro chamber system used here is closed and in static conditions. It is thus likely that the creation of an extra acidic milieu was formed and caused more death, which may not be the case in the joint environment.
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5. Conclusions
This study presents an alternative means of producing biphasic composites with three different biodegradable materials for the purpose of repairing subchondral defects. Comparatively, Col-HA constructs were superior than P(L)LA and P(D,L)LA, in terms of cell viability, construct shape and cellular integration. It is thought that these biphasic implants have great potential to support the on-going development of the overlying neo-cartilage while allowing for the repair of the underlying subchondral bone. The potential of using these constructs for repair of osteochondral lesions and large articular cartilage defects needs to be further validated in vivo.
Acknowledgements
We wish to thank the support of Professor Roland Jakob (Hôpital Cantonal de Fribourg, Switzerland), Professor Robert Jérôme (University of Liège, Belgium) and Dr Peter Bittmann (Centerpulse, Switzerland). A special acknowledgement to Mr. Johannes Schittny for his technical assistance in the scanning electron microscopy work (Institute of Anatomy, University of Bern, Switzerland). Véronique Maquet “Postdoctoral Researcher” was funded by the “Fonds National de la Recherche Scientifique” (F.N.R.S). CERM is indebted to the “Services Fédéraux des Affaires Scientifiques, Techniques et Culturelles” for financial support in the frame of the “Pôles d’Attraction Interuniversitaires: PAI 5/03”. Centerpulse Biologics is also acknowledged for their financial and scientific support. This project was largely supported by the Swiss Commission for Technology and Innovation (CTI:4716.1) and the Swiss National Science Foundation (SNF:4046-58623).
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Chapter 4
Chapter 4 - Disease-Modifying Effects of N-butyryl
Glucosamine in a Streptococcal Cell Wall (SCW)
Induced Arthritis Model in Rats
Susanne X. Wang 1, Annie Cherian 1, Mircea Dumitriu 1, Marc D. Grynpas 1 John
Carran2,4, Dan Wainman2, Tassos Anastassiades 2,3*
1 Department of Laboratory Medicine and Pathobiology, University of Toronto and Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada
2Department of Medicine, Arthritis Center, Queen’s University, Kingston, ON, Canada 3Dept of Biochemistry, Queen's University, Kingston ON, Canada
4Department of Chemistry, Queen’s University, Kingston, ON, Canada
*Corresponding author: Division of Rheumatology, Dept of Medicine, Rm 2050 Etherington Hall, Kingston ON, K7L4B4. Tel: 613-544-2623. E-mail: [email protected]
J Rheumatol. 2007 Apr;34(4):712-20.
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Abstract
Objective. To evaluate the effect of different doses of N-butyryl glucosamine (GlcNBu) on joint preservation and subchondral bone density and quality in a streptococcal cell wall (SCW) induced arthritis model in Lewis Rats.
Methods. Chronic arthritis was induced in 36 female Lewis rats by a single intra-peritoneal (IP) injection of SCW antigen. The groups studied were: (a) no arthritis, no treatment; (b) arthritis, no drug treatment; (c) arthritis, oral GlcNBu 20mg/kg/day; (d) arthritis, oral GlcNBu 200mg/kg/day. Inflammation (ankle swelling) was quantified throughout the clinical course; bone mineral density (BMD) was measured by dual energy x-ray absorptiometry (DEXA) on dissected distal femurs and proximal tibias, in user defined regions of interest (ROI). Qualitative and quantitative three-dimensional bone architecture changes were determined using micro-computed tomography (micro-CT) on the left tibias. Subchondral plate thickness and trabecular bone connectivity were studied on the proximal tibia epiphyses from the central coronal sections of each scanned tibia.
Results. GlcNBu inhibited inflammatory ankle swelling both at 20 and 200 mg/kg/day, the latter being statistically significant, with an average reduction of 33%. GlcNBu preserved or enhanced BMD, bone connectivity and prevented further bone loss at both the high and the low dose. Comparisons of the isosurfaces and the architectural parameters in the different groups demonstrate that GlcNBu effectively protects the joint surfaces from further erosion in this model of chronic inflammatory arthritis. For some of the bone measurements, increasing doses of GlcNBu showed increasing protective effects, while for other measurements, effects were maximal at the lower dose.
Conclusion. These data indicate that GlcNBu provides anti-inflammatory and anti-erosive effects and preserves BMD, joint integrity and bone architecture, in involved joints of the SCW model.
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1. Introduction
Rheumatoid arthritis (RA) and osteoarthritis (OA) both demonstrate prominent bone remodeling, although the histopathology of the subchondral bone, its relation to damaged cartilage and the rates of remodeling are considerably different (1-3). A number of cytokines, biochemical markers and gene expression alterations, reflecting cartilage or bone turnover and disease progression, have been reported some of which may be seen very early (4-6).
In RA, damage to articular cartilage begins at the cartilage–pannus interface, but there are also progressive erosions occurring into the subchondral bone. The pattern of bone damage in the joint includes focal erosions and juxta-articular osteopenia. The impact of RA on bone, however, is also observed systemically in the axial and appendicular skeleton, with reductions in bone mineral density causing osteoporosis and, as a consequence, increased risk of fracture (7;8). Many patients with RA have radiographic evidence of substantial joint damage within the first 2 years of disease (9), and even in the first few months, evidence of bone erosion may be seen with magnetic resonance imaging (10;11). Further, bone loss is a typical pathological feature of RA. The skeletal target most exposed to inflammatory damage is the subchondral bone adjacent to inflamed synovial tissue. Early after disease onset, this particular area faces rapid destruction, which results in the typical radiologic signs of RA, manifested as local bone erosion and periarticular demineralization (12). However, erosive changes also occur more centrally which are related to inflammatory changes in the subchondral marrow spaces, and remodeling of the subchondral bone is a feature of disease progression (13).
Destruction of bone has become a synonym for irreversible tissue damage and poor functional outcome in RA patients (14-16) and its prevention underlies an important therapeutic principle of antirheumatic drug therapy. The gold standard for evaluating bone destruction in patients with RA is the visual scoring of radiographs of the hands for joint erosions and narrowing (17;18), The scores obtained correlate highly with the quantitative measurement of metacarpal cortical width, which declines with RA progression due to accelerated bone loss at the endosteal surface (19). Computed tomography (CT) has been shown to be more sensitive than radiography in detecting bone erosions in RA patients, due to its 3-dimensional (3-D) perspective (20). Micro-CT has proven to be a powerful technique to analyze bone structure and density in small animals.
Evaluation of current therapies for RA by meta-analysis of randomized clinical trials (RCT) demonstrates still significant toxicity with currently used anti-rheumatic drugs although risk/benefit ratios appear to be improving compared to previous treatments (21). Efficacious therapies with very low toxicity, are not available for RA. In OA, glucosamine (GlcN) or glucosamine sulfate (GlcN.S), a non-covalently bound sulfate
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salt of GlcN, are considered to constitute very low toxicity therapies. However, metanalysis of available RCTs (22), as well as clinical studies where GlcN was withdrawn (23), renders the question of efficacy of these compounds controversial for OA. The efficacy of GlcN compounds has not been evaluated in RA.
A different and novel approach to drug development for diseases of joints consists of utilizing an abundant, non-toxic naturally occurring parent carbohydrate, such as GlcN, and chemically modifying it to yield products that are similar, but not identical, to those that occur in nature. GlcN occurs in matrix and cellular glycoconjugates largely in the N-acetylated form, N-acetyl GlcN (GlcNAc). Chemical N-acylation of glucosamine leads to a class of compounds that exhibit biological activity which is markedly different from their parent GlcN (24). Among these GlcNAcyls, which were initially developed as chondroprotective agents, N-butyryl glucosamine, N-(2,4,5-Trihydroxy-6-hydroxymethyl-tetrahydro-pyran-3-yl)-butyramide, (GlcNBu) is the most studied and preferred compound of the synthetic GlcNAcyls. This is because of its chemical properties, lack of toxicity in both animals and cell culture and genotoxicity studies, and the biological activity profile (25). Compared to GlcN, which was found to have suppressive effects on bovine chondrocyte proliferation and proteoglycan (PG) synthesis, GlcNBu demonstrated stimulatory effects. The differences between GlcN and the GlcNAcyls were marked under high concentrations, were more prominent under anchorage-dependent than anchorage independent culture conditions. Also, GlcNBu up-regulated a number of genes in human OA chondrocytes, compared to GlcN (25). These effects were seen both in the presence and absence of the cartilage and bone regulatory peptide TGF-β, which is related to the bone morphogenetic protein super-gene family. In addition, GlcNBu increased type II collagen expression by rat chondrocytes (26). GlcNBu and similar GlcNAcyls are good substrates for glycosyltransferases from bone and cartilage cells (27), although their mechanism of action for the biological effects has not been established. Recently, a high performance liquid chromatography method for quantification of GlcNBu in rat plasma has been developed (28). The method is linear over the range of 0.2-200 μg/mL, and was applied to oral administration of GlcNBu in the rat.
For pre-clinical evaluation of GlcNBu a number of animal models have been considered. Some osteoarthritis models depend on altering joint biomechanics by disrupting the anterior cruciate ligament of the knee, as in the dog or rabbit (29). Such models are time consuming and relatively expensive, partially because of the number of animals required to overcome variability. The rabbit model (29) has provided preliminary results of interest with oral feeding of GlcNBu, which are being histologically analyzed. However, inflammatory stimuli can provide rapid remodeling of bone and cartilage in the joint (1). The Lewis rat (LEW/N) model has been well studied and is particularly susceptible to induction of arthritis by a single intra-peritoneal injection of SCW (30). In this model there is an immediate onset of acute arthritis,
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affecting primarily hind leg distal joints, and this is followed after 2-3 weeks, by a secondary chronic arthritis, where severe destruction and remodeling of the initially affected joint occurs. The arthritis can be reversed by an injection of anti-transforming growth factor-β antibody (31) and attenuated by splenectomy (32). Dual energy x-ray absorptiometry (DEXA) for the assessment of bone density and 3-D computed tomography (33;34) had not been previously applied to the study of drug effects on the bone and cartilage changes in this model.
The purpose of this study is to determine the effects of orally administrated GlcNBu, on the inflammation, bone mineral density and subchondral bone quality measurements in a SCW-model in the rat. Micro computed tomography was employed to quantify the cancellous bone architectural and connectivity changes in rat tibiae.
2. Materials and Methods
2.1 Animal model and experiment design.
The animal protocol was approved by the Queen’s University Animal Care Committee service. There was active supervision of the arthritis model by a veterinarian from that service, with respect to ethical treatment of the animals. Forty-two inbred female Lewis rats weighing at least 150g were purchased from Charles River Laboratories, through Queen’s University, and acclimated for at least 1 week prior to use. The rats were housed in filter-capped polycarbonate cages and maintained under constant environmental conditions (average 22°C, humidity 50%). Rats were kept on a 12h /12h light–dark cycle and had unrestricted access to purified bottled drinking water and standard chow.
Chronic arthritis was induced in 36 female Lewis rats by a single IP injection of SCW fragments (15µg/g) as previously described (30). The purified peptidoglycan-polysacharide polymers (PG-PS 10S, Lee Laboratories, Grayson GA) were pre-sonicated for 10-20 seconds prior to injection. The rats were divided in to 4 groups according to the induction of disease and supplementation of GlcNBu. The GlcNBu, or glucose (for controls with arthritis) was administered in a small amount of peanut butter once a day and was readily ingested by the rats. The groups were (a) Control: no arthritis, no treatment, n=6; (b) SCW induced arthritis, no drug treatment (oral glucose 200mg/kg/day), n=12; (c) SCW induced arthritis, low dose oral GlcNBu (20mg/kg/day), n=12; (d) SCW induced arthritis, high dose oral GlcNBu (200mg/kg/day), n=12.
Inflammation of the ankles was quantified daily by measuring the ankle diameter using a standard caliper method from disease onset to day 28 after initiation of arthritis. Three measurements were done on each ankle, for each time point, to an accuracy of 0.1 mm and averaged. Animal weights were also monitored daily. At the end of 4 weeks, the
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animals were sacrificed and both left and right femurs were dissected from soft tissue and fixed in 10% natural buffered formalin for BMD and micro CT analysis.
2.2 Bone mineral density (BMD) measurement.
All the dissected femurs and tibias were scanned with dual energy x-ray absorptiometry (DEXA) using the PIXImus™ densitometer (GE Lunar Corp.). After calibration with an aluminum/lucite phantom (Lunar Corp/GE), the excised bones were placed on a polystyrene tray to mimic soft tissues for scanning. The PIXImus™ automatically calculated the areal bone mineral density (aBMD) from the bone mineral content (BMC) and the measured region of interest (33). The bone mineral density (aBMD, g/cm2) and bone mineral content (BMC, g) were measured at the distal femur, and proximal tibia using a user defined region of interest (ROI).
2.3 MicroCT imaging protocol.
The fixed left tibias were scanned with micro-CT scanner (Explore Locus SP micro CT Scanner, GE Healthcare). The eXplore CT acquisition software from General Electric Medical Systems was used to set the scan at 16-µm isotropic voxel size, with 499 projections, and total scanning time 150min. The tibias were placed in a water filled specimen tube in a firmed position such that the main axis was kept as parallel as possible to the Z-axis of the micro-CT image coordinate system. This minimized any beam-hardening effects, since the x-ray beam crossed a minimal volume of bone tissue. The scan was corrected following image acquisition using data acquired from an empty scan in which bright and dark images are gathered. Correction accounts for variations in temperature and attenuation of the x-rays (35). The reconstructed tibia images were analyzed with MicroView 2.0 software (GE Healthcare, London, ON) in two and three dimensions.
2.4 Threedimensional trabecular bone measurements.
Three dimensional bone analyses were conducted using three selected regions of interest (ROIs) using MicroView 2.0 analysis software. Two of the ROIs were rectangular with dimensions 1.65mm x1.65mm x 0.55mm and positioned in the middle of the lateral and medial epiphysis to measure the 3-D micro architecture of the secondary spongiosa (Figure 1a and 1b). A third ROI (Figure 1c), a cylinder 2mm x 2mm x 1mm in size, was placed 1mm below the growth plate to measure the 3-D micro architecture of the secondary spongiosa.
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On 3D analysis, the tissue volume (3D-TV, mm3) and trabecular bone volume (3D-BV, mm3) were measured directly, and the fractional trabecular bone volume (3D-BV/TV, %) was calculated. The trabecular thickness (3D-Tb.Th, µm) and trabecular number (3D-Tb.N, N/mm) were measured directly on 3D images (36). Another parameter obtained is the structure model index (SMI). Established by Hildebrand and Ruegsegger (37) the SMI represented the plate-rod characteristics of trabecular structure. In this measure, a negative number is indicative of a plate-like structure containing holes, 0 represents an ideal plate-like structure while 3 represents the ideal rod (cylindrical) structure. Larger SMI values indicate that trabecular structure contains a more rod-like structure in older or diseased bone.
2.5 Threedimensional isosurface.
A 3-D isosurface image of each tibia was obtained by a volume-rendering function in MicroView to qualitatively examine the erosive damage in the bone surface. Since the bone destruction was mainly observed in the subchondral bone region adjacent to the inflamed synovial tissue, we compared the proximal surface of the tibia.
2.6 Twodimensional subchondral bone analysis.
Three coronal sections of proximal tibia were obtained from each 3-D reconstructed tibia image: anterior, central, and posterior (1mm apart). The subchondral trabecular bone structure and connectivity in the epiphyseal region of the proximal tibia was
Figure 1:
Figure 1. Locations of the regions of interest analyzed using the bone analysis application of Micro View. Regions highlighted in (a) and (b) have dimensions of 1.65x 1.65 x 0.55 mm and are placed in the medial and lateral proximal epiphysis respectively. The line in (c) is 1mm in length and marks the start of the cylindrical metaphyseal ROI analyzed. This ROI has dimensions of 2mm x 2mm x 1 mm.
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analyzed using an image processing and analysis system (Quantimet 570, Leica) on the 2-D micro-CT images. After thresholding, a binary image was obtained at the medial and lateral side of the epiphyseal region to analyze the structure parameters according to the ASBMR guidelines (38): fractional trabecular bone volume (2D-BV/TV, %), trabecular thickness (2D-Tb.Th) and trabecular number (2D-Tb.N, n/mm).
The connectivity parameters of trabecular bone were measured on the skeletonized binary images using an in-house program based on the techniques described by Parisien et al in 1992 (39). The connectivity parameters obtained in the analysis include: Number of multiple points (1/mm2), Number of end points (1/mm2), Lengths of node-node struts (mm/mm2), Lengths of free-free struts (mm/mm2), and Total strut length (mm/mm2). Figure 2a illustrates the area of cancellous bone analyzed by strut analysis and Figure 2b defines strut connectivity parameters.
The subchondral bone plate thickness (SCP.Th, mm) was also measured on the central section of the 2D micro-CT proximal tibia images using individual point-to-point distance measures. The thickness was calculated by averaging 12 measurements per tibia. All measurements were made in a standardized viewing area by a single observer who was blinded to the experiment.
2.7 Statistical analysis.
All the data were analyzed with SPSS 11.0 for Windows statistical package (SPSS Inc., Chicago, IL). One-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) post-hoc test was used to compare means. Significance was considered at p < 0.05. All data are presented as mean ± standard error of the mean (SEM).
3. Results
3.1 Effects of GlcNBu on the SCWinduced inflammation.
All the groups increased in weight equally over the 4 weeks experiment period (data not shown). The ankle size measurements demonstrated that all of the animals receiving the
Figure 2.
Figure 2. (a) Illustrates the area of cancellous bone analyzed by strut analysis and (b) defines strut connectivity parameters
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IP SCW injection entered the acute phase immediately (Figure 3). Between days 6 and 12, the acute inflammatory response tended to subside. Approximately 80% of animals in each group entered the chronic phase, at about 2 weeks, partially accounting for the larger standard errors in the ankle size measurements between days 14 and 26. Orally administered GlcNBu at 200mg/kg/day significantly decreased the swelling compared to the group without treatment (top and middle lines respectively in Figure 1, *p<0.05). Low dose GlcNBu treatment (20mg/kg/day) generated an intermediate response, with a decline in ankle size measurements, which did not reach statistical significance (data not shown).
3.2 Decline of BMD by the arthritis and reversal by GlcNBu.
Four-week treatment with GlcNBu significantly preserved BMD in the ROI of the tibias and femurs of rats with SCW-induced arthritis (Figure 4a). This effect occurred in a
Figure 3.
Figure 3. Inhibition of inflammatory ankle swelling in rats with SCW‐induced adjuvant arthritis by GlcNBu. The arthritic joint presented a constant swelling in comparison to the controls (top and bottom lines respectively). High dose GlcNBu at 200mg/kg/day significantly decreased the swelling compared to the no treatment group (top and middle lines respectively, *p<0.05).
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dose-dependent manner. The lowest BMD were seen in the SCW-induced arthritis without drug treatment (i.e, with glucose control). For the rat tibias from the arthritic rats, both the high and low dose GlcNBu treatment showed significant increases in BMD (p<0.05), compared to the group without drug treatment. For the femurs, there were trends for both GlcNBu doses, compared to no drug treatment. Overall, GlcNBu at 200mg/kg/day increased BMD compared to SCW-induced arthritis in animals treated with glucose, up to a level that shows no statistical difference from the normal joints.
Figure 4a
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For volumetric bone mineral density measurements (3D-BMD), rats with SCW-induced arthritis and treated with GlcNBu generally demonstrated lower bone loss than those that were untreated (Figure 4b). There was increased 3D-BMD with treatment of GlcNBu in all three regions examined, by the three-dimensional analysis. The largest effects (statistically significant) were noted in the BVFs of the metaphyses , which were essentially normalized in the SCW-arthritis groups treated with both doses of GlcNBu. For the BVFs of the metaphyses the maximal increase was seen with the lower GlcNBu dosage (20mg/kg/day) and no significant difference was observed by increasing the dose.
Figure 4b
Figures 4 (a, b). Bone densitometry evaluation of the distal femur and proximal tibia in rats treated with various doses of GlcNBu. Designations for the various groups are given in the text.
(a). Bone mineral density (BMD) measured by DEXA. (b). Bone volume fraction (BVF) from the micro‐CT scanned tibia at 3 regions of interest: epiphyseal lateral, epiphyseal medial and metaphyseal.
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3.3 Microarchitecture of the trabecular bone.
Trabecular bone volume, trabecular number and thickness were significantly decreased in the arthritic subchondral compared to the controls. Similar change was observed in both 2D and 3D images. These changes were improved by GlcNBu treatment in a dose dependent manner, so that trabecular bone subjected to high dose GlcNBu treatment was similar to the controls. The metaphyseal region illustrated a less marked increase in trabecular volume between low and high dose groups; however there was a significant increase of 12% from the SCW group to the GlcNBu treated groups (p < 0.05). The metaphyseal bone structure changes suggest that GlcNBu functions systemically.
Subchondral Plate Thickness was decreased in the SCW group, and this change was minimized by GlcNBu treatment, especially in the high dose treatment group. Table 1 presents the mean trabecular volume (3D-Tb.V, mm3), trabecular number (3D-Tb.N), trabecular thickness (3D-Tb.Th, µm), and subchondral plate thickness (SCP.Th, mm) data for each of the arthritis-induced groups in the epiphyseal and metaphyseal regions. Each of these parameters increased with the dose of GlcNBu.
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Table 2 compares the potency of the high and low doses of GlcNBu based on the significance of the changes observed. The arrows indicate whether the groups displayed an increase or a decrease when compared to the mean values exhibited by untreated
Table 1: Micro‐architecture changes
Treatment Regions of interest
Bone Parameters
3D‐BV/TV (%)
3D‐Tb.N (n/mm3)
3D‐Tb.Th (µm) SCP.Th
(mm)
Control
Lateral epiphysis
42.2± 0.9 5.3 ± 0.2 68 ± 4 0.11 ± 0.013
Medial epiphysis
40.5 ± 1.2 4.4 ± 0.1 83 ± 4 0.07 ± 0.014
Metaphysis 26.5 ± 1.5
6.0 ± 0.3 35 ± 1
SCW
Lateral epiphysis
32.6 ± 3.0 5.5 ± 0.4 50 ± 4 0.07 ± 0.01
Medial epiphysis
32.7 ± 4.2 4.8 ± 0.5 60 ± 7 0.07 ± 0.03
Metaphysis 22.5 ± 7.4 6.8 ± 1.8 27 ± 3
SCW + GlcNBu Low
Lateral epiphysis
33.7 ± 2.7 5.8 ± 0.5 52 ± 8 0.10 ± 0.04
Medial epiphysis
33.1 ± 3.0 5.0 ± 0.4 62 ± 11 0.07 ± 0.03
Metaphysis 34.4 ± 9.4 7.7 ± 1.3 34 ± 5
SCW + GlcNBu High
Lateral epiphysis
36.9± 3.7 6.0 ± 0.6 54 ± 8 0.11 ± 0.04
Medial epiphysis
36.3 ± 4.8 5.1 ± 0.7 66 ± 14 0.07 ± 0.04
Metaphysis 32.6 ± 4.7 8.2 ± 1.5 34 ± 4
Table 1: Micro‐architecture changes among the various groups in three different regions: trabecular bone volume (3D‐BV/TV,%), trabecular number (3D‐Tb.N), trabecular thickness (3D‐Tb.Th, mm), and subchondral plate thickness (SCP.Th) [mean±SEM]
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SCW injected rats. Both doses gave positive changes and the more significant bone changes occurred in the higher dose group. The metaphysis was significantly positively affected in both the high and low dose groups.
3.4 Cancellous bone connectivity.
Strut analysis showed a significant increase in end points and a decrease in multiple points, node-node struts, and total strut length (table 3) in the untreated arthritis-induced group compared to the controls. This loss of connectivity was partially corrected by the GlcNBu treatment. The improvement of connectivity was dose dependent with GlcNBu treatment. Table 3 shows the cancellous bone strut analysis of various groups at three coronal sections of the proximal tibia: anterior, central, and posterior.
3.5 Sitespecific structure change.
Regional differences were observed in both 2D and 3D analysis. In 3D analysis, the metaphysis showed significant change over the epiphysis region. Comparing the 2D analysis among the three sections, the posterior side presented the highest bone volume, thickness and connectivity. Moreover, the higher bone mass and more connected cancellous bone is more apparent in the medial side rather than the lateral side, but the lateral region exhibits more significant disease change and appears to have responded to the treatment more than the medial side. These changes suggest that the more weight bearing region is related to higher bone mass and connectivity and probably strength and is less affected by the disease. The anterior section was the site most affected by the disease, there were 45% loss in bone volume 33% loss in 2D-Tb.Th., 34% reduce in total skeleton length, 54% decrease in multiple points and as much as 80% decrease in node-node struts (table 3). A possible reason that the anterior area is more affected could be due the wide contact with the synovium.
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Table 2: Comparison of bone changes in the proximal tibia resulting from high and low dosages of GlcNBu
Dosage Group Compared
Region of Proximal Tibia
Bone Parameters
Trabecular Bone Volume
Structure Model Index
Trabecular Separation
Trabecular Number
Trabecular Thickness
Subchondral Plate Thickness
GlcNBu High Dose vs. SCW
lateral epiphysis ↑↑ ↓ ↓↓ ↑↑ ↑ ↑↑
medial epiphysis ↑ ↓ ↓ ↑ ↑ ↑
metaphysis ↑↑ ↓↓ ↓↓ ↑↑ ↑↑
GlcNBu Low Dose vs. SCW
lateral epiphysis ↑ ↓ ↓↓ ↑ ↑ ↑
medial epiphysis ↑ ↓ ↓↓ ↑ ↑ ↑
metaphysis ↑↑ ↓↓ ↓ ↑ ↑↑
The arrows indicate a bone change when compared to untreated SCW‐arthritis induced rats. Changes are considered significant when p values < 0.05. ↑↑: significant increase ↑: increase ↓↓: significant decrease ↓: decrease
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Table 3.
Regions Anterior/ lateral Anterior/medial Middle section/lateral Middle section/medial Posterior/lateral Posterior/medial
Parameters GROUP Mean ±
SEM %
Loss Mean ±
SEM %
LossMean ±
SEM % Loss Mean ± SEM % Loss Mean ± SEM % Loss Mean ± SEM % Loss
2D-BV/TV, % SCW 21.8 ± 1.5 45 24.8 ± 1.6 39 21.4 ± 1.3 25 28.7± 1.6 32 27.6 ± 1.4 13 34.1 ± 1.3 25
Low dose 23.7 ± 2.3 40 27.2 ± 1.4 33 24.9 ±1.6 13 29.2 ± 2.3 31 24.9 ± 1.5 21 36.2 ± 1.6 20
High dose 24.8 ± 3.4 37 27.8 ± 2.7 31 24.1 ± 2.1 15 35.1 ± 4.1 17 23.2 ± 1.0 27 35.9 ± 2.2 21
Control 39.5 ± 0.3 0 40.4 ± 0.3 0 28.5 ±0.4 0 42.1 ± 5.2 0 31.6 ± 0.7 0 45.3 ±0.5 0
2D-Tb.Th.µm SCW 38.2 ± 2.6 33 45.3 ± 3.0 34 49.3 ±1.9 20 59.6 ± 2.4 34 53.4 ± 1.8 17 57.0 ± 1.8 26
Low dose 38.7 ± 1.7 32 48.6 ± 1.6 29 54.3 ± 2.8 11 63.0 ± 4.0 30 51.8 ± 1.6 19 59.3 ± 2.3 23
High dose 39.7 ± 2.9 30 49.5 ± 4.3 28 51.3 ± 2.8 16 68.5 ± 6.3 24 49.8 ± 1.7 22 61.0 ± 3.6 21
Control 56.7 ± 4.3 0 68.9 ± 3.9 0 61.3 ± 3.1 0 90.4 ± 5.8 0 64.2 ± 7.7 0 77.2 ± 4.4 0
2D-Tb.N./mm SCW 5.7 ± 0.3 23 5.6 ± 0.3 13 4.3 ± 0.2 7 4.8 ± 0.2 -4 5.2 ± 0.2 -1 6.0 ± 0.2 6
Low dose 5.8 ± 0.4 21 5.6 ± 0.3 12 4.6 ± 0.2 2 4.6 ± 0.2 1 4.8 ± 0.2 7 6.2 ± 0.2 3
High dose 6.0 ± 0.7 20 5.7 ±0.6 10 4.7 ± 0.3 0 5.1 ± 0.2 -9 4.5 ± 0.2 13 6.1 ± 0.2 5
Control 7.4 ± 0.5 0 6.4 ± 0.6 0 4.7 ± 0.2 0 4.6 ± 0.3 0 5.2 ± 0.5 0 6.4 ± 0.5 0
End points SCW 40.5 ± 5.6 -161 30.0 ± 4.1 -123 21.9 ± 2.6 -112 15.8 ± 2.2 -217 16.3 ± 1.2 -62 20.8 ± 1.9 -306
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Low dose 36.2 ± 2.5 -134 22.5 ± 1.7 -67 17.5 ± 1.9 -69 14.1 ± 2.3 -184 20.0 ± 1.8 -100 16.2 ±2.1 -216
High dose 34.1 ± 4.1 -120 26.7 ±5.7 -99 19.8 ± 1.7 -92 12.2 ± 2.1 -145 21.4 ± 1.7 -114 17.1 ±1.8 -233
Control 15.5 ± 6.2 0 13.4 ±3.8 0 10.3 ± 2.5 0 5.0 ± 1.1 0 10.0 ± 1.3 0 5.1 ± 1.5 0
Multiple points SCW 7.5 ± 1.1 54 8.5 ± 1.2 47 6.8 ± 1.3 31 9.0 ± 1.0 21 9.8 ± 0.7 44 13.7 ± 0.8 30
Low dose 9.3 ± 1.6 43 9.2 ±1.0 42 8.3 ± 1.1 16 9.9 ± 1.1 13 7.8 ± 0.8 55 12.6 ± 1.4 36
High dose 10.3 ± 2.7 37 10.7 ± 1.7 32 9.5 ± 1.9 2 12.6 ± 1.8 -11 7.1 ± 0.7 59 13.9 ± 1.8 29
Control 16.4 ± 4.5 0 15.8 ± 1.3 0 9.8 ± 0.2 0 11.3 ± 0.3 0 17.4 ± 2.2 0 19.7 ± 8.5 0
Node-node struts SCW 0.7 ± 0.2 80 0.9 ± 0.2 61 0.7 ± 0.2 29 1.2 ± 0.2 41 1.3 ± 0.2 -1 1.5 ± 0.1 46
Low dose 0.8 ± 0.2 75 1.2 ± 0.2 47 1.0 ± 0.2 3 1.2 ± 0.2 42 0.9 ± 0.1 30 1.7 ± 0.3 37
High dose 1.2 ± 0.4 64 1.3 ±0.3 45 0.9 ± 0.2 15 2.0 ± 0.3 4 0.7 ± 0.1 46 2.1 ± 0.3 23
Control 3.3 ± 0.4 0 2.3 ±0.6 0 1.0 ± 0.1 0 2.1 ± 0.0 0 1.3 ± 0.4 0 2.7 ± 0.8 0
Total strut length SCW 4.9 ± 0.2 34 4.8 ±0.2 19 3.8 ± 0.2 17 4.3 ± 0.2 10 4.8 ± 0.2 -1 5.5 ± 0.2 14
Low dose 5.3 ± 0.4 30 5.1 ±0.3 14 4.0 ± 0.2 14 4.2 ± 0.3 11 4.3 ± 0.2 11 5.7 ± 0.3 10
High dose 5.4 ± 0.7 28 5.1 ± 0.5 15 4.2 ± 0.2 9 4.9 ± 0.3 -2 4.0 ± 0.2 16 5.6 ± 0.3 12
Control 7.5 ± 0.9 0 5.9 ±0.7 0 4.6 ± 0.3 0 4.7 ± 0.3 0 4.8 ± 0.1 0 6.3 ± 1.0 0
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3.6 Joint integrity.
Isosurfaces generated from the scan of diseased bone show clear bone erosion at the joint margin. However, protection against bone erosions was observed in the GlcNBu treated group, especially in high dose. Figure 5 shows the isosurfaces of proximal rat tibia in a) No arthritis, no treatment; (b) arthritis, no treatment; (c) arthritis + low dose GlcNBu; (d) arthritis + high dose GlcNBu groups. Comparisons of the isosurfaces in the studied groups demonstrate that GlcNBu effectively protects bone from further erosion in this model of chronic inflammatory arthritis.
Figure 5:
Figure 5: Isosurfaces of proximal rat tibiae show the preservation of subchondral bone, a direct effect of GlcNBu treatment. A smooth intact surface showed in a healthy rat tibia (a), severe subchondral bone erosions were evident in the pannus‐bone interfaces of the epiphysis in the SCW‐arthritis induced no treatment group (b). These erosions were diminished in the GlcNBu‐treated rat at low dose (c) and at high dose (d).
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3.7 Structure model index (SMI).
The untreated SCW group had significantly higher structure model index values (p<0.05) in both epiphyses and metaphysis regions compared to the Control, indicating that the arthritic trabecular bone was becoming a rod-like structure. Whereas the GlcNBu treated groups have 15-33% difference in SMI values compared to the untreated group in the three tested areas. Correspondingly, the trabecular separation was increased in the SCW group and this change was significantly reduced by GlcNBu treatment.
4. Discussion
In this study, we found that oral administration of GlcNBu, administered at two doses, resulted in disease modification in an SCW-induced arthritis rat model. There was significant improvement in direct measurement of joint inflammation, BMD and bone micro-architecture with administration of GlcNBu. The protection afforded by GlcNBu is considerable. For the inflammatory measurements most bone parameters, the effect is maximal at the relatively high single oral dose (200mg/kg/day), but for others the maximal effect is seen at the relatively low single dose (20mg/kg/day). Our pharmacokinetic studies in the rat (28) indicate that similar orally administered doses of GlcNBu (233 mg/kg) resulted in concentrations in the venous circulation that rise early and slowly decline.
In the SCW-arthritis model bone loss (osteoporosis) and marginal bone destruction are the common form of bone change (40;41),. A number of studies have described the pathological and structural changes in human arthritic joints (42-44). However, the trabecular bone connectivity alterations and site-specific micro-architecture changes in inflammatory models have not been reported before. Specifically, we report for the first time that trabecular connectivity is significantly decreased in the arthritic bone and the decreased connectivity was considerably reversed or prevented by GlcNBu treatment. Another unique finding was the 3-dimensional site-specific micro-architecture change. It is important to note that the posterior part of the joint showed higher bone mass but not necessarily higher connectivity than the middle or anterior parts. Similarly, the medial side of a coronal section has higher bone volume and thickness but no differences in connectivity compared to the lateral side. The SCW-mediated arthritis also demonstrated site-specific changes, namely, the anterior part (particularly in the lateral side) showed most arthritic changes compared to other sites.
Distinct evidence for disease-modifying activity for the compound was provided by the obvious improvement observed in the micro-CT images, in which the arthritic joints were significantly protected by treatment at the 200mg/kg/day dose, and were partially protected by treatment at the 20mg/kg/day dose (table 2). In addition, micro-CT technology, which is uniquely suited for comprehensive 3-dimensional analysis of bony
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joint surface integrity (figure 5) changes in the SCW-induced arthritis in the rat, showed clear protection with GlcNBu at both high and low dose. Connectivity evaluation showed that GlcNBu treatment resulted in protection of the subchondral cancellous bone connectivity and architecture. Improvement in bone density by DEXA was observed, this was particularly apparent in the distal tibia. Similar bone density protective effects have also been observed by treatment with cytokine inhibitor in a model of adjuvant-induced arthritis in the rat (45).
Our data from the current experiments demonstrates the bone-protective efficacy of GlcNBu in the Lewis rat model of SCW chronic destructive arthritis. It is not clear if the preventive changes in bone degradation are as a result of the apparent anti-inflammatory effect and decreased synovitis or if there is an independent effect on bone. The anti-inflammatory effect can be modified somewhat by different feeding protocols, such as adding the same total daily dose to crushed chow, so that the lower amounts are consumed continually (unpublished data).
A possible avenue of future investigation of the anti-inflammatory effects of GlcNBu is through a glycobiology perspective. Synthetic fibronectin peptides are known to suppress SCW-induced arthritis by interfering with leukocyte cell adhesion (46). A large molecular weight hyper-glycosylated fibronectin isoform, as well as a relatively under-glycosylated isoform, are induced by treatment of bovine chondrocytes by TGF-β-1 (47). As indicated in the Introduction, anti-TGF-β antibodies reverse the SCW-induced arthritis (31). Preliminary work from one of our Labs (T.A.) indicates that the hyperglycosylated fibronectin isoform does not bind antibodies such as rheumatoid factor, which are readily bound by the smaller MW under-glycosylated isoform, and that glycosylation of fibronectin may be affected by the addition of GlcNBu in some cell culture systems. A variety of antibodies have been identified in SCW-induced arthritis (48), so antibody binding to hyperglycosylated and under-glycosylated fibronectin, in animals treated and not-treated with GlcNBu, could provide some insight into a mechanism of action for the anti-inflammatory effects of this compound.
Also, it is possible that the preventive effect of GlcNBu on bone degradation in this model was mediated through directly inhibited osteoclast numbers and function or by increasing the number of osteoblasts at sites of bone erosion. The comparison of the isosurfaces and the architectural parameters demonstrate that GlcNBu effectively protects bone from further erosion in this model. However, the metaphyseal bone structure changes suggest that GlcNBu functions systemically.
Taken together, our findings indicate that GlcNBu has potential for development either as a stand-alone for inflammatory joint diseases with bone loss or in combination with other anti-inflammatory and/or bone growth-promoting agents. The findings also suggest that further evaluation of GlcNBu in a non-inflammatory animal model with bone loss, such as the ovariectomised rat, might provide further insight as to whether the
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effects observed were dependent on suppression of the inflammatory response or whether this drug affects bone metabolism directly.
Acknowledgements
This work was supported by a CIHR Proof of Principle Grant # 20020PPP (to T. Anastassiades) and a Strategic NSERC Grant # STPG 246039-01 (to T. Anastassiades).
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40. Chan JM, Villarreal G, Jin WW, Stepan T, Burstein H, Wahl SM. Intraarticular gene transfer of TNFR:Fc suppresses experimental arthritis with reduced systemic distribution of the gene product. Mol Ther 2002; 6(6):727-736.
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Chapter 5
Chapter 5 - The Effect of Glucosamine Hydrochloride on
Subchondral Bone Changes in an Animal Model of OA
Susanne X. Wang1, Sheila Laverty2, Mircea Dumitriu1, Anna Plaas3, and
Marc D. Grynpas1
1University of Toronto, Toronto, Ontario, Canada;
2 Universite´ de Montre´al, Montre´al, Quebec, Canada;
3 University of South Florida, Tampa.
Arthritis and Rheumatism, 2007 May;56(5):1537-48.
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Abstract
Objective: To quantify periarticular subchondral bone changes in a rabbit model of experimental osteoarthritis (OA), and to determine the effects of continuous administration of a clinically relevant dose of glucosamine HCl on subchondral bone changes in this model. Methods: Anterior cruciate ligament transection (ACLT) was performed on the left femorotibial joints of 16 rabbits to induce OA. Ten rabbits that did not undergo ACLT served as unoperated controls. Eight rabbits that underwent ACLT and 6 control rabbits were treated with 100 mg of glucosamine daily, and the others were given a placebo. The articular cartilage was evaluated macroscopically and graded at the time of necropsy, 8 weeks after ACLT. Bone mineral density (BMD) was measured using dual-energy x-ray absorptiometry on the dissected distal femur and proximal tibia. Subchondral trabecular bone turnover, architecture, and connectivity, as well as subchondral plate thickness and mineralization were studied on the undecalcified tibia sections from each animal.
Results: Eight weeks after ACLT, most of the operated joints had various degrees of cartilage damage and fibrillation. Compared with the control group, the ACLT group had significantly increased subchondral bone turnover and lower BMD, bone volume, connectivity, and bone mineralization. The high bone turnover was significantly reduced in glucosamine-treated animals that underwent ACLT. In fact, there were no significant differences between the ACLT/glucosamine group and the control/glucosamine group in most of the bone parameters studied.
Conclusion: This study shows that subchondral bone turnover, structure, and mineralization are significantly altered in the early stages of experimental OA, and that these changes are attenuated by glucosamine treatment.
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1. Introduction
Osteoarthritis (OA) is one of the most common joint diseases affecting aged humans. It is characterized by the progressive destruction of articular cartilage and concomitant changes in subchondral bone. It is estimated that 68% of Americans older than age 55 years have radiographic evidence of OA ([1]), with almost 85% of the population affected by age 75 years ([2]). The prevalence of OA is expected to increase ([3]). In the past, OA research focused mostly on alterations of cartilage, because cartilage degeneration was believed to be the most important change in OA. More recently, OA was recognized as a disease that involves the entire organ, i.e., the joint, not just cartilage. Subchondral bone changes in patients with OA are now attracting more interest ([4-8]). Twenty years ago, Radin and Rose ([9]) had already pointed to the possible role of subchondral bone in the initiation and progression of cartilage degeneration. Many studies have shown the dynamic morphologic changes of subchondral bone during the evolution of OA, which seem to be a part of disease progression ([10][11]). Several reports pointed out that the subchondral bone remodeling in OA involves both bone resorption and formation. In the early stage of OA, bone resorption was described as the primary feature of the bone remodeling process ([11-13]). In contrast, bone formation was predominant in the more advanced stage of the disease ([11][14][15]).
Moreover, evidence is now accumulating that certain types of primary OA might initially be a bone disease rather than a cartilage disease ([16]), although studies led to the conclusion that subchondral bone changes may be secondary to cartilage damage and proceed deeper into subchondral bone with increasing cartilage degeneration ([17]).
Recent studies addressed the changes that occur in the bone during the early stage of experimental OA in animal models ([18][19]). By suppressing bone turnover, calcitonin ([20-22]) and bisphosphonates ([23][24]) have been shown to reduce the severity of cartilage lesions in the early stages of experimental OA in animals. A cross-sectional study showed a significantly decreased prevalence of knee OA-related subchondral bone lesions in elderly women who received antiresorptive drugs, such as alendronate and estrogen, compared with elderly women reporting no use of these medications ([25]), underpinning the concept of the joint organ (cartilage and bone) being involved in the OA disease process.
Glucosamine is a naturally occurring aminomonosaccharide in the human body and is one of the principal substrates used in the biosynthesis of glycosaminoglycans, proteoglycans, and hyaluronan, all of which are fundamental components of articular cartilage. In several European double-blind studies in the early 1980s, investigators reported that oral glucosamine decreases pain and improves mobility in human OA and had no side effects ([26-31]). Conversely, a recently updated meta-analysis of
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glucosamine therapy in OA revealed that glucosamine failed to show benefit in terms of pain and functional outcomes ([32]). However, it was also noted that studies evaluating a European preparation of glucosamine sulfate did show more favorable effects ([32]), indicating that the effectiveness may depend on the brand of glucosamine used. Similarly, investigators in the Glucosamine/Chondroitin Arthritis Intervention Trial concluded that glucosamine did not reduce pain effectively in patients with knee OA, but that a combination of glucosamine and chondroitin sulfate may be effective in a subgroup of patients with moderate-to-severe knee pain ([33]).
Recently, 2 large, well-conducted, prospective 3-year randomized controlled trials showed that long-term administration of glucosamine had both structure-modifying (slowed radiographic progression) and symptom-modifying effects in knee OA ([34][35]). The results of these studies suggest that glucosamine is a suitable treatment for OA and may actually prevent joint damage. Differences in the results from various studies may likely be attributable to differences in the products studied, the study design, and the study populations ([36]).
In addition to the persisting controversy on the effects of glucosamine therapy in OA, there is also a lack of complete understanding of the mechanisms leading to the therapeutic effects of glucosamine. Several recent in vitro studies of cultured chondrocytes have shown that glucosamine sulfate stimulates glycosaminoglycan synthesis ([37-39]) and reduces its catabolism ([40][41]). In vitro studies have also shown that glucosamine was highly effective in preventing the interleukin-1 (IL-1 )-mediated suppressive effects on cartilage matrix synthesis ([37][38][42]). In a study using human OA chondrocytes, glucosamine was shown to inhibit the synthesis of proinflammatory mediators induced by IL-1 through an NF- B-dependent mechanism ([43]). However, these effects were observed with levels of glucosamine much higher than those attainable in vivo, in serum or synovial fluid, with currently used doses ([44-46]).
Recent in vitro studies of bovine and equine cartilage/chondrocytes have shown that glucosamine concentrations close to those attainable in vivo may regulate expression of matrix-degrading enzymes, their inhibitors, and inflammatory mediators at the transcriptional level, and this has provided a possible mechanism for their chondroprotective properties ([47-50]). Another study with OA cartilage explants showed that glucosamine could reduce the enzymatic breakdown of the extracellular matrix in human cartilage ([51]). Furthermore, glucosamine up-regulated aggrecan and type II collagen chondrocyte gene expression at physiologically relevant doses. Interestingly, up-regulation of transforming growth factor 1 was also observed at these concentrations, which could account for some of the proanabolic effects observed ([52]). Conversely, in another study using biologically relevant doses of glucosamine, no
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change in aggrecan expression or proteoglycan synthesis was detected when bovine chondrocytes were studied ([53]).
Glucosamine is widely used by patients with OA, for pain relief and to improve joint function, even though its mechanism of action is still not well understood and its effectiveness remains controversial. Animal models of OA provide a wide range of possibilities for understanding the effects of glucosamine on cartilage and bone structure, remodeling, and degeneration. In a chymopapain-induced model of articular insult in the rabbit, glucosamine increased glycosaminoglycan content in injured knees compared with control knees ([54]). The findings from animal experiments suggested that glucosamine, alone or in combination with other compounds, might act to modulate articular cartilage matrix metabolism, stimulating glycosaminoglycan synthesis in vivo ([39][55][56]). To date, the effects of glucosamine on subchondral bone changes in OA have not been evaluated.
The purpose of this study was to determine the magnitude of subchondral bone changes in the early stage of OA and to evaluate the effect of orally administered glucosamine on subchondral bone in a rabbit ACLT model of OA. Cartilage changes, bone mineral density (BMD), subchondral bone structure, bone turnover, and bone mineralization were studied.
2. Materials and Methods
2.1 Animals and OA model.
Twenty-six 9-month-old male NZW rabbits (Charles River Canada, St. Constant, Quebec, Canada) with no clinical or radiographic evidence of joint disease were used for the study. The mean ± SD weight of the rabbits was 4.60 ± 0.47 kg. All experiments were conducted according to the Canadian Council on Animal Care guidelines and with the approval of the Animal Care and Use Committee of the Faculty of Veterinary Medicine, University of Montreal.
Fifteen rabbits underwent unilateral (left knee joint) ACLT under general anesthesia. Briefly, a medial parapatellar skin incision was made, the patella was subluxated laterally, and the knee was placed in full flexion. The anterior cruciate ligament was transected, and the incision was sutured in a standard manner. A preoperative antibiotic (Tribrissen 24%, 15 mg/kg subcutaneously) (Schering Canada, Pointe-Claire, Quebec, Canada) and an analgesic (Buprenex, 0.03 mg/kg subcutaneously) (Reckitt & Colman, Richmond, VA) were administered. A fentanyl patch (25 g/hour) was also applied for pain relief for 72 hours. Following surgery, animals were maintained in cages, with unrestricted activity. Eight animals that underwent ACLT and 6 unoperated animals received orally administered glucosamine HCl (Sigma, St. Louis, MO), 100 mg in a 5-gm wafer (Bio-Serve, Frenchtown, NJ), daily for a period of 8 weeks, starting the day
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after surgery. The rest of the animals received a 5-gm placebo wafer daily for 8 weeks. The glucosamine dose was based on efficacy studies of glucosamine administration in human OA ([57]). The wafer administration protocol was described previously ([56]).
The experimental animals were divided into 4 groups, as follows: the control group comprised 4 rabbits that did not undergo ACLT or receive glucosamine treatment; the ACLT group comprised 8 rabbits that underwent ACLT of the left knee and received placebo; the control/glucosamine group comprised 6 unoperated rabbits that received glucosamine at a dosage of 100 mg daily for 8 weeks; and the ACLT/glucosamine group comprised 7 rabbits that underwent ACLT and received glucosamine treatment. One rabbit in the ACLT/glucosamine group was excluded from the study because the ACL section was observed to be incomplete on post mortem examination.
2.2 Macroscopic examination of cartilage.
Eight weeks after surgery, all animals were killed, and the left distal femurs and proximal tibias were excised, with the knee joint intact. The knee joint was then opened, and soft tissue was removed to expose the articular surfaces of the distal femurs and proximal tibias.
Four femorotibial joint compartments (the medial femoral condyle, the lateral femoral condyle, the medial tibial plateau, and the lateral tibial plateau) in all animals were examined for gross morphologic changes of the articular cartilage and graded, following the application of India ink. The grading system was modified from that described previously ([56][58-60]). Briefly, cartilage changes were graded on a scale from 1 to 6, where grade 1 = no uptake of India ink, indicating an intact surface; grade 2 = minimal focal uptake of India ink, indicating mild surface irregularity; grade 3 = evident large dark focal patches of ink uptake, showing overt fibrillation; and grades 4-6 = increasing size of cartilage ulceration. Details of the cartilage grading system are presented in Table 1. The lesions were mapped as a schematic representation of the femorotibial articular surfaces and were also documented digitally using a D1 Nikon camera (Nikon, Tokyo, Japan). Figure 1 shows typical cartilage lesions.
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Figure 1:
Figure 1. A, Typical cartilage lesions in the femur of a rabbit that underwent anterior cruciate ligament (ACL) transection. White arrow shows a grade 6 lesion; grey arrow shows grade 2 fibrillation. B, Osteophyte formation (grey arrows) in the tibial plateau of an ACL‐transected joint.2.3 BMD measurement.
Table 1. Results of macroscopic evaluation of cartilage 8 weeks after surgery*
Compartment/groupControl (n = 4)
ACLT (n = 8)
Control/GlcN (n = 6)
ACLT/GlcN (n = 7)
Medial tibial plateau 1.0 ± 0 2.9 ± 0.8 1.0 ± 0 1.7 ± 0.6
Lateral tibial plateau 1.0 ± 0 2.1 ± 0.6 1.0 ± 0 1.7 ± 0.4
Medial femoral condyle
1.0 ± 0 3.8 ± 0.8 1.0 ± 0 3.1 ± 0.5
Lateral femoral condyle
1.0 ± 0 3.1 ± 0.5 1.0 ± 0 2.7 ± 0.2
* Values are the mean ± SEM. Cartilage was scored on a 1‐6‐point scale, where 1 = intact surface normal in appearance; 2 = minimal fibrillation; 3 = overt fibrillation, distinguished surface irregularity, or cracks; 4 = erosion >0 mm and 2 mm; 5 = erosion >2 mm and 5 mm; 6 = erosion >5 mm. ACLT = anterior cruciate ligament transection; GlcN = glucosamine.
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Both the tibia and femur from the left knee of each animal were scanned by dual x-ray absorptiometry (DXA) (PIXImus; GE Lunar, Madison, WI). After calibration with an aluminum/lucite phantom (GE Lunar), the excised bones were placed on a polystyrene tray to mimic soft tissue, and the BMD (gm/cm2) and bone mineral content were measured at the distal femur and proximal tibia. One investigator (SXW) analyzed the BMD of the femur and tibia.
2.4 Processing of undecalcified tissue.
The proximal tibias were cut coronally, from the mid line into the anterior and posterior parts, and fixed in 10% neutral buffered formalin. Fixed specimens were dehydrated in ascending grades of acetone (70%, 90%, and 100%) and then infiltrated with increasing strengths of Spurr's resin (Marivac, Halifax, Nova Scotia, Canada)/acetone solutions (dilutions of 50:50, 80:20, 90:10, to 100%). Anterior and posterior proximal tibia specimens were then embedded in freshly made 100% Spurr's resin. Five-micrometer sections were cut using a Reichert-Jung 2050 motorized microtome (Reichert-Jung, Nussloch, Germany) and stained with modified Goldner trichrome for static histomorphometry. The remaining Spurr's resin blocks containing the mid sections of the tibias were polished, up to a 1- m diamond finish, using a Phoenix Beta Grinder/Polisher (Buehler, Lake Bluff, IL) and carbon-coated. Backscattered electron (BSE) images were obtained using a scanning electron microscope (model XL300ESEM; FEI, Hillsboro, OR) with a backscattering Centaurus detector (K.E. Developments, Cambridge, UK). Beam conditions were set at 20 kV accelerating voltage, a load current ranging from 5,887 microamperes. Contrast was set in the condition in which nonmineralized tissue and the plastic matrix appeared black, and mineralized tissue appeared in shades of gray (gray levels). A set of 2 BSE images were obtained at 50× magnification (50× BSE images) in the proximal tibia region and used for bone architecture and connectivity analysis of the subchondral trabecular bone and also for measurement of subchondral bone thickness. Another set of 6-8 BSE images was obtained at 200× magnification (200× images) at the subchondral plate of each proximal tibia and used to study bone mineralization.
2.5 Histomorphometric analysis.
Static histomorphometry was performed on undecalcified Goldner trichrome-stained mid sections (5 m) of the proximal tibia. Measurements of the trabecular bone area were obtained from the central epiphyseal subchondral bone region, which was within 1 mm beneath the cortex. The region was enclosed by the subchondral bone plate, both cortices, and the growth plate. Bone formation and resorption parameters were measured using BQ Nova Prime version 6.50.10 software (BioQuant Image Analysis, Nashville, TN). The osteoid volume, osteoid surface, osteoid thickness, and eroded surface were
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measured and calculated according to American Society for Bone and Mineral Research (ASBMR) guidelines ([61]).
2.6 Bone structure.
The subchondral trabecular bone structure in the epiphyseal region of the proximal tibia was analyzed using a Quantimet 570 image processing and analysis system (Leica, Richmond Hill, Ontario, Canada) on the 50× BSE images. After thresholding the gray-level images, binary images were obtained to analyze the structure parameters. The structural and connectivity parameters of trabecular bone were measured on BSE images and calculated using the ASBMR guidelines ([61]). The following structural parameters were obtained from the image: trabecular bone volume/total volume, trabecular thickness, trabecular number, and trabecular separation.
2.7 Bone connectivity.
A skeletonized binary image was used to measure the bone connectivity parameters. The image analysis was done by using an in-house program based on the techniques described by Parisien et al ([62]). The following connectivity parameters were obtained in the analysis: number of multiple points (1/mm2), number of end points (1/mm2), length of node-free struts (mm/mm2), length of node-node struts (mm/mm2), length of free-free struts (mm/mm2), ratio of end points:multiple points, and total strut length (mm/mm2).
2.8 Assessment of subchondral bone plate thickness.
The subchondral bone plate thickness of the tibia was measured on the 50× BSE images using individual point-to-point distance measures. The thickness was measured from the top of the calcified cartilage to the deep surface of the subchondral bone plate by averaging 10 measurements per tibia. All measurements were made in a standardized viewing area by a single observer who was blinded to the experiment.
2.9 Bone mineralization assessment.
A set of 6-8 BSE images was obtained at 200× magnification for each tibia in the region of the subchondral bone plate, at a working distance of 15 mm. Calibration of the machine was performed with silicon dioxide before and after obtaining the 200× images for each sample. The mineralized bone exhibited a range of gray-scale values (range 0-255), and the area at each gray level was measured from the BSE images using the Quantimet 570 image analysis system (Leica Imaging Systems, Cambridge, UK). In a BSE image, gray levels represent varying degrees of bone mineralization, where brighter intensities indicate higher degrees of mineralization. The percentage area of pixels, excluding the plastic region, was calculated. The mineralization profile of a specimen is represented by a histogram of gray-level intensities. The gray-level intensity
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distribution was described using the logit function, or cumulative log ratio ([63]), and is defined as follows: logit = ln (proportion >125 to proportion <125).
2.10 Statistical analysis.
All data were analyzed with the SPSS version 11.0 for Windows statistical package (SPSS, Chicago, IL). One-way analysis of variance with Fisher's post hoc analysis of least significant difference was used to compare means. P values less than 0.05 were considered significant. All data are presented as the mean ± SEM.
3. Results
3.1 Macroscopic features of cartilage.
Cartilage from the left femorotibial joints of rabbits in the control and control/glucosamine groups (10 joints) was unremarkable and had macroscopic grade 1 at every assessed compartment. No cartilage ulcerations were observed in any of the joint compartments in the contralateral unoperated joints of the 16 rabbits that underwent ACLT (data not shown).
Cartilage lesions were observed in the ACLT model, as previously observed in our laboratory ([54]). Briefly, in all animals that underwent ACLT, lesions in the femoral condyles were more severe than those in the tibial plateau. The fibrillation and ulceration of articular cartilage occurred focally in most of the femurs from rabbits that underwent ACLT and at similar sites in the condyles. It is also interesting to note that cartilage ulcerations in the tibias from rabbits that underwent ACLT occurred on the caudal (posterior) tibial articular surface. This was probably attributable to the biomechanical alterations induced by cutting the ACL.
The control and control/glucosamine groups had the lowest scores for macroscopic cartilage damage, and the ACLT group had the highest score. The ACLT/glucosamine group tended to have lower grades for the severity of cartilage lesions in all 4 compartments when compared with the ACLT/placebo group, but this difference was not statistically significant (Table 2).
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3.2 DXA findings.
The results of measurement of BMD of the left femur and tibia are shown in Figure 2A. BMD was significantly lower in the femurs and tibias of animals in the ACLT/placebo group compared with control joints. However, there were no significant differences in BMD between the ACLT/glucosamine and the control/glucosamine groups (Figure 2A).
Table 2. Results of macroscopic evaluation of cartilage 8 weeks after surgery*
Compartment/groupControl (n
= 4) ACLT (n
= 8) Control/GlcN (n
= 6) ACLT/GlcN (n
= 7)
Medial tibial plateau 1.0 ± 0 2.9 ± 0.8 1.0 ± 0 1.7 ± 0.6
Lateral tibial plateau 1.0 ± 0 2.1 ± 0.6 1.0 ± 0 1.7 ± 0.4
Medial femoral condyle
1.0 ± 0 3.8 ± 0.8 1.0 ± 0 3.1 ± 0.5
Lateral femoral condyle
1.0 ± 0 3.1 ± 0.5 1.0 ± 0 2.7 ± 0.2
* Values are the mean ± SEM. Cartilage was scored on a 1-6-point scale, where 1 = intact surface normal in appearance; 2 = minimal fibrillation; 3 = overt fibrillation, distinguished surface irregularity, or cracks; 4 = erosion >0 mm and 2 mm; 5 = erosion >2 mm and 5 mm; 6 = erosion >5 mm. ACLT = anterior cruciate ligament transection; GlcN = glucosamine.
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Figure 2. Bone mineral density (BMD) and structural changes. A, BMD of the left distal femur and proximal tibia after anterior cruciate ligament transection (ACLT) and/or glucosamine (GlcN) treatment. B, Bone turnover parameters. Bone formation (osteoid volume) and resorption (eroded surface) were significantly greater in the ACLT group compared with all other groups. Conversely, the ACLT/glucosamine group
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demonstrated significantly less change in bone turnover parameters than the ACLT group. C, Average subchondral plate thickness (Avg subchon plate Th.) in the various groups. Subchondral bone thickness was significantly decreased in the ACLT group compared with the control (Ctrl) group but not the ACLT/glucosamine group. Data are shown as box plots. Each box represents the 25th to 75th percentiles. Lines outside the boxes represent the 10th and the 90th percentiles. Lines inside the boxes represent the median.
3.3 Bone static histomorphometric findings.
Results of histomorphometry of the left proximal tibias from the control and control/glucosamine groups were unremarkable (similar to the cartilage change), with higher trabecular bone volume and thickness, but lower osteoid volume, surface, and thickness and less eroded surface (Table 2).
There were remarkable increases in osteoid volume (P = 0.001), surface (P = 0.001), and eroded surface (P = 0.032) but decreased trabecular bone volume (P = 0.006) and thickness (P = 0.002) in the ACLT/placebo group compared with the control group. This indicates that ACLT induced high bone turnover and consequently lower bone mass.
The ACLT/glucosamine group showed relatively higher osteoid volume (P = 0.041) but no significant change in eroded surface (P = 0.96), trabecular bone volume (P = 0.29), and thickness (P = 0.008) compared with the control/glucosamine group (Figure 2B and Table 2).
It is important to note that there was a significant reduction in the osteoid volume (P = 0.042) in the ACLT/glucosamine group compared with the ACLT/placebo group and a trend toward smaller osteoid surface (P = 0.055). This indicates that glucosamine treatment suppressed the high bone turnover observed in the ACLT group, preventing or significantly reducing the subchondral bone structure changes caused by ACLT.
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3.4 Subchondral bone thickness.
Subchondral bone thickness was decreased considerably in the ACLT/placebo group compared with the control group, but less so in the ACLT/glucosamine group. The statistical analysis showed a significant decrease in subchondral bone thickness in the ACLT/placebo group compared with the control group, but no difference between the ACLT/glucosamine group and the control/glucosamine group (Figure 2C). The decreased subchondral bone thickness in the operated groups could be the result of higher subchondral bone turnover after surgery.
Table 2. Results of histomorphometry and connectivity analysis by backscattered electron imaging
Control ACLT P Control/GlcN ACLT/GlcN P
Structure
Trabecular bone volume, % 33.26 ± 3.61 23.41 ± 0.36* 0.006 29.84 ± 1.62 27.66 ± 1.08 0.29
Trabecular thickness, m 150.93 ± 1.40 114.84 ± 4.29 0.002 153.17 ± 9.65 127.13 ± 4.07 0.008
Trabecular number, 1/mm2 159.6 ± 12.3 122.1 ± 4.4 0.002 163.2 ± 9.9 136.5 ± 4.2 0.01
Trabecular separation, mm 2.20 ± 0.14 2.06 ± 0.07 0.335 1.98 ± 0.14 2.18 ± 0.05 0.129
Connectivity
No. of multiple points, 1/mm2 1.66 ± 0.36 2.38 ± 0.24 0.039 1.42 ± 0.17 2.00 ± 0.12 0.060
No. of end points, 1/mm2 310.8 ± 36.8 375.58 ± 12.7 0.034 353.5 ± 23.6 334.1 ± 10.8 0.451
Length of node-node struts, mm 0.50 ± 0.05 0.60 ± 0.04 0.143 0.41 ± 0.03 0.57 ± 0.04 0.014
Length of free-free struts, mm 1.45 ± 0.23 1.13 ± 0.06 0.059 1.44 ± 0.09 1.16 ± 0.08 0.055
End point:multiple point ratio 2.07 ± 0.32 2.03 ± 0.09 0.888 2.10 ± 0.31 2.23 ± 0.18 0.653
Total strut length, mm/mm2 0.037 ± 0.009 0.010 ± 0.003! 0.086 0.038 ± 0.013 0.034 ± 0.01 0.772
Values are the mean ± SEM. * P = 0.033, ACLT versus ACLT/GlcN group. ! P = 0.068, ACLT versus ACLT/GlcN.
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3.5 Bone architecture and connectivity.
Bone architecture changes affected by ACLT and glucosamine treatment were observed in BSE images of the mid sections of coronally cut tibias. Representative BSE images from the 4 groups are shown in Figure 3. The BSE image from the control group illustrates the general bone structure and connectivity in tibias from healthy rabbits of the same age as animals in the other groups. Compared with trabecular bone of animals in the control group, trabecular bone of rabbits in the ACLT/placebo group had the lowest volume, thickness, and connectivity. In contrast, animals in the ACLT/glucosamine group had thinner trabeculae but no significant decrease in trabecular bone volume when compared with animals in the control/glucosamine group.
Table 2 shows trabecular bone structure and connectivity data for the proximal tibia. Similar to the results from histomorphometry, there was a significant loss of trabecular bone in the ACLT/placebo group, as indicated by a decrease in trabecular bone volume, trabecular thickness, and trabecular number compared with the control group. It is important to note that the ACLT/glucosamine group had significantly higher trabecular bone volume than the ACLT/placebo group (P = 0.033), indicating that glucosamine protects against trabecular bone loss in joints from animals that underwent ACLT. The numbers of end points and multiple points in the ACLT/placebo group were
Figure 3
Figure 3. Representative backscattering electron images of proximal tibias, showing changes in bone volume, trabecular connectivity, and mineralization in the different study groups. Compared with control (Ctrl) bone, bone from animals that underwent anterior cruciate ligament transection (ACLT) showed fewer and thinner trabeculae. ACLT combined with glucosamine (GlcN) treatment partially prevented the changes in trabecular bone thickness and volume caused by surgery.
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significantly higher than those in the control group; this reflects higher bone resorption and formation (turnover) in bones from animals that underwent ACLT. However, there was no statistically significant difference in connectivity between the ACLT/glucosamine group and the control/glucosamine group, reflecting the protective effect of glucosamine on bone connectivity. Also, importantly, bone from animals in the ACLT/placebo group showed the lowest total strut length among all groups (P < 0.086), and the ACLT/glucosamine group did not differ from the 2 control groups; this correlates with the bone volume and connectivity results.
In summary, joints from animals in the ACLT/placebo group showed significant bone loss and disconnectivity; conversely, joints from animals in the ACLT/glucosamine group did not show significant bone loss and disconnectivity when compared with unoperated control limbs.
3.6 Mineralization (BSE microscopy)
Decreased bone mineralization was observed in joints from rabbits that underwent ACLT compared with control joints. The mineralization profile of rabbits in the ACLT/placebo group showed an 8% decrease in bone mineralization compared with that in the control group (Figure 4). However, there was only a 4% decrease in the ACLT/glucosamine group compared with the control/glucosamine group (Figure 4). The logit functions calculated from the mineralization profiles did not show statistically significant differences between groups.
Figure 4.
Figure 4. Mineralization profile of the subchondral bone plate from the proximal tibia, showing the percentage area versus the gray level (a higher gray level represents a higher degree of mineralization). There was an 8% decrease in the mineralization profile of the group that underwent anterior cruciate ligament transection (ACLT) compared with the control (C) group. However, there was only a 4% decrease in the ACLT/glucosamine group compared with the control/glucosamine
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4. Discussion
ACLT in various animal models has been widely used to study the pathogenesis of OA and to examine disease-modifying treatments of OA ([23][64-66]). ACLT-induced experimental OA in rabbits is one of the animal models most commonly used to study new treatment options for OA ([56][66-72]).
This study demonstrated that in the rabbit model of ACLT-induced OA, excessive subchondral bone turnover occurred early. This phenomenon is associated with decreased BMD, mineralization, trabecular bone volume, and thickness, lower connectivity, and thinner subchondral bone plate. The lower connectivity observed in the setting of ACLT is consistent with increased bone turnover. However, fewer differences were observed between the ACLT/glucosamine group compared with the control/glucosamine group than between the ACLT/placebo group compared with controls, which indicates that glucosamine partially inhibits the high bone turnover caused by ACLT. A decrease in BMD was also recently reported in the rabbit model of OA, using microfocal computed tomography (micro-CT) analyses at 8 weeks post surgery, but BMD returned to normal levels at 12 weeks ([73]).
Mansell and Bailey studied bone collagen metabolism in the femoral heads of patients with OA and observed increased collagen metabolism in OA joints, with the greatest changes occurring within the subchondral zone ([74]). Several clinical studies also demonstrated that bone resorption is increased in patients with progressive hip and knee OA ([6][75]). In the model in our study, the ACL-transected bones presented activated bone resorption and consequently decreased trabecular bone volume, thickness, and trabecular connectivity. Many other studies also confirmed that increased bone resorption occurs in earlier stages of experimental OA ([12][20][76-78]). A longitudinal study in cruciate-deficient dogs with OA showed a loss of trabecular bone by micro-CT analysis ([11]). Similarly, this has been observed in a rabbit model of OA ([73]). Decreased trabecular bone volume and trabecular thickness and higher trabecular separation have been observed in other OA models as well ([18][77]). All of these structural changes are associated with increased bone turnover.
In addition to structural changes, hypomineralization (another feature of subchondral bone change) has also been observed in patients with OA. Grynpas et al ([79]) reported that thickening of the subchondral bone with an abnormally low mineralization pattern was pronounced in samples obtained from human OA hips. Later, Mansell and Bailey ([74]) confirmed the hypomineralization of deposited collagen in the subchondral zone of OA femoral heads, supporting the notion of a greater proportion of osteoid in diseased tissue. In our study, an 8% decrease in subchondral bone mineralization was observed in the ACL-transected joints compared with control joints. Our results are consistent with the above-mentioned decreased mineralization observed in human OA.
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DXA has been used extensively as a simple tool to assess the density of subchondral bone in OA ([6][7][75][80-82]). Although increased BMD in patients with OA has been reported many times ([75][80][83]), low BMD and high bone turnover appear to occur in the affected regions ([4][8][81]) and are associated with more rapid progression of OA ([6][82][84]). In this study, decreased BMD was present in the knee joints of animals with an early stage of experimental OA, predominantly in the distal femur. Our results are consistent with the findings in other animal models ([73][76]).
In the present study, we investigated early subchondral bone changes in various ways, measuring changes in BMD, subchondral bone plate thickness, trabecular bone structure and connectivity, bone mineralization, and bone histomorphometry. This approach provides a unique way to demonstrate the complicated subchondral bone alterations related to disease progression. We observed that ACL-transected joints exhibited higher bone turnover and more severe subchondral bone changes and cartilage damage. By inhibiting high bone turnover, glucosamine protected most of the subchondral bone structure and partially prevented mineralization and cartilage changes.
The search for disease-modifying drugs to treat OA has become a priority in arthritis research. For many years, the nutritional supplement glucosamine has been used for the treatment of OA, but the mechanisms of its palliative and potential disease modification effects remain unknown. The effect of glucosamine in animal models of OA has been studied extensively in the past to investigate the therapeutic efficacy of glucosamine and to understand the underlying mechanism of its action ([38][39][42][54-56][66]). These studies were mainly focused on the effect of glucosamine on OA cartilage. The effects of glucosamine on subchondral bone change in OA have not been reported previously.
Our study demonstrated that the high bone turnover in the joints of animals that underwent ACLT and received placebo was significantly reduced in the joints of rabbits that underwent ACLT and received glucosamine. The associated changes in joints of animals in the ACLT/placebo group, such as decreased BMD, bone volume, and subchondral bone plate thickness, were partially corrected in the ACLT/glucosamine group. The gross cartilage morphologic changes induced by ACLT were partially decreased by glucosamine treatment in this study; it should be noted that these results were not statistically significant but do represent a trend consistent with our other results. The cartilage changes were assessed macroscopically in this study, to preserve the underlying bone tissue for assessment. Histologic evaluation may have permitted identification of more subtle effects of glucosamine on cartilage, as in a previous study ([56]).
The presumed underlying basis for the disease-modifying activity of glucosamine could include effects on osteoclast and osteoblast function, which could indirectly preserve cartilage from degeneration. In 1999, Manicourt and colleagues used calcitonin, an antiresorptive drug, to suppress the response of bone, cartilage, and synovium in the
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early stages of canine experimental OA and reduced the severity of the cartilage lesions ([20]).
We chose to evaluate glucosamine HCl in this study for the following reasons: both its quantity and purity were known, its effects on cartilage were previously assessed in this model ([56]), and its pharmacokinetics and bioavailability were established using a similar dose in another monogastric animal, the horse ([44]). In a pilot study in rabbits (not published), following the administration of glucosamine orally, serum concentrations comparable with those reported in the horse were attained. The maximum serum concentrations in both species were similar to those recently reported in humans following oral administration of a similar dose of the patented crystalline glucosamine sulfate formulation, indicating similar absorption characteristics of glucosamine formulations in monogastrics ([45]).
A potential limitation of the current study, when extrapolating findings to human patients with OA, is that glucosamine was administered very early in the disease process, and our protocol would therefore reflect preventive therapy. However, the findings are relevant because they would favor the use of glucosamine in OA-susceptible patients, e.g., immediately after joint injury. Furthermore, other factors should be kept in mind when extrapolating the results to therapy for human OA and include biologic variations between species and the difference in the speed of development of OA in this animal model (weeks) when compared with naturally occurring disease in humans (usually many years).
In this animal model, ACLT altered the distribution of mechanical forces to the various tissues to which bone is a sensitive responder. The decreases in subchondral bone volume, BMD, and mineralization, as well as increased connectivity in the injured knee due to increased bone turnover, may be related to altered loading and kinematics in the knee. The bony changes that develop after ACLT also could result in abnormal transmission of stress to the overlying cartilage and thereby contribute to the progression of cartilage degeneration. This could be an important factor in the pathogenesis of posttraumatic OA. Subchondral bone changes are clearly important in the pathogenesis of OA, and changes in the subchondral bone region at the cellular level warrant further study.
In summary, this study provides new and interesting findings on subchondral bone structure, mineral properties, and morphologic changes, as well as their relationship to cartilage lesions. Glucosamine protects against subchondral bone change during the development of OA. These inhibitory effects of glucosamine on subchondral bone change in the early phase of surgically induced OA suggest a possible novel mechanism of glucosamine to partially protect cartilage from degeneration. These data suggest a disease-modifying effect of glucosamine HCl on subchondral bone in OA.
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Chapter 6
Chapter 6 - Stereological Study of Cartilage and Subchondral
Bone Changes in Spontaneous Knee Osteoarthritis: Results from Two Strains of Guinea
Pig Susanne X. Wang1, David Eyre2, Ryan H. Tam3, Larry Arsenault4, Ernst B.
Hunziker1
1University of Bern, Bern, Switzerland;
2University of Washinton, Seattle, WA, USA
3 Shiley Center for Orthopaedic Research & Education, La Jolla, California, USA
4 McMaster University, Hamilton, ON, Canada
(To be submitted)
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Abstract
OBJECTIVE: The aim of the study was to characterize the three-dimensional properties of bone and cartilage in a spontaneous GP OA model and to investigate possible correlations between changes in the different tissues. We also explored the possible connection between the serum biomarker, bone and cartilage changes in this model of OA.
METHODS: 6 Dankin Hartley and 6 GOHI strain guinea pigs were used in the study. Serum and urine samples were collected at 6, 6.5, 7, 7.5, 8 and 8.5m old of age and the level of collagen type II C-telopeptide degradation product (CTX-II) was measured by ELISA. Animals were sacrificed at 11 month of age. 24 proximal tibias were embedded in MMA and cut at 5 coronal planes from anterior to posterior and then analyzed using stereological method. 24 femurs were scanned with micro CT. A quantitative analysis of the three-dimensional subchondral bone microarchitecture was performed in the regions of anterior medial (AM), anterior lateral (AL), and posterior medial (PM), posterior lateral (PL) epiphysis.
RESULTS: At 11 months of age, structural changes in both strains of guinea pig were similar to those seen in human OA. Compared to the GOHI strain of guinea pig, the DH strain exhibited a significantly larger lesion volume (3.8% vs. 1.5%), a thicker articular cartilage (0.042 vs. 0.035mm), but a thinner calcified cartilage (0.008 vs. 0.01 mm) and subchondral cortical plate (0.035 vs. 0.039 mm). The thickness of calcified cartilage in DH guinea pigs is significantly thinner than that of GOHI.
The micro-CT analysis showed lower bone mineral density in the DH strain compared to GOHI strain, but the subchondral trabeculae bone were thicker and more distinctly separated in the DH strain. These changes were most severe on the medial side of the joint, particularly in the anterior region.
CONCLUSION: Results from this study concludes that biomarkers are not as sensitive as 3-D structural measurements from cartilage stereology and micro CT, which provides significantly more qualitative details over their two-dimensional counterparts. The qualitative three-dimensional data provided by the present study should facilitate and support future investigations in this research area.
Chapter 6: Stereological Study of Spontaneous OA Joints
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1. Introduction
Osteoarthritis (OA) is a slowly progressive disorder, characterized morphologically by the destruction of cartilage, sclerosis of subchondral bone, formation of cysts, and the presence of osteophytes at the joint margins. It is divided into two main types: primary (idiopathic) and secondary (due to disease or injury). Although much effort has been put into studies on primary OA, little is known of its etiology and pathogenesis. Studies of early primary OA in humans is difficult because the course of the degenerative process is slow and subtle. By the time it is usually detected via radiographic evidences of joint space narrowing, the disease has already advanced toward the late stages of its development.
While the symptoms of OA are well-characterized and pronounced (i.e. erosion of the cartilage layer, formation of osteophytes, and sclerosis of subchondral bone), the causes and initiating factors remain largely unknown. The complexity in both the development and the progression of OA can be attributed to the highly intimate relationship between cartilage, subchondral bone, and neighboring tissues and the complex level of organization necessary for normal joint function. The interdependent relationships between the tissues of the joint organ (cartilage, calcified cartilage, bone, synovium and ligaments) create a delicate system of mutual feedback and support. When one element falls out of balance, it inevitability triggers multiple physiological and biochemical responses from the other components as the joint system seeks to rebalance itself. Multiple biochemical and biomechanical pathways, both cell and non-cell mediated, exist to potentially initiate and to fuel the progression of the disease. This problem is only compounded by the tissues’ significant remodeling in response to the increased levels of stress, especially in the subchondral bone, and completely changes both the physiology and the loading dynamic of the joint with disease progression. In addition, pain and limitation in range of motion with the progression of OA may also affect the way diseased individuals move and load their joints, adding yet another level of complication to disease development.
At the microscale, changes to the tissue are as remarkable, if not even more profound than the macro-changes. In the cartilage alone, there exist numerous highly interdependent organization and pathways that can affect the progression and development of the disease. Its two primary matrix molecules, collagen and proteoglycan, have are intimately linked in their organization, synthesis, and repair, as well as their biomechanical functions. A compromise in the function of one constituent can elicit a biomechanical and potentially damaging effect on the other. The quality and quantity of their content in the ECM in turn has both a direct biochemical and an indirect biomechanical effect on the metabolism and production of the native chondrocytes. The resulting high stress level can lead to cell death or abnormal cell
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metabolism, both with evident influence on the matrix maintenance and the remaining cell population.
Bone, while it normally retains a higher capacity for healing than cartilage, does not necessarily respond in a manner that benefits the entire joint. In response to the higher stress levels, bone remodeling begins with an increase in bone metabolism, which initially decrease the level of mineralization in the tissue coupled with an increase in bone mass, followed by an increase in mineralization at later stages. The physiological change in the subchondral bone has a direct biomechanical impact on the cartilage tissue layer on top, perceived as an initial drop in stiffness and then a significant decrease in shock absorption. Other changes associated with bone remodel such as increase in vascularity and biochemical changes in the microenvironment may also have a significant effect on all tissue of the joint, in particular, perhaps playing a crucial role in the formation of osteophytes.
Because of the high level of complexity in the initiation and progression of OA, understanding the mechanics and the specific pathways of the disease progression at different stages is critical for the development of therapeutic and preventative treatments. New evidences have shed new light on the significance of bone as not only a mediator but a potential initiator of OA. The relationship existing between calcified cartilage, subchondral cortical and trabecular bone are thought to play an important role in the pathogenesis of OA. Analysis of their influence on OA progression, however, has largely been based upon 2 dimensional radiographic data. As a structural tissue, the 3 dimensional properties of bone (such as trabecular thickness, connectivity, and separation) have a profound influence on its biomechanical properties. This study believes that 3 dimensional evaluations of subchondral bone changes in an OA joint will yield unique insights on the disease progression.
One other major obstacle to the study of early OA is the lack of human subjects and unavailability of tissue samples. Researchers, however, noticed that osteoarthritis-like conditions occur naturally in many animals such as horses, pigs, dogs, and guinea pigs, and have developed many animal models for experimental OA. The Dunkin-Hartley guinea pig, is an especially well-recognized model for experimental OA and is known to present more osteoarthritic cartilage changes than other strains of guinea pig (ref). The aim of the present study was to investigate the structural properties of these tissues in the primary osteoarthritic joints of two strains of guinea pig.
2. Materials and Methods
2.1 In vivo study
Six GOHI/SPF guinea pigs were purchased from RCC Ltd., Switzerland. Six Dunkin-Hartley guinea pigs were imported from Charles River Laboratories, Germany. At the
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beginning of the investigation, guinea pigs were 6 months old; at its termination (5 months later), they were 11 months old. Series of four blood samples and four urine samples per animal were collected every 2 weeks between the animal ages of 6 to 8 month old.
Blood was withdrawn from the Angulus Venosus under general anesthesia and collected into EDTA-containing tubes and was centrifuged for 10 minutes at 2000 g at room temperature to isolate the plasma samples. Urine samples were collected in pharmalogical cages, which were specially designed for this study. Urine samples were always collected at the same hour of the day.
2.2 Biomarker measurement
Urine and plasma samples collected from all time points were stored at -70°C until their evaluation at the end of the study. Type II collagen is the major structural component of hyaline cartilage, and almost exclusively localized in this tissue. Therefore fragments derived from this protein should represent a specific index for cartilage degradation. The serum and urinary concentration of collagen type II C-telopeptide degradation products (CTX-II) was measured by enzyme linked immunosorbent assay (ELISA) and normalized to creatinine (at the Orthopaedic Research Laboratories of the University of Washington).
2.3 Tissue processing
Twelve hind limbs from each group were amputated and the knees were dissected free of soft tissues. Total of 24 left and right distal femurs were removed and fixed in 10% natural buffered formalin. The 24 proximal tibias together with the medial and lateral meniscus were fixed by immersion in 2.5% (volume per volume) glutaraldehyde (Merck, Dusseldorf, Germany) and 2.5% (volume per volume) formaldehyde (Merck) buffered (pH 7.4) with 0.1 mol sodium cacodylate (Merck) for 2–4 days at room temperature. They were then dehydrated in series of ascending concentrations of ethanol and embedded in methylmethacrylate. A series of five 1.2 mm thick tissue slices from each tibia were coronally cut using a diamond band-saw (Exact Medical Instruments, Oklahoma City, OK), glued onto polished Plexiglas object holders (Altumax France SA, Paris La Défense, France), milled to a thickness of approximately 100–150 μm with a Polycut E (Reichert-Jung, Heidelberg, Germany), polished, surface-stained with McNeil's tetrachrome, toluidine blue O, and basic fuchsin[E.B. Hunziker, Growth-factor-induced healing of partial-thickness defects in adult articular cartilage. Osteoarthritis Cartilage 9 (2001), pp. 22–32.], and examined in a Vanox AH-2 light microscope (Olympus, Tokyo, Japan).
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2) The mean defect depth, which was evaluated systematically by taking linear measurements.
2.5 3D histological analysis:
All 5 sections were used for semiquantitative lesion volume/cartilage volume analysis. Three dimensional cartilage lesion volume measurements were performed using the method descried previously. [Quantitative structural organization of normal adult human articular cartilage. E.B. Hunziker. Osteoarthritis and Cartilage (2002) 10, 564-572]
2.6 2D histological analysis:
All the thickness measurements of the tibia were using individual point-to-point distance measures. Total of 12 -15 lines vertically to the cartilage surface were drawn with equal distance on each tibia. The thickness of articular cartilage and calcified cartilage was measured from the top surface to the bottom surface, while the subchondral bone plate thickness was measured from calcified cartilage to the deep surface of the subchondral bone plate by averaging measurements per tibia. All measurements were made in a standardized viewing area by a single observer who was blinded to the experiment (figure 2).
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2.7 MicroCT imaging analysis
Twenty-four femurs (12/group) were scanned with micro-CT scanner (Explore Locus SP micro CT Scanner, GE Healthcare) at 16-µm isotropic voxel size. The reconstructed tibia images were analyzed with MicroView 2.0 software (GE Healthcare, London, ON) in two and three dimensions.
Figure 2
Figure 2: A. point‐to‐point measures of articular cartilage, calcified cartilage and subchondral bone plate thickness. B. A micrograph of Cartilage and subchondral bone, showing the cartilage lesion, articular cartilage (AC), calcified cartilage (CC), subchondral bone plate (SBP), old bone (OB), new bone (NB), trabecular bone (Tr.B) and bone merrow (BM).
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2.8 Threedimensional trabecular bone measurements
Three dimensional subchondral trabecular bone analyses were conducted using four selected regions of interest (ROIs) that were positioned in the middle of the anterior medial (AM), anterior lateral (AL), and posterior medial (PM), posterior lateral (PL) epiphysis. All four ROIs were rectangular shape with dimensions 1.5mm x1.5mm x 0.5mm (Figure 3).
On 3D analysis, the tissue volume (3D-TV, mm3) and trabecular bone volume (3D-BV, mm3) were measured directly, and the fractional trabecular bone volume (3D-BV/TV, %) was calculated. The trabecular thickness (3D-Tb.Th, µm) and trabecular number (3D-Tb.N, N/mm) were measured directly on 3D images [Hildebrand T, Laib A, Muller R, Dequeker J, Ruegsegger P. Direct three-dimensional morphometric analysis of human cancellous bone: microstructural data from spine, femur, iliac crest, and calcaneus. J Bone Miner Res 1999; 14(7):1167-1174].
2.9 Threedimensional isosurface
A three dimentional isosurface image of each distal femur was obtained by a volume-rendering function in MicroView to exam the osteophyte formation (figure 4).
Figure 3:
Figure 3: A 1.5mm x1.5mm x 0.5mm region of interest was placed in the central of anterior medial condyle to collect the 3‐D trabecular bone measurements.
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2.10 Statistical analysis.
All the data were analyzed with SPSS 11.0 for Windows statistical package (SPSS Inc., Chicago, IL). One-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) post-hoc test was used to compare means. Significance was considered at p < 0.05. All data are presented as mean ± standard error of the mean (SEM).
3. Results:
3.1 Stereology Results:
At 11 months of age, structural changes in both strains of guinea pig were similar to those seen in human osteoarthritis, especially those occurring on the central medial plateau. In general, tibia plateau cartilage was thicker on the medial than on the lateral side. The small, superficial lesions were usually associated with a thickening of the cartilage layer. Compared to the GOHI strain of guinea pig, the DH strain exhibited a significantly larger lesion volume (3.8% vs. 1.5%) and a thicker articular cartilage - AC (0.042 vs. 0.035mm), however a thinner calcified cartilage - CC (0.008 vs. 0.01 mm) and subchondral cortical plate - SCP (0.035 vs. 0.039 mm). Although the total thickness of the cartilage did not differ between the two groups, the thickness of calcified cartilage in DH guinea pigs is significantly thinner than that of GOHI.
Figure 4:
Figure 4: An isosurface image of distal femur showing osteophyte formation in color.
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3.2 MicroCT data
The micro-CT analysis revealed the DH strain to have a significantly lower bone mass than the GOHI one: a lower bone mineral density, a smaller bone volume fraction and bone surface area. However, the subchondral bone trabeculae were thicker and more distinctly separated in the DH than in the GOHI strain. Each of the aforementioned changes was most severe on the medial side of the joint, particularly in the anterior region (See Table 1 for details).
Figure 5
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Micro-CT analysis of 3D structure changes during the development of spontaneous OA in two strains of guinea pigs. The DH strain showed significantly lower bone mass than the GOHI strain and those changes are site-specific.
Figure 6:
Figure 6: Osteophyte formation in GOHI strain (left) and DH strain (right) guinea pigs. DH strain guinea pig showed significantly larger osteophyte formation (red circle).
Table 1:
Table 1: Site‐specific bone properties of GOHI vs. DH guinea pig
STRAIN Anterior Posterior Anterior Posterior Average
Volume of Bone (mm^3) GOHI 0.46±0.02 0.50±0.03 0.46±0.03 0.44±0.03 0.46±0.02
DH 0.36±0.02 0.45±0.02 0.43±0.02 0.44±0.02 0.42±0.01P -value 0.005 0.228 0.498 0.845 0.042
Bone mineral density (mg/cc) GOHI 504±13.6 541±12.1 497±21.6 494±17.5 509±9.7
DH 439±24.9 501±13.0 481±16.3 487±15.8 477±12.5P -value 0.033 0.033 0.566 0.761 0.055
Bone volume fraction GOHI 0.47±0.03 0.51±0.03 0.46±0.03 0.45±0.02 0.47±0.02
DH 0.37±0.02 0.47±0.02 0.44±0.02 0.45±0.02 0.43±0.01
P -value 0.009 0.251 0.674 0.915 0.077
Surface area (mm^2) GOHI 6.88±0.22 6.51±0.19 6.78±0.18 6.93±0.13 6.77±0.10
DH 5.19±0.23 5.68±0.13 5.95±0.17 5.55±0.13 5.59±0.07P -value 0.000 0.002 0.002 0.000 0.000
Trabecular thickness (pixels) GOHI 3.42±0.24 3.97±0.25 3.50±0.28 3.22±0.20 3.53±0.16
DH 3.67±0.23 4.07±0.23 3.78±0.29 4.11±0.26 3.91±0.09P -value 0.470 0.774 0.494 0.013 0.048
Trabecular seperation (pixels) GOHI 5.5±0.4 5.2±0.4 6.0±0.6 5.7±0.4 5.6±0.3
DH 10.2±1.1 6.7±0.4 6.9±0.4 7.4±0.5 7.8±0.3P -value 0.000 0.024 0.197 0.012 0.000
Medial Lateral
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The osteophyte formation was mainly observed in the joint surface edge where cartilage surface meets bone surface. The DH strain presented more osteophyte formation (8 out of 12 vs. 2 out of 12 joints) and larger osteophyte volume (figure 6).
3.3 Biomarker Results
There is no significant difference in the serum levels of biomarker between the two guinea pig groups. Urinary levels of Col2CTx, however, show a higher trend in DH guinea pigs throughout the entire study (figure 7).
4. Discussion:
As early as 1986, it was proposed that subchondral bone may play a role in the initiation and progression of OA (Radin et al). From a physiological and mechanical standpoint, it is evident that subchondral bone’s structure properties would affect the functions of the joint organ. Clinically, it is shown that remodeling of the subchondral bony tissues is a well-defined characteristic of advanced OA. However, due to previous limitations in imaging technology, early studies of subchondral bone changes rely primarily on two-dimensional radiographic images. Nevertheless, as biomechanical tissues, cartilage, calcified cartilage, and subchondral bone all have distinct three-dimensional structural properties that affect their functions in the joint.
Figure 7:
Figure 7: Urinary levels of Col2CTx in two strains of guinea pigs between the age of 6 to 9 month old. DH strain revealed higher urinary Col2CTx level than GOHI strain guinea pig.
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This study aimed to characterize the three-dimensional properties of bone and cartilage in developing OA and investigate possible correlations between changes in the different tissues. Because OA is a progressive disease that involves multiple tissues, understanding the interactions between them is crucial for the development of treatment and effective therapies. The authors postulate that the identification of key links in the remodeling of tissues would provide entryways for potential drug or surgical intervention to slow or half disease progression.
[Differences between the two GP groups] In this study, structural changes similar to those observed in human osteoarthritis were present in both strains of guinea pig. At 11 months of age, however, the two guinea pig models employed showed a markedly different rate of OA development. Compared to the GOHI strain of guinea pig, the DH strain exhibited a significantly larger lesion volume (3.8% vs. 1.5%). This data is consistent with the higher rate of OA progression previously reported for DH guinea pigs (Huebner JL, Hanes MA, Beekman B, TeKoppele JM, Kraus VB.A comparative analysis of bone and cartilage metabolism in two strains of guinea-pig with varying degrees of naturally occurring osteoarthritis. Osteoarthritis Cartilage. 2002 Oct;10(10):758-67.). Total cartilage thickness, however, is not significantly different between the two groups. On closer examination, it is revealed that the layer of calcified cartilage is much thinner in DH than that in GOHI guinea pigs (0.008 vs. 0.01 mm), resulting in a much thicker layer of articular cartilage (0.042 vs. 0.035mm). One possible explanation for this phenomenon is that with the onset of OA, both bone and cartilage response to the elevated levels of stress and enters a phase of increased remodeling. Unlike many other tissues, the growth and remodeling of bone begins first with a resorption phase where the bone volume is actually lost through the actions of activated osteoclasts. This hypothesis is supported by the thinning of subchondral bone plate in DH vs. than their GOHI counterpart (0.035 vs. 0.039 mm). The reduction in the thickness of the calcified cartilage layer may either be attributed to the systematic demineralization processes in the activated joint, or due to the subchondral bone invasion, or both. The demineralization process associated with rapid bone remodeling results in a net loss in bone stiffness and support for the cartilage, which in turns reacts with increased levels of matrix production. It is known that chondrocytes in OA patients is associated with the production of lower quality matrix. Whether this is purely a result of aging cells or if the cells are ‘worn out’ in a period of accelerated matrix production is unknown.
[MicroCT bone data] The micro-CT analysis revealed marked differences in the bone structure and material properties between the DH and GOHI guinea pig strains. This difference mirrors the changes measured in stereology and confirms the correlations between bone and cartilage morphology in OA progression. The DH strain has a significantly lower bone mass than the GOHI one: a lower bone mineral density (477 vs. 509 mg/cc), a smaller bone volume fraction (43% vs. 47%) and bone surface area (5.59
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vs. 6.77 mm2). This data confirms the loss of bone mass in the early stages of OA and can be attributed to the unique mechanisms of bone remodeling. Bone remodeling is typically separated into four distinct phases: activation, resorption, matrix production, and mineralization. On the other hand, the subchondral bone trabeculae were thicker and more distinctly separated in the DH than in the GOHI strain. While the general theory of bone remodeling accounts for the initial loss of bone mass, it does not adequately explain the concomitant thickening and separation of the bone trabeculae. The authors propose that another mechanism exists in the early stages of bone remodel where bone mass is increased in selected area even during the resorption phase of remodeling. The presence of preexisting osteoids in the bone is likely to repel osteoclasts actions and to continue, perhaps even in an accelerated rate, their mineralization process. This hypothesis accounts for the thickening of the remaining trabeculae while the degree of separation is increased (unprotected trabeculae are destroyed by the activated osteoclasts).
[Site Specificity of Changes] One other important finding of this study is the site specificity of the OA development. In general, plateau cartilage was thicker on the medial than on the lateral side. In the bony tissue, it was found that differences between the two strains were more pronounced on the medial side of the joint, particularly in the anterior region. This indicates that OA development is highly site-specific, and presumably under the influence of varied loading dynamics in different joint regions. It is important to note that changes in the cartilage tissue are always mirrored by a corresponding change in the bony tissue, indicative of a direct link between the remodeling mechanisms.
[Biomarkers] Biomarkers offer new options for the detection of OA tissue changes by tracking byproducts of matrix degradation. This study monitored the levels Col2CTx, a known product of collagen II degradation in both serum and urine. However, no significant difference was found in the serum levels of biomarkers between the two groups, even though there were unmistakable differences in their rates of OA progression. Stereology and microCT data showed marked structural differences such as the volume fractions of cartilage lesions, subchondral bone thickness, and the presence of osteophytes. The urine data do show a consistently trend of higher levels of biomarkers in the DH vs. GOHI guinea pig. It is unclear why there is a difference in the sensitivity between serum and urine levels of Col2CTx. This lack of sensitivity in serum levels of Col2CTx may be attributed to the age of the animals, where
[Conclusion] In conclusion, subchondral bone changes are invariably linked to cartilage changes in the development and progression of OA. While biomarkers hold tremendous potential for the prediction and detection of tissues changes in the joint during OA development, this study did not find them as sensitive as three-dimensional measurements from stereology and microCT. Stereological histology holds a significant
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advantage over two-dimensional histology by providing qualitative details on the structural organization of the cartilage and subchondral tissue. Likewise, microCT analysis is able to presents three-dimensional data on both the structural and material properties of bone when compared to two-dimensional radiographs. This sensitivity of the imaging technique also revealed many site-specific differences in the OA tissue. The high level of complexity of tissue structure and organization in the joint continues to make characterization of OA progression a great challenge. The qualitative three-dimensional data provided by the present study should facilitate and support future investigations in this research area.
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15. Pastoureau PC, Chomel AC, Bonnet J. Evidence of early subchondral bone changes in the meniscectomized guinea pig. A densitometric study using dual-energy X-ray absorptiometry subregional analysis. Osteoarthritis Cartilage 1999; 7: 466-473.
16. Watson PJ, Hall LD, Malcolm A, Tyler JA. Degenerative joint disease in the guinea pig. Use of magnetic resonance imaging to monitor progression of bone pathology. Arthritis Rheum 1996; 39: 1327-1337.
17. Manicourt DH, Altman RD, Williams JM, Devogelaer JP, Druetz-Van Egeren A, Lenz ME, et al. Treatment with calcitonin suppresses the responses of bone, cartilage, and synovium in the early stages of canine experimental osteoarthritis and significantly reduces the severity of the cartilage lesions. Arthritis Rheum 1999; 42: 1159-1167.
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Chapter 7
Chapter 7 - Progressive Change of Cartilage and Subchondral
Bone in Partial Medial Menisectomy and Accelerated Spontaneous OA Models - A 6
Month Study with Dunkin Hartley Guinea Pigs
Susanne X. Wang1, Mircia Dumitriu1, Ryan H. Tam2, Rita A. Kandel1, Richard
Reulund1, Marc D. Grynpas1
1University of Toronto, Toronto, Ontario, Canada;
2 Shiley Center for Orthopaedic Research & Education, La Jolla, California, USA
(To be submitted)
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Abstract
OBJECTIVE: The present study not only investigates cartilage structure and biochemistry, but also tracks the progressive changes in bone density, subchondral trabecular bone architecture, bone turnover, mechanical and material properties of bone, and bone mineralization of both traumatic and accelerated spontaneous OA in a single animal model from time 0 to 10, 20, 30, 90, and 180 days after surgery. METHODS: Sixty guinea pigs were used in this study. Fifty 2-months old male Dunkin-Hartley guinea pigs underwent partial medial meniscectomy (pMNX) in the right knee. Ten animals were euthanized at 0, 10 and 20days, 1, 3, and 6 months time points postsurgery OA cartilage morphological changes were validate/evaluated by Mankin grading. Cartilage biochemistry, bone mineral density (BMD), subchondral bone plate thickness, bone histomorphometry and osteophyte formation were studied.RESULTS: Animals underwent partial medial meniscectomy displayed significantly higher degree of cartilage degeneration, increased subchondral bone turnover, early decrease followed by an increase in bone mineral density and bone volume and subchondral bone plate area. RESULT: In this guinea pig model, we have observed spontaneous OA-like changes in the un-operated knee, and accelerated progressive changes in the operated knee. Cartilage structure changes include: surface destruction, chondrocyte hyper/hypo-cellularity, tidemark duplication, and loss of cartilage. Changes in cartilage biochemistry include: loss of proteoglycan, collagen, and DNA. Subchondral bone changes include: calcified cartilage thickening, changes in subchondral bone plate thickness and mineralization, changes in trabecular bone remodeling, architecture, and material properties. CONCLUSION: The accelerated nature of this surgery-enhanced spontaneous OA model has been confirmed by the rapid occurrence of bone and cartilage changes in the operated knees while similar changes were observed in the contralateral joint only at later times. Therefore, this is a suitable model to study the epidemiology, disease progression and treatment of OA.
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1. Introduction:
Osteoarthritis (OA) is the most common form of arthritis. It has become second only to heart disease as the leading cause of work disability in the general population [1], and is the primary cause of disability among people over the age of 65 [2]. It is estimated that 68% of Americans over the age of 55 have radiographic evidence of OA, with almost 85% of the population affected by age 75. Clinically, OA is characterized by pain, inflammation, deformity, and limited range of motion. Pathological changes include progressive articular cartilage loss and destruction, subchondral bone sclerosis, synovial inflammation, formation of cysts, and the presence of osteophyte at the joint margins.
However, despite its prevalence among the elderly and extensive documentations on its late stage pathology, little is known about OA pathogenesis. Initially believed to be a cartilage disease, recent evidences highlight subchondral bone’s active involvement in the initiation and progression of OA, as first suggested by Radin et al in 1970 [3, 4]. Because normal articular joint function depends on both the excellent frictional properties of cartilage and the shock-absorbing support of the mineralized tissues, changes in the structural or biochemical properties of any tissue in the joint organ, however subtle, can potentially disrupt this delicate balance. Microscopic structural changes in the tissues, biochemical and biological changes at the cellular levels are likely to be responsible for the subtle yet significant steps in OA development [5, 6].
OA is typically diagnosed by radiographic detection of joint space narrowing and visible cartilage destruction, but recent studies suggest that its pathogenesis occurs long before the appearance of any gross morphologic transformation [5, 7]. Bone exhibits higher levels of metabolism and remodeling than cartilage and responds rapidly to external stresses. Such changes in the subchondral mineralized tissues would have strong influences on OA pathogenesis by altering the biomechanical and biochemical microenvironment of the entire joint organ. Bone metabolism has also been linked to the formation of cartilage lesions, may provide a potential route of early detection for OA. As a highly complex disorder involving many tissues, OA pathogenesis and progression can be understood only if the relationship between cartilage and subchondral mineralized tissue is fully appreciated.
The complexity in OA pathogenesis can be attributed to the highly intimate relationships between cartilage, subchondral bone, and neighboring tissues as well as the complex level of organization necessary for normal joint function. Multiple biochemical pathways, both cell and non-cell mediated, and biomechanical pathways exist to potentially initiate and to fuel the progression of the disease. This problem is only compounded by the tissues’ significant remodeling in response to the increased levels of stress, especially in the subchondral bone, which completely changes both the physiology and loading dynamics of the joint with disease progression. In addition, pain
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and limitations in range of motion with the progression of OA also affect the way diseased individuals move and load their joints, adding yet another level of complexity to disease development.
As such, once OA progression reaches a critical stage, its development is usually accelerated by multiple positive feedback mechanisms that exist between the many joint components. Disease-modifying and preventative treatments, therefore, are far more likely to be found at the early stages of disease development. It is believed that the early progression of OA involves an ordered cascade of biological events linked to various changes in the different tissues in the joint organ. Characterizing tissue changes in early OA development, especially in subchondral bone, will help us gain a better understanding of pathogenesis and early progression of OA.
However, studies of early osteoarthritic changes in humans are difficult because of ethical considerations concerning tissue sampling. The alternative therefore is to study OA development in available animal models. A variety of animal models has been developed and utilized for the study of OA disease pathogenesis. Methods to induce experimental OA in animals include anterior cruciate ligament transection [8-10], meniscectomy [11-14], and repetitive impulsive loading [15]. Each animal model has its own disadvantages and advantages [16]. A common drawback to all trauma-induced models is their rapid rate of disease progression, and thus can only represent some aspects of secondary, but not primary OA in humans.
Spontaneous OA, on the other hand, has a much slower rate of disease progression and most closely mimics the natural pathology of primary OA in humans. Spontaneous OA is known to occur naturally in guinea pigs, Syrian hamsters, and nonhuman primates. In particular, the guinea pig model of knee osteoarthritis is one of the most fully described models of spontaneous of osteoarthritis [11, 17, 18]. It shares many characteristics of human knee OA development and progression, being spontaneous and involving the medial tibio-femoral compartment with histopathology and biochemical alterations [18]. A common tradeoff in employing spontaneous models is that they include much variability between animals and are therefore less predictable than their surgically-induced counterparts [16].
Dunkin-Hartley (DH) guinea pigs are a particularly popular model of spontaneous OA among guinea pigs due to their relatively predictable disease development. DH guinea pigs develop a slowly progressing arthropathy, morphologically resembling human primary osteoarthritis. Qualitative light microscopic studies have shown that moderate to severe degeneration of the tibial plateau develops in these animals by the age of 9 months and that the changes regularly progress to severe osteoarthritis in old age [16, 17].
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It is shown that performing a partial medial meniscectomy in this model further reduces variability between animals and accelerates the rate of OA progression [19-22]. By performing partial medial meniscectomy on one knee and tracking OA progression in both joints, this study explores the possibility of examining two OA models in a single animal: 1. the first 6 months of OA development in the partial medial meniscectomized knee joints – this model mimics secondary OA; 2. the progression of spontaneous guinea pig OA between age 3 to 9 months in the contralateral knees - this model mimics primary OA, although the natural OA development could be slightly accelerated by the favored usage of the non-surgical leg.
The present study not only investigates cartilage structure and biochemistry, but also tracks the progressive changes in bone density, subchondral trabecular bone architecture, bone turnover, mechanical and material properties of bone, and bone mineralization of both traumatic and accelerated spontaneous OA in a single animal model from time 0 to 10, 20, 30, 90, and 180 days after surgery.
The study results will provide useful information about the dynamics of disease progression in the earlier stage of OA, especially the insight on the relationship between articular cartilage and subchondral bone. This study might answer the following question - during the progression of OA, what changes first, subchondral bone or cartilage?
2. Materials and methods
2.1. Animal model
Sixty 2-months old Male Dunkin-Hartley guinea pigs obtained from Charles River (US) were housed in single cages and maintained at standard condition. Animals were fed a normal guinea pig chow. The surgery performed when the animals at 3-months of age and weighed about 680±20g.
Partial medial meniscectomy was done on the right knee of 50 guinea pigs anesthetized by inhalation of 5% isoflurane in a mixture of 50% oxygen and 50% nitrous oxide. Fifty guinea pigs received partial medial meniscectomy (pMNX) in the right knee. The technical aspect of the operation followed previously published surgical procedures by Bendele {Bendele, 1987 #21}. Minor adjustments of the surgical protocol were used to make this model less invasive, therefore analogous to spontaneous OA. In the surgery, the joint capsule was opened in the medial side and meddle 1/3 part of the meniscus was removed without transection of the medial collateral ligament. The capsule was then sutured and the skin incision closed. No considerable inflammation from the operation was observed in any of the animals.
During the study, all the guinea pigs were healthy as indicated by maintenance of normal body weight and food consumption.
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2.2. Samples collection
Ten guinea pigs were sacrificed at 0, 10, 20, 30, 90, and 180 days after surgery. Animal weight were monitored upon arrival (average animal age 60 days), at surgery, and 10, 20, 30, 60, 90, 120, 150 and 180 days post-surgery. Ten days and three days before sacrifice, the animals received injections of tetracycline, a bone marker, in order to label the newly formed bone and to study dynamic histomorphometry. All the right femurs and tibias (the operated knee) were stripped of tendon and musculature. Cartilage sample were harvest from femur chondyles and groves immediately after dissection, and subjected to cartilage biochemistry study. The remaining femurs were placed in 70% ethanol for histomorphometric analysis. All the tibias were placed in 10% formalin for the cartilage morphometric analysis.
2.3. Dual Energy Xray Absotiometry (DEXA):
The excised right and left femur and tibia of each animal were scanned with DEXA, the bone mineral density (BMD) was measured in the distal femur and proximal tibia.
2.4 Cartilage Biochemistry:
Articular cartilage slices were collected from the femoral groove and condyle. The frozen tissues were lyophilized and weighed. The wet weight and dry weight of the sample was measured before and after lyophilization. Lyophilized samples were digested at 65°C for 48 hours in 40μg/ml papain solution. The glycosaminoglycan (GAG), collagen (hydroxiproline), and DNA contents were measured by biochemical methods [23-25]. Replicate measurements were taken for all of the assays.
2.5 Histological Grading of Cartilage:
The tibias were fixed in 10% formalin and cut coronally, with the first half of the tibias decalcified, embedded in paraffin, and used for cartilage grading. Safranin-O and Toluidine Blue stained 5μm histological sections were evaluated using a modified Mankin grading to quantify OA disease severity.
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2.6 Subchondral bone plate mineralization and thickness change
The second half of the tibia was fixed in 10% formalin and not decalcified. All tibias were processed with ascending concentrations of spurr resin/acetone, and finally embedded in spurr resin. The embedded tibia blocks were then polished, carbon coated, and examined using backscattering scanning electron (BSE) microscopy. BSE images of subchondral bone plate were taken at 200x magnification to determine the degree of mineralization of subchondral bone plate and calcified cartilage. Five parameters were analyzed on the distal tibia (epiphysial region): subchondral bone plate thickness, porosity, percentage bone volume, percentage calcified cartilage, and mineralization (grey level) distribution of the bone in the subchondral plate. Subchondral bone plate thickness was measured using 50x BSE tibia images by averaging 15 individual point-to-point distance measures.
2.7 Histomorphometry analysis using Bioquant Nova Pro System
Femurs were fixed in 70% ethanol and processed with ascending concentrations of Spurr’s Resin/acetone, and embedded in Spurr’s Resin. Five-μm undecalcified coronal middle plane sections of each femoral block were cut and stained with Goldner’s Trichrome for static histomorphomic analysis. Bioquant Nova Pro System (Bioquant, US) was used to assess bone structure and formation parameters of the distal femur in
Table 1: Modified Mankin scale evaluation parameters
Test Parameter Condition Score
Structure Normal 0 Surface Irregularities 1 Pannus and Surface Irregularities 2 Clefts to Transitional Zone 3 Clefts to Radial Zone 4 Clefts to Calcified Zone 5 Complete Disorganization 6 Cells Normal 0 Diffuse Hypercellularity 1 Clonning 2 Hypocellularity 3 Proteoglycan Content Normal 0 Slight Reduction 1 Moderate Reduction 2 Severe Reduction 3 No Dye Noted 4 Tidemark Integrity Intact 0 Crossed by Vessels 1
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studying subchondral bone structure, connectivity and histomorphometry. Seven-μm sections were also cut, and left unstained for dynamic histomorphometry analysis.
2.8 Femoral subchondral bone structure and connectivity analysis using BSE images
The remaining femoral blocks were then polished, carbon coated, and examined using backscattering scanning electron (BSE) microscopy. The BSE images were collected at 50X and analyzed for subchondral trabecular bone structure and connectivity, as well as growth plate volume/thickness. Structure and connection analysis were done using method described in Wang et al [26].
Briefly, binary images from 50X BSE images were used to analyze structural and connectivity parameters of trabecular bone using the ASBMR guidelines [27]. The following structural parameters were obtained from the image: trabecular bone volume/total volume, trabecular thickness, trabecular number, and trabecular separation.
The subchondral trabecular bone structure in the epiphyseal region of the proximal tibia was analyzed using a Quantimet 570 image processing and analysis system (Leica, Richmond Hill, Ontario, Canada) on the 50× BSE images. After thresholding the gray-level images, binary images were obtained to analyze the structure parameters. The structural and connectivity parameters of trabecular bone were measured on BSE images and calculated using the ASBMR guidelines [27]. The following structural parameters were obtained from the image: trabecular bone volume/total volume, trabecular thickness, trabecular number, and trabecular separation.
2.9 Bone connectivity
A skeletonized binary image was used to measure the bone connectivity parameters. The image analysis was done by using an in-house program based on the techniques described by Parisien et al [28]. The following connectivity parameters were obtained in the analysis: number of multiple points (1/mm2), number of end points (1/mm2), length of node-free struts (mm/mm2), length of node-node struts (mm/mm2), length of free-free struts (mm/mm2), ratio of [end points]:[multiple points], and total strut length (mm/mm2).
2.10 Microhardness
The plastic tibia blocks were polished and micro-hardness of the cortical and trabecular bone will be tested with a Knoop diamond indenter under a 20 grams load for 10 seconds. The micro-hardness is determined from the length of the indentation and the applied force, the method was described by Chachra et al {Chachra, 1999}. The Hardness of bone is linked to the degree of mineralization and is proportional to bone stiffness.
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2.11 Data analysis
All data were analyzed by one-way ANOVA using SPSS statistical software package and significance assigned at p≤0.05.
3. Results:
3.1 Animal Weight Change
Animal weights increased with time in both treated and untreated groups with the same scale, from average of 600g at 2 month of age to 750g at surgery (3month age), to a final weight of roughly 1080g at sacrifice (figure 2).
3.2 Histology
Mankin grading was 6.4 ±1.4 at 1 month and showed a significant progression in OA severity to 7.5 ±1.3 at 3 months and 9.9 ±1.4 at 6 months after surgery. Table 2 shows the comparative Mankin scores between the operated and control joints. Some representative histological graphs from various time points are showing in Figure 3 to illustrate the progressive change in OA cartilage.
Figure 2:
Figure 2: Guinea pig weight change during the experiment period
Surgery Date
0
200
400
600
800
1000
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60 90 120 150 180 210 240 270
Wei
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Guinea Pig Age (Days)
Body Weight of Guinea Pigs
Dunkin Harley Guinea Pig Surgery Date
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3.3 Cartilage Biochemistry:
The contents of DNA, proteoglycan, and collagen were determined for each time point during the 6-month study. Cartilage matrix changes in operated right knee joints
Figure 3:
Figure 3: Cartilage degeneration as the disease progresses
Table 2.
Mankin Score time 0 10 days 20 days 30 days 90days 180days
Operated leg 4.5±1.4 5.6±1.5 6.4 ±1.4 7.6 ±1.3 9.9 ±1.4
Control leg 0.6±0.6 1.2±0.7 1.6±0.4 2.1 ±0.5 3.9 ±0.8 5.9 ±1.7
Table 2. OA Severity in the partial medial meniscectomized guinea pigs
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3.6 Bone structure analysis on femoral BSE images
Bone structure parameters: The trabecular bone volume of operated femurs exhibited an initial increase followed by a steep decline in the first 30 days, and trabecular thickness decreased more than 10% in the first month. In the surgical leg, both trabecular bone volume and thickness reached their lowest point at 30 days and increased thereafter. Both were drawn back to baseline level at 90 days and kept climbing until the end of the experiment (Figure 10A&B).
On the other hand, trabecular number of the operated femurs had their highest peak in the 20 day time point. In addition, it is important to note that the trabecular number in the surgery group exceeds that of the control group at this time point. In contrast, trabecular separation exhibited a sharp decrease in the same period. Both trabecular number and separation dropped back to their respective baseline levels at the 90-days
Figure 9
A
B C
Figure 9: Osteoid Surface, Osteoid volume and thickness change with time in the femoral condyle of the surgery and controlateral knee
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time point and remained unchanged through the reminder period of the study (Figure 10C&D). All the structural parameters of contralateral femurs remained relatively stable with small deviation.
3.7 Bone connectivity analysis on femoral BSE images
Connectivity parameters Trabecular bone strut analysis of the operated femurs revealed a significantly higher number of end point and free-free struts, but a lower level of multiple points and note-note struts compared to the contralateral femurs during the progression of the disease. A rapid change was observed in the first month after surgery (see figure 11). The operated
Figure 10:
A.
B.
C. D.
Figure 10: trabecular bone volume, thickness, and trabecular number and separation changes with time during the progression of OA
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limbs demonstrated a higher disconnectivity in the subchondral bone in the early stage of OA progression.
3.8 Subchondral bone plate change:
The subchondral plate of the osteoarthritic joints exhibits higher calcified cartilage and porosity but lower mineralized bone volume compared to the contralateral joints (Figure 12). The medial tibia of the operated limbs (most affected by OA) presented thicker and denser subchondral bone plate than the lateral side of the same bone. When comparing these parameters longitudinally, from 10 days to 90 days the operated medial tibia shows a continuous increase in calcified cartilage content and subchondral bone thickness. Figure 13 is showing the progressive change of subchondral bone plate mineralization and organization change in OA. Future analyses will focus on the
Figure 11:
A.
B.
C.
D.
Figure 11: bone connectivity parameters of femoral condyles from BSE image
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quantitative study of subchondral plate mineralization, in order to understand how subchondral bone mineralization effects the OA progression.
Figure 12:
Figure 12: subchondral bone plate composite change during the progression of OA.
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3.9 Osteophyte formation in the osteoarthritic joint:
The osteophyte formation has been extensively observed in the joint margin of the osteoarthritic joints. The histological appearance of the osteophyte is a kind of osteochondral excrescence (figure 14 and 15). In our guinea pig model, cartilaginous osteophyte was observed since 20 after surgery (figure 14), the bonny osteophyte started seen at 90 days after surgery (figure 15), the size and number of osteophyte increases with time after surgery.
Figure 13:
A. 10 days after surgery
B. 90 days after surgery
C. 180 days after surgery
Figure 13: bone mineralization change during the progression of OA, presented by BSE images
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Figure 15:
Figure 15: backscattered electron images showing mineralized osteophyte in the medial tibial plateau of the operated joint.
Figure 14:
Figure 14: histological appearance of osteophyte formation in partial medial meniscectomized guinea pig tibia
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4. Discussion:
Due to the subtle nature of OA progression and the current lack of early detection technology, the onset of OA is usually first diagnosed with the radiographic evidence of reduced joint space [30-32]. However, detectable narrowing of the joint gap equates a significant loss cartilage tissue, indicative of only late or advanced stages of OA. As a result, many animal models were developed and have proved to be invaluable in the study of OA development and progression. The Dunkin Harley guinea pig model, in particular, is a well-recognized model of spontaneous OA, expressing many symptoms and a rate of progression parallel to the human pathology [11, 17, 18].
While the Dunkin Harley (DH) guinea pig spontaneous OA model is a fairly good representation of idiopathic OA development in humans, it, like its human complement, is highly susceptible to variability between individuals [16]. By performing a partial medial meniscectomy to the DH guinea pig, this study aims to develop a more predictable model with a slower progression than other more invasive secondary OA models (ACLT, total menisectomy). A slower disease progression is especially critical for the investigation of the series of complex tissue interactions in the relatively narrow window of disease initiation and early development. Because the surgical procedure and resulting OA in one leg invariably impose biomechanical (mechanical compensation) and biochemical (growth factors and cytokines) pressure on the other, this study also employed the contralateral joint as a separate model to conduct parallel investigation of accelerated spontaneous OA, a phenomenon also frequently observed in humans [33].
Cartilage together with the underlying subchondral bone is an inhomogeneous multi-property tissue. It is composed of several different tissues in a multi-layered organization (cartilage, calcified cartilage, subchondral plate - cortical bone, and subchondral trabecular bone). These tissues all have different material and biological properties and respond to force, damage, and drugs in different ways. Because stimulus on one tissue may elicit a reaction that have both biomechanical and biochemical effects [34, 35] on every other constituent, the interactions between tissues within a joint organ determines the complicated nature of the joint and its disease progression.
4.1 Histology
In this guinea pig model, we have observed significant and progressive OA changes in the operated knee, with parallel OA progression also detected un-operated knee at a lesser severity. Changes of the joint in this animal model are continuous and involve many different tissues. Cartilage structure changes include: surface destruction, chondrocyte hyper/hypo-cellularity, tidemark duplication, and loss of cartilage. Changes in cartilage biochemistry include: loss of proteoglycan, collagen, and DNA. Subchondral bone changes include: calcified cartilage thickening, changes in
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subchondral bone plate thickness and mineralization, changes in trabecular bone remodeling, architecture, and material properties.
4.2 Biochemistry DNA
Early DNA content increase in the control group may be attributed to a cell proliferation response to the surgery (increased loading stimulus from mechanical compensation) followed by a drop as the animals quickly recovered from the operation. The relatively fast recovery period (within 10 days) indicates a short lasting reaction in the cell population when driven by a mechanically dominated stimulus. The second increase of cell population found at the 30-days time point in the control group may also be a product of a similar loading mechanism, although the higher peak and longer duration suggest the involvement of additional factors, most probably a change in the general blood chemistry attributable to the onset of OA in the operated leg. The progressive drop in the control cell population after the 30-days time point can be a combination effect of tissue’s desensitization to the elevated levels of cytokines and/or natural relaxation towards its normal baseline level.
Although there is a similar long-term trend in the population density in both the control and surgery group, it is important to note that the changes in the DNA content is composed of a combination effect of both the cell death from mechanical/biochemical stress and an increase in cell proliferation from mechanical/biochemical stimulus. Thus, the lower fluctuation in the DNA content of the surgery leg in the early stage (0-30 days) does not necessarily reflect a reduced responsiveness of the operated joint to the surgical stimulus. Rather, the relatively steady level of DNA content probably denotes a high level of cell death coupled with a high rate of cell proliferation. This hypothesis is supported by the histological data, with the level of change and tissue remodeling being significantly more severe in the operated joint.
In addition, DNA content may not accurately reflect sudden drops in the live cell population, as dead cells may also be counted prior to the full degradation of their DNA content. Therefore, drops in actual cell population may be even sharper than the DNA content data may suggest.
4.3 Biochemistry GAG
The GAG content (normalized by DNA content) in the control group generally follows the pattern of cell population change as measured by DNA content, suggesting a mirrored activation in both cell proliferation and productivity in response to mechanical stimulus. A notable exception is found in the early stage (20-days time point), where an evident peak in the cell population is not reflected in a similar increase in GAG content. It is speculated that when the cells are expose to a threshold level of stimulus, they will switch to a short period of proliferation mode with reduced matrix production.
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On the other hand, the surgical group exhibits significant fluctuation in its GAG content in period following surgery: dropping sharply to a minimum at the 10-days time point, recovering to a peak at the 20-days time point before declining steadily towards another minimum at the 90-days time point. To understand their significance, the changes in GAG levels must be examined as a combined product of several distinct factors: pre-existing levels of GAG in the matrix, active production rate of GAG by cells over time, and active (cell-mediated) and passive (non cell-mediated) degradation/loss of GAG from the matrix.
Due to the size of the proteoglycan molecules and the way it is embedded in the complex collagen network, the rate of loss of proteoglycan through diffusion or hydrostatic action is relatively low [36]. Thus, the loss of GAG production from even severe levels of cell injury or death in the tissue is unlikely to be responsible for a sudden drop in matrix GAG content. The cause for the initial drop in GAG content is probably due to a combination effect of direct damage to the collagen network by mechanical overload and cell-mediated degradation of the matrix via cytokines and enzymes. The rapid recovery of the level of GAG content is likely to be a reaction of the cells to the mechanical load/overload. From the 90-days time point on, GAG content of the surgical group progressively increased while that of the control group decreased. This trend continued through the end of the study (180-days time point) where GAG content of the surgical group exceeded that of the control group.
4.4 Biochemistry Hydroxproline
Examination of the hydroxproline data would show almost identical trends both experimental groups in the rate of degradation as in the GAG content. However, there was no decrease of hydroxyproline content in the control leg even by the end of the study. Since hydroxyproline exists in all types of collagen, this could be explained by the increased collagen I level in the self repair tissue of cartilage defect. Another possible explanation for the persistence and endurance of collagen in the degrading cartilage is that, unlike proteoglycan, its anchorage in the matrix is not directly dependent on the quality or presence of the other. Thus, while a drop in the collagen meshwork integrity would increase the diffusion and loss of proteoglycan from the matrix, the reverse isn’t necessarily true. At 9 month of age (180 days post-surgery), most guinea pigs developed early to moderate stage OA, but this is probably reflected more accurately by the quality rather than quantity of the collagen meshwork.
4.5 Bone Mineral Changes:
The BMD of the operated legs decreased significantly at 1 month after surgery and increased thereafter. This pattern of change closely correlates this model with the progressive bone change in OA patients (REF). The BMD of contralateral leg exhibited a trend of fluctuation mirroring that of the operated leg, but did not drop below the
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baseline BMD initially observed at any time during disease progression. This phenomenon suggests that the control leg experience a similar biomechanical and/or biochemical stimulus as the surgery leg throughout the progression of the study, albeit in significantly reduced levels. It is also important to note that the detected BMD value is a combined product of a number of different factors,
including: 1) existing mineralized bone, 2) BMD increase from the mineralization of existing osteoid, 3) BMD decrease from osteoclast activities resulting bone loss. Taken together, this suggests that a threshold level of stimulus (both biomechanical and biochemical) must be reach to increase sufficient levels of bone loss to overcome the effects mineralization processes and to induce an overall mineralization ‘loss’.
4.6 Bone formation:
Bone formation change in the operated femurs revealed a similar pattern of the BMD changes – an initial decrease followed by a progressive increase. In the operated medial condyles, Osteoid Volume (OV) significantly increased in the early on (between 10 and 30-day) and then slowly decreased at later stages. This data is consistent with the hypothesis that the initial BMD increase observed immediately after surgeries not from an overall increase of bone content, but rather a temporary increase in mineralization from the maturation of the osteoids. The site specificity of this mineralization event also suggests that the process is highly sensitive to the levels of mechanical loading.
Following an initial drop at the 20-days time point, the osteoid volume significantly increased in the sites with greatest mechanical loading (surgical medial condyles). This escalation in bone remodeling is consistent with the histological data concerning the severity of cartilage changes in the same areas, notably formation of cartilage lesions and loss of proteoglycan density.
4.7 Bone Structure Analysis
The trabecular bone volume of operated femurs exhibited an initial increase followed by a steep decline in the first 30 days. Trabecular thickness, on the other hand, showed a steady decrease of more than 10% in the first month. In contrast, the total trabecular number of the operated femurs had their highest peak in the 20 day time point. This suggests that early bone remodeling initiates with the mineralization of pre-existing osteoids, which often bridge gaps between mineralized trabeculae. This speculation is further substantiated by how the number of trabeculae in the surgery group exceeds that of the control group at this time point.
In the surgical leg, both trabecular bone volume and thickness reached their lowest point at 30 days and increased thereafter. Both were drawn back to baseline level at 90 days and kept climbing until the end of the experiment. The bone in contralateral femurs
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remained relatively stable with small steady increase in all structural parameters, presumably due to biomechanical and/or biochemical effect from the surgical leg. The overall changes in the surgical OA model reflect a fast bone turnover period (induced bone loss in the early stage of disease progression), followed by slowly increased subchondral bone mass. All these findings are consistent with clinical progression of OA in humans.
Further investigation of site-specific bone changes in the operated femurs reveals greater bone loss and slower recovery in the medial than in the lateral condyle. This demonstrates the sensitivity of this animal model for study of site-specific interaction during initiation stages of OA. This research area should prove to be fruitful for the investigation of OA development and possible treatments to slow its progression.
4.8 Bone connectivity analysis
The bone connectivity result adds yet another dimension to this study. Trabecular bone strut analysis of the operated femurs revealed a significantly higher number of end point and free-free struts, but a lower level of multiple points and note-note struts compared to the contralateral femurs during the progression of the disease; this correlates to the high bone turnover found in the BMD and bone structure analysis. A rapid change was observed in the first month after surgery (see figure 11). The operated limbs demonstrated a higher disconnectivity in the subchondral bone in the early stage of OA progression. This effect may be attributed to how osteoid mineralization form new struts that have yet to thicken and to be more fully integrated into the existing trabecular network.
4.9 Subchondral bone plate change
The subchondral plate of the osteoarthritic joints exhibits higher calcified cartilage and porosity but lower mineralized bone volume when compared to the contralateral joints. Subchondral bone change proved to be site-specific, with the medial tibia of the operated limbs (most affected by OA) presenting a thicker and denser subchondral bone plate than the lateral side of the same bone. When comparing these parameters longitudinally, from 10 days to 90 days the operated medial tibia shows a continuous increase in calcified cartilage content and subchondral bone thickness. Future study will include a quantitative investigation of subchondral plate mineralization, in order to better understand how subchondral bone mineralization effect the OA progression.
4.10 Osteophyte formation
The osteophyte formation has been consistently observed in the joint margin of the osteoarthritic joints. The histological appearance of the osteophyte is a kind of osteochondral outgrowth (figure 14 and 15). In our guinea pig model, cartilaginous osteophyte was observed since 20 after surgery (figure 14), the bonny osteophyte started
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seen at 90 days after surgery (figure 15), the size and number of osteophyte increases with time after surgery.
Osteophyte/osteochondrophyte formation, being one of the most remarkable and consistent features of OA, was observed consistently in our experimentally induced OA model. The presence of osteophyte as a pathological feature distinguishes OA from many other forms of arthritis. The mechanical factor seems played an important role in stimulating osteochondrophyte formation, with possibility that the osteochondrophyte may serve to slow down the further development of OA by stabilizing the joint. The results demonstrated the competence of this animal model for the future study of sequential osteophyte development.
In this guinea pig model, we have observed spontaneous OA-like changes in the un-operated knee, and accelerated progressive changes in the operated knee. Changes of the joint in this animal model are continuous and involve many different tissues. Cartilage structure changes include: surface destruction, chondrocyte hyper/hypo-cellularity, tidemark duplication, and loss of cartilage. Changes in cartilage biochemistry include: loss of proteoglycan, collagen, and DNA. Subchondral bone changes include: calcified cartilage thickening, changes in subchondral bone plate thickness and mineralization, changes in trabecular bone remodeling, architecture, and material properties.
The accelerated nature of this surgery-enhanced spontaneous OA model has been confirmed by the rapid occurrence of bone and cartilage changes in the operated knees while similar changes were observed in the contralateral joint only at later times. Therefore, this is a suitable model to study the epidemiology, disease progression and treatment of OA.
In this study we have summarized some evidence for the role of subchondral bone and calcified cartilage in the progression of OA. Our result has confirmed that the interaction of subchondral bone to the cartilage is one of the most important driving forces in OA progression. In this model, the high bone turnover, which induces subchondral bone changes may not be responsible for the initiation of cartilage damage but most likely could enhance the cartilage damage seen during the progression of OA. Therefore, we studied alendronate treatment in this animal model and the OA progression was delayed by stopping bone resorption. The drug study is included in a separate paper.
We are the first group showing the time course of the change in subchondral bone and calcified cartilage the in guinea pig OA model in great details. Further study is needed to characterize dynamic histomorphometry, to characterize subchondral bone material and mechanical properties, and to quantify subchondral bone mineralization.
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Chapter 8
Chapter 8 ‐ The Effects of Alendronate on
the Progression of OA in Cartilage and Subchondral
Bone Tissues using a Partial Medial Menisectomy Guinea
Pig Model Susanne X. Wang1, Mircia Dumitriu1, Ryan H. Tam2, Rita A. Kandel1, Richard
Reulund1, Marc D. Grynpas1
1University of Toronto, Toronto, Ontario, Canada;
2 Shiley Center for Orthopaedic Research & Education, La Jolla, California, USA
(To be submitted)
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Abstract
OBJECTIVE: Subchondral bone remodeling plays an important role in the progression of osteoarthritis (OA). The aim of the present study is to investigate whether alendronate treatment can delay the progression of OA by suppressing subchondral bone turnover using alendronate (ALN) treatment during a six-month period.
METHODS: Ninety guinea pigs were used in this study. Eighty 3-month old male Hartley-Dunkin guinea pigs underwent partial medial meniscectomy in the right knee, and 30 guinea pigs received alendronate treatment twice a week (0.35mg/kg, subcutaneous injection) starting from the day after surgery. Ten animals from the untreated group were euthanized at 0, 10 and 20days, 1, 3, and 6 months postsurgery, and the treated groups were euthanized at 1, 3, and 6 months postsurgery. OA cartilage morphological changes were validate/evaluated by Mankin grading. Cartilage biochemistry, bone mineral density (BMD), subchondral bone plate thickness, bone histomorphometry and osteophyte formation were studied.
RESULTS: Animals underwent partial medial meniscectomy displayed significantly higher degree of cartilage degeneration, increased subchondral bone turnover, early decrease followed by an increase in bone mineral density and bone volume and subchondral bone plate area. However, the animals treated with alendronate revealed less severe osteoarthritic change (lower Mankin score), all the subchondral bone changes were corrected in significant degrees.
CONCLUSION: In this study alendronate prevents bone loss by suppression of subchondral bone turnover early in the disease and this correlated with a lesser (or delayed) cartilage damage, as reflected by Mankin grading. By preventing the bone resorption occurring early in OA the rate of bone turnover/remodeling is reduced and the progression of OA is delayed. This is a potential mechanism for chondroprotection and suggests a role for bisphosphonate treatment in OA.
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1. Introduction
Osteoarthritis (OA) is the most common form of arthritis, characterized morphologically by destruction of cartilage, sclerosis of subchondral bone, formation of cysts, and the presence of osteophytes at the joint margins. Studies of early primary osteoarthritis in humans are difficult because of the subtle progression of the disease, and by the time it is discovered, patients are mostly at the late or advanced stages of OA. Therefore, animal models are commonly used in the study of OA pathogenesis and potential therapeutic option of disease. Spontaneous OA occurs in the knee joints of guinea pigs, mice, dogs and nonhuman primates. The advantage of naturally occurring OA in animal models is that they possess similar pathology and pathogenesis to that of humans. However, such models usually exhibit slow disease progressions and are susceptible to large variability between individuals. Surgically induced OA models have also been developed in various animal species, using such methods as anterior cruciate ligament transection in dogs [1] and rabbits [2, 3], total or partial meniscectomy in guinea pigs [4] and rabbits [5], and the combination of meniscectomy and ligament transection [6]. Most of the surgically induced models showed rapid disease progression, which resembles secondary osteoarthritis after trauma.
Dunkin-Hartley guinea pig is a widely used spontaneous OA animal model, and a partial meniscectomy could make the arthritis change occurring earlier and more predictable; the cartilage histopathological change and disease progression in this model was well documented by A. Bendele in 1987 [4]. We have modified this model by dissecting out middle 1/3 of the medial meniscus to induce a chronic osteoarthritis, the histopathological change of this model is similar to that of spontaneous guinea pig OA but in an accelerated pace [7].
Osteoarthritic change involves more than just cartilage degeneration; subchondral bone change was described in many clinical studies, including subchondral sclerosis, cyst and osteophyte formation [8]. The importance of the bone changes in the initiation and progression of OA is still being debated. The mechanical properties of the subchondral bone have a direct effect on the functional integrity of the overlying articular cartilage. Increased subchondral bone stiffness reduces its ability to dissipate the load and evenly distribute the strain generated within the joint, resulting in higher stress in articular cartilage during impact loading, and accelerating cartilage damage over time [9]. Accordingly, cartilage damage progress into full-thickness cartilage loss only upon repetitive loading over an already stiffened subchondral bone plate [10].
The role of subchondral bone changes in the initiation and progression of OA has been extensively studied in the past decade. Increased subchondral bone resorption and turnover was observed both in patients [11-14] and in experimental OA [15, 16]. Calcitonin, an anti-resorptive bone drug, was reported to reduce the severity of the bone,
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cartilage, and synovium change in the early stages of canine ACLT experimental OA by either subcutaneous injection [17] or nasal spray [18]. Workers have shown that Zoledronate has a partial chondroprotective effect in the Carrageenan rabbit model of inflammatory arthritis by preventing bone resorption [19].
Alendronate (ALN), a second generation of bisphosphonate, is a potent inhibitor of osteoclast-mediated bone resorption. At present, ALN is used clinically for the treatment and prevention of osteoporosis[20, 21], Paget's disease [22], and for reducing osteolysis around prostheses to prevent implant loosening [23, 24]. Patients receiving prolonged ALN therapy have shown histologically normal bone [25]. ALN has demonstrated to improve the mechanical properties and microstructure of trabecular bone after either short-term [26], mid [27] or long term [28] treatment in canines. Local alendronate treatment can increase early implant osseointegration and biomechanical fixation of implants [29].
Current therapies of OA are largely aimed at the symptoms of OA and do not modify the course of the disease. The development of chondroprotective/disease modifying drugs is one of the major aims of OA drug development. In our concurrent study, we characterized the accelerated spontaneous guinea pig OA model in cartilage histology and biochemistry, in bone mineral density, subchondral bone structure, bone turnover, mechanical properties, material properties and bone mineralization. We have shown that subchondral bone change developed simultaneously with cartilage changes.
We hypothesize that bisphosphonate may suppress the subchondral bone change in osteoarthritis by preventing bone resorption and turnover. Furthermore, bisphosphonate may delay cartilage degeneration promoted by subchondral bone change. The aim of the present study was to investigate whether suppression of subchondral bone changes with alendronate treatment can delay the progression of in the partial medial meniscectomised guinea pig model for a six-month period.
2. Material and method
2.1 OA Model and Experiment Design
Ninety male albino Dunkin-Hartley guinea pigs purchased from Charles River laboratory (Montreal, Canada) were housed in the environmentally controlled single cage and fed standard guinea pig chow. The guinea pigs were acclimated for a period of 1 month before initiation of the study. The animals were 3 months old and weighted 737±35 g at the beginning of the study. The guinea pigs were randomly divided into 9 groups (n=10). Physical well-being and activity were checked daily, and body weight was measured at every experiment time point. The weights of each animal were recorded at the start and at the end of the experiment. The groups and average weight of each group of animals at ending point were listed in Table 1.
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Partial medial meniscectomy was done on the right leg of 80 guinea pigs (8 groups). The animals were preinjected with 0.03mL Atropine and anesthetized with constant inhalation of 2-3% isofluorine in oxygen. A subcutaneous injection of 0.5mL bupernorphine diluted 1:9 with saline was administered for post-operative pain relief. Under sterile condition, the surgical approach was made through a medial longitudinal parapatellar incision. The synovial capsule was incised along the direction of the skin incision in the medial aspect of the joint. The subcutaneous (s.c.) dissection is carried out to. The medial collateral ligament was partially transected to expose the middle part of the medial meniscus, approximately 1/3 of the meniscus was excised in the middle portion without any contact with the articular surfaces. The medial collateral ligament was sutured and joint capsule was closed with interrupted suture ligatures using 6-0 PDSII (Polydioxanone) suture (Johnson& Johnson). The subcutaneous and skin incisions were closed sequentially with 4-0 dexon sutures.
A group of 10 animals were sacrificed at 3-months old without surgery and used as time 0 control. Five groups of partial medial meniscectomized guinea pigs were sacrificed at 10, 20, 30, 90 and 180 days after surgery consequently to study the disease progression in this animal model. Other three groups of animals received subcutaneous injection of alendronate (Aln, Merck and Co., West Point, PA) twice a week at 0.35mg/kg/week, starting two days after surgery. Ten animals were sacrificed at 30, 90, and 180 days after surgery respectively to investigate the effect of alendronate in this animal model of OA. Ten and three days before sacrifice the animals received a 35mg/kg intraperitioneal injection of Oxytetracycline Hydrochloride - a bone label. Animals were killed by intraperitioneal injection of 1.7-2.0 ml sodium pentobarbital (40mg/kg, Sigma) followed by 0.3 ml T-61 (Intervet Canada Ltd.) I.V. injection. The experimental design and animal weight at the end of each experiment points were presented in Table 1.
2.2 Sample Collection
The right hind leg of each guinea pig was excise upon sacrifice, femur and tibia were further dissected free of soft tissue. The hyaline cartilage was shaved off the femur condyle and grove surface with a #11 scalpel right after opening of the joint capsule. Cartilage sample was weighted immediate after collection (wet weight) and lyophilized
Table 1:
Groups (age, months) Time 0 (3m) 10 days 20 days 30 days (4m) 90 days (6m) 180 days (9m)
OA (weight, g) 10(733.1) 10(747.1) 10(768.7) 10(874.1) 10(925.7) 10(1062.1)
OA+Aln (weight, g) 10(865.7) 10(944.3) 10(1097.8)
Table 1. Experimental design and animal weight at the end of the experiment
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overnight, and then the dry weight was determined. Cartilage samples were stored at -20°C until biochemical analysis was performed. The femurs were then fixed in 70% v/v ethanol. Gross morphology of the meniscus and articular cartilage surface of the tibia plateau was examined, documented after opening the joint. Meniscus and proximal tibia were then fixed in 10% neutral buffered formalin.
2.3 Bone Mineral Density Measurement
Both tibia and femur from the left knee of each animal were scanned by dual energy x-ray absorptiometry (DEXA) (PIXImus™, Lunar Corp/GE). After calibration with an aluminum/lucite phantom (Lunar Corp/GE), a single femur or tibia was placed on a polystyrene tray to mimic soft tissues, and the bone mineral density (BMD, g/cm2) and bone mineral content (BMC, g) were measured at the distal femur, medial and lateral femoral condyle, proximal tibia, and the medial and lateral tibial plateau.
2.4 Histology, Mankin grading
After DEXA scan, the proximal tibias were coronally cut from the middle line into anterior and posterior parts. Posterior portions were decalcified and embedded in paraffin. Safranin-O and Toluidine Blue stained 5μm histological sections from the middle of a tibia were evaluated the histopathological OA changes. A semiquantitative histopathologic grading was performed according to a modified Mankin scoring system (Table 2) to quantify OA disease severity.
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2.5 Cartilage Biochemistry
The cartilage samples from distal femur were digested with papain (Sigma; 40 µg/mL) for 48 hours at 65°C. The DNA content was determined from aliquots of the papain digest using the Hoechst 33258 dye binding assay (Polysciences Inc.) and fluorometry (SpectraMAX Gemini Fluorometric Plate reader, Molecular Devices) with an emission wavelength of 365 nm, and an excitation wavelength of 460 nm [30]. The DNA content was calculated from a standard curve using calf thymus DNA (Sigma) as a standard.
The proteoglycan content was estimated by determining the amount of sulphated glycosaminoglycans (S-GAG) in the papain digests using the dimethylmethylene blue dye binding assay (Polysciences Inc.) and spectrophotometry (MicroQuant UV/VIS Plate Reader, Bio-Tek Instruments) at 525 nm [31]. The S-GAG content was calculated from a standard curve using bovine chondroitin sulphate (Sigma) as a standard.
The collagen content was calculated from the hydroxyproline content of the cartilage, by multiplying the later by 8.33. Aliquots of the papain digests were hydrolyzed in 6N HCl for 18 hours at 110°C, after which the hydroxyproline content was determined using the chloramine-T/Ehrlich’s reagent assay and spectrophotometry (MicroQuant UV/VIS
Table 2:
Table 2: Modified Mankin scale evaluation parameters
Test Parameter Condition Score Structure Normal 0 Surface Irregularities 1 Pannus and Surface Irregularities 2 Clefts to Transitional Zone 3 Clefts to Radial Zone 4 Clefts to Calcified Zone 5 Complete Disorganization 6 Chondrocytes Normal 0 Diffuse Hypercellularity 1 Clonning 2 Hypocellularity 3 Proteoglycan Content Normal 0 Slight Reduction 1 Moderate Reduction 2 Severe Reduction 3 No Dye Noted 4 Tidemark Integrity Intact 0 Crossed by Vessels 1
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Plate Reader, Bio-Tek Instruments) at 560 nm [32]. The hydroxyproline content was then calculated from a standard curve using L-hydroxyproline (Sigma) as a standard.
2.6 Undecalcified Sample Processing
After DEXA scan, the fixed distal femurs were coronally cut into posterior and anterior parts. The posterior femurs and anterior tibias were dehydrated in ascending grades of acetone (70%, 90% and 100%) and then infiltrated with increasing strengths of Spurr (Marivac Ltd., Halifax, N.S. Canada)/acetone solutions (50:50, 80:20, 90:10, to 100%), and then embedded in freshly made 100% Spurr’s resin. Two 5μm and two 7μm undecalcified sections were cut continuously from the middle of each femur. The 5μm sections were stained with Goldner’s Trichrome for static histomorphometric analysis, and the 7μm sections were left unstained for dynamic histomorphometric analysis.
2.7 Bone Static and Dynamic Histomorphometry
Measurements of the trabecular bone area were taken from the central epiphyseal subchondral bone region, which was within 1mm beneath the cortex. The region was made up of the subchondral bone plate, both sides of cortical bone, and growth plate. To further quantify the effects of surgery and treatment in the local area, the medial and lateral femoral condyles were evaluated separately and also combined together to analyze the distal femur as one area. Bone formation and resorption parameters were measured using BQ Nova Prime V6.50.10 software (BIOQUANT Image Analysis Corporation, US) connected to a digital camera (Retiga 1300, QImaging). The osteoid volume (OV), osteoid surface (OS), osteoid thickness (O.Th), and eroded surface (ES) were measured and calculated according to ASBMR guidelines [33]. The mineralizing surface, mineral apposition rate, bone formation rate, and mineralization lag time were calculated according to the measures of dynamic versus static histomophometry.
2.8 Backscattered Electron Microscopy
The remaining femoral and tibial Spurr resin blocks containing the middle part of the femur and tibias were then polished up to 1μm diamond finish using the phoenix BETA Grinder/Polisher fitted with the VECTOR Power Head (Buehler) and carbon-coated. Backscattered electron (BSE) images were obtained using a scanning electron microscope (Philips, XL300ESEM) with a backscattering detector (Centaurus Detector). Beam conditions were set at 20kV accelerating voltage, a load current ranging from 58-87 microamperes, and a spot size of 7. Contrast was set in the condition that non-mineralized tissue and the plastic matrix appeared black, and mineralized tissue appeared in shades of grey (greylevels).
One set of BSE images was obtained at 50x magnification in the epiphyseal region of the distal femurs, and used for bone architecture and connectivity analysis of the
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subchondral trabecular bone, and also for the measurement of the subchondral bone plate and growth plate volume/thickness. Another set of 200x BSE images was obtained at the subchondral plate region of the proximal tibia that contains calcified cartilage and subchondral cortical plate, and used to study bone mineralization.
2.9 Femoral Subchondral Bone Structure and Connectivity Analysis
The subchondral trabecular bone structure in the epiphyseal region of the proximal tibia was analyzed using an image processing and analysis system (Quantimet 570, Leica) on the 50x femoral BSE images. After thresholding the grey-level images, binary images were obtained to analyse the structure parameters. The structural and connectivity parameters of trabecular bone were measured on BSE images and calculated using the ASBMR guidelines [33]. The following structural parameters were obtained from the image: Trabecular Bone Volume -BV/TV (%), Trabecular Thickness -Tr.Th (mm), Trabecular Number -Tr.N (1/mm2), and Trabecular Separation -Tr.Sp (mm).
A skeletonized binary image was used to measure the connectivity parameters. The image analysis was done by using an in-house program based on the techniques described by Parisien et al [34]. The listed connectivity parameters were obtained in the analysis: Number of multiple points (1/mm2), Number of end points (1/mm2), Lengths of node-free struts (mm/mm2), Lengths of node- node struts (mm/mm2), Lengths of free-free struts (mm/mm2), Ratio of end points/multiple points, Total strut length (mm/mm2).
2.10 Subchondral Bone Plate Thickness
The subchondral bone plate area and thickness of tibia was measured on the 50x tibial BSE images using individual point-to-point distance measures. The thickness was measured from the top of the calcified cartilage to the deep surface of the subchondral bone plate by averaging 15 measurements per tibia. All measurements were made in a standardized viewing area by a single observer who was blinded to the experiment.
2.11 Subchondral Bone Plate Mineralization Assessments
Another set of six to eight BSE images at 200x magnification were taken from each tibia in the region of the subchondral bone plate at a working distance of 15mm. Calibration of the machine was performed with silicon dioxide (SiO2, Z=30) before and after taking the 200x images from each sample. The mineralized bone exhibited a range of greyscale values (0 to 255) and the area at each grey level was measured from the BSE images using Quantimet 570 image analysis system (Leica Imaging Systems, Cambridge, UK). In a BSE image, greylevels represent varying degrees of bone mineralization, where brighter intensities indicate higher degrees of mineralization. The percentage area (% area) of pixels excluding the plastic region was calculated. The mineralization profile of a specimen is represented by a histogram of greylevel
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intensities. The grey level intensity distribution was described using the logit function, or cumulative log ratio [35], and is defined as follows: logit = ln (proportion >125 proportion <125).
2.12 Osteophyte Incidence and Area
Osteochondrophyte incidence was evaluated from the Safranin-O stained tibia sections. Total mineralized osteophyte numbers of each knee joint were examined from the middle femur and tibia 50x BSE images. The surface area of each osteophyte was measured according to the method of Baddeley et al. [36], the total osteophyte area of each joint was the sum of the measures the 50x femur and tibia BSE images of the same joint.
2.13 Statistical Analysis
All data analyses were performed using SPSS 11.0 for Windows statistical software (SPSS Inc., Chicago, IL, USA). Independent sample T-test was used to compare differences between the OA and the treatment groups. One-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) post-hoc test was also used to examine differences between the various time points. Significance was considered at p≤0.05. All data are presented as mean ± standard error of the mean (SEM).
3. Results
3.1 Gross and Microscopic Morphology
Gross findings at 10 days post-surgery did not show changes consistent in all the tibia plateaus. The surface of the medial tibia plateau became less smooth at 20 days post-surgery, and 4 out of 10 tibias in the untreated group had small osteophyte formation in medial periphery of the tibia plateau. At 30 days post-surgery, the medial tibia plateaus became fibrillated, and osteophytes grew to be apparent in 80% of the untreated medial tibia plateaus (Figure 1A), interestingly the osteophyte formation always located in the central of the medial margin where the segment of meniscus removed by the surgery (between the arrowheads). After 90 days post-surgery, each of the untreated medial tibia plateaus had osteophyte formation and more profound cartilage gross change.
Histomorphological changes of the partial medial meniscectomized tibia were similar to those of human osteoarthritis. The cartilage lesions were predominantly on the central-medial region of the medial tibial plateau, and progressed deeper and peripherally with time. The superficial fibrillation, focal lesions and thickening of the cartilage were first observed at 10 days post-surgery, and developed to mild lesions around 20 days post-surgery, with some lesions accompanied by small chondrophyte formation (3 out of 10). Cartilage lesions became more apparent after 30days post-surgery, as fibrillations turned to clefts. After the cartilage tissue was worn away, the lesions became deep and wide
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erosions, and most of the erosions accompanied by osteophytes formation (Figure 1B). By the end of the experiment every medial plateau had remarkable cartilage lesion up to the deep zone and tidemark, along with evidences of osteophytes formation. The lateral tibial plateau developed much less severe degenerative cartilage change than the medial side.
The disease severity was evaluated by a Modified Mankin scale at the medial tibial plateau of each joint (see table for grading scale). The Mankin score of the operated joints indicated a rapid OA progression, especially in the first month, from 0.7±0.6 increased to 6.4 ±1.4. It continuous increased to 7.5 ±1.3 at 3 months and 9.9 ±1.4 at 6months after surgery. However, the progression of OA severity in the treated surgery joint showed smaller increases, from 6.1±1.2 at 1 month to 6.9±1.3 and 8.1±1.5 at 3 and 6 months. A significant difference was found between the 3-month surgery group and the 3-month treatment-surgery group (marked as *), as well as between the 6-month surgery and treatment group. Figure 2 is the comparison of Modified Mankin score in the treated and untreated groups.
Figure 1:
A. B.
Figure 1: Osteophyte formation accompanied cartilage degeneration in the meniscectomized tibia.
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The alendronate treated group had less evident gross morphological change than the untreated group. The microscopic changes of the treatment group were not as pronounced as the untreated group and the occurrence of cartilage lesions was considerably delayed. At 180 day after surgery, more than half of the lesions of untreated group had reached calcified cartilage (Figure 3A), when, at the same time, the lesions of the ALN treated group progressed only to the transitional or deep zone (Figure 3B). Osteophyte formation was only observed after 30 days post-surgery in the treatment group, with fewer incidences and smaller sizes when compared to the untreated group throughout the course of this study.
Figure 3:
A. B.
Figure 3: Representative photomicrograph of Safranin‐O stained central sections of medial tibial plateau at 180 days post‐surgery. A. The lesions of untreated group had reached subchondral bone in the untreated group, B. Aln treatment delayed cartilage degeneration, and the lesions were mostly developed up to middle zone.
Figure 1:
Figure 2: Cartilage degeneration was delayed in the treatment group reflected by the Mankin grading. Mankin score in the alendronate treated group were significantly lower than those in the untreated group at 90 and 180 days post‐surgery (* P < 0.05).
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3.2 Cartilage Biochemistry:
The biochemistry results were presented as per-unit dry weight when comparing the concentrations of DNA, proteoglycan (GAG) and collagen (hydroxyproline) content in cartilage specimens from untreated and alendronate treated femurs (Figure 4). The effects of alendronate on those molecules were quantified at 1, 3 and 6 month after surgery. At a weekly dose of 0.35mg/kg, alendronate significantly increased DNA, proteoglycan and collagen contents of the cartilage in the operated joint, especially at early stage of the disease (30 days post-surgery, P<0.01).
In the nontreatment group, the DNA, GAG and hydroxyproline contents all experienced a pattern of initial decrease followed by an increase in the first month and then constantly decreased thereafter (Fig.4 A-C soft lines). This pattern disclosed the cartilage metabolic changes during the OA progression. Comparison between the alendronate treated and untreated groups revealed a positive effect of the treatment. The treatment group presented a 64% increase in DNA content at 30 days, and a 25% and 27% increase at 90 and 180 days respectively after surgery when compared to the untreated group (Figure 4, B). The alendronate-treated group also exhibited increased GAG content by 44%, 25% and 17% at 30, 90 and 180 days after surgery when compared to the untreated group (Figure 4, B). The effects of treatment in the hydroxyproline changes are similar to that of the GAG content with a 33% increase at 30 days a 29% increase at 90 days and a 23% increase at 180 days after surgery. (Figure 4, C).
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3.3 Bone Mineral density
The bone mineral density of the osteoarthritic joints is characterized by an early drop in BMD followed by a trend constant increase. However this pattern was not observed in the treatment group. The treated group showed a constant increase in BMD at all time points after surgery. The distal femur and proximal tibia BMD changes before and after treatment are shown in Figure 5 A and B. The most significant change observed in the medial femoral condyle (Figure 5 C).
Figure 4:
A B
C
Figure 4. Parameters of the biochemical composition of cartilage specimens collected from the distal femur of the right knees (operated) of untreated (soft line) and treated group (solid line). A. DNA content change with or without alendronate treatment. B. GAG content, and C. Hydroxyproline (Collagen) content.
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3.4 Bone structure and connectivity analysis
Analyses of the untreated femoral trabecular bone structure and connectivity revealed a rapid increased bone mass and connectivity within the first 20 days post surgery. This peak is followed by an exponential decline through 90 days post surgery. In the later stage of this experiment OA (between 90 to 180 days), the untreated group showed higher bone volume, thicker and more connected trabeculae that the treated group (Figure 6 & 7, dotted lines). Although anabolic bone change was in the lead at the early stage of this experimental OA, bone resorption was actually existed from the onset of the disease, as confirmed by decreased trabecular thickness and increased number of end points. In the early to mid stage of the OA progression in this experiment, higher bone resorption was the main feature of the subchondral trabecular bone change.
Figure 5:
A B
C
Figure 5: The BMD change in distal femur (A), proximal tibia (B) and medial femoral condyle (C) with or without alendronate treatment.
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Treatment with alendronate (antiresorptive drug) has significantly increased trabecular bone volume, number and thickness but decreased trabecular separation between 30 to 90 days post-surgery (Figure 6, solid lines), which displayed an improved trabecular bone structure than the untreated bones. The connectivity parameters revealed a more connected trabecular bone in the alendronate-treated group when compared to the untreated ones during 30 to 90 days post surgery (Figure 7, dotted lines).
By 180 days post surgery, measured bone structure and connectivity parameters of alendronate treated bones were not significantly different from the untreated ones. A more stable bone status was observed in the treatment group throughout the total experiment period.
Figure 6:
Figure 6: The trabecular bone structure change with or without alendronate treatment: (A) Trabecular bone volume, (B) Trabecular thickness, (C) Trabecular number, and (D) Trabecular separation. (* p<0.05, † 0.05<p<0.1)
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3.5 Subchondral Bone Plate thickness
The subchondral bone plate thickness of tibiae significantly decreased in the first 20 days post surgery (p=0.024), and then followed by continuous increase through the end of the study. Before 90 days post surgery, the subchondral bone plate thickness was always below the baseline. The thickness at 180 days was significantly higher than all other time points studied (p<0.05).
Alendronate treatment significantly increased subchondral plate area and thickness at 30 and 90 days after surgery when compared to the control (Figure 8). However, there was no difference in the subchondral plate thickness between the treated and untreated group
Figure 7:
Figure 7: The trabecular bone connectivity change with or without alendronate treatment: (A) Length of free‐free struts, (B) Length of node‐node struts, (C) Length of total skeleton, and (D) Number of Multiple points. (* p<0.05, † 0.05<p<0.1)
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at 180 days post surgery. Alendronate treatment appeared to have a stabilizing effect on change in the subchondral plate thickness throughout the study.
3.6 Static and dynamic histomophometry
Subchondral bone static and dynamic histomophometry were analyzed on the middle sections of the operated distal femur in both treatment groups. A significant increased bone formation rate was observed in the non-treatment group throughout the 180 days experiment time, with the highest point at 90 days after surgery (Figure 9, A). Osteoid volume was also quickly elevated up to a considerably high level within 30days and remained in a higher than normal level through the end of the study (Figure 9, B). Combining the bone surface (BS/BV) and adjusted apposition rate, the discrepancy between the two and with the bone formation rate and osteoid volume revealed a significant level of resorption in the trabecular bone within the first 90 days post-surgery (Figure 9, A-E).
Alendronate treatment significantly reduced the bone turnover in the first month of the treatment and almost fully stopped bone turnover after one-month treatment. This confirmed by the zero bone formation and apposition rate and very low osteoid volume between 30-180 days post surgery (Figure 9, blue lines).
Figure 8:
Figure 8: Subchondral plate area (A) and thickness (B) change of the tibiae with or without alendronate treatment. (* p<0.05, † 0.05<p<0.1)
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Figure 9:
A B
C D
E
Figure 9: Subchondral bone static and dynamic histomophometry of the femora with or without alendronate treatment. (A) Bone formation rate, (B) Osteoid volume, (C) Bone surface/bone volume, (D) Apposition rate, and (E) Percent quiescent surface.
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3.7 Inhibition of osteophyte formation by alendronate
Osteophyte formation was observed in the joint margin of all osteoarthritic joints. It is important to note that osteophyte formation were fewer in number and smaller in size in alendronate-treated osteoarthritic joints when compared to the untreated ones. Table 3 summarizes the rate of osteophyte formation in both treatment groups from the histological sections and backscattered electron images.
4. Discussions
The importance of subchondral bone was being recognized as early as 1962, when Johnsson [37] suggested that bone remodeling could lead to osteoarthritis. Radin, et al studied subchondral bone changes in OA patients [38] and experimental animals [9, 39], suggested that stiffness of the subchondral bone leads the cartilage change. The changes of subchondral bone in OA has been more recognized recently and studied in human [40], experimental animals [41], and in the human osteoblasts isolated from healthy and osteoarthritic subjects [42]. Although it is not yet clear that whether subchondral bone changes in OA are preceding, concurrent with, or subsequent to cartilage degeneration, the increased subchondral bone resorption and formation and their enhancement effect in OA progression is a commonly recognized [43].
High resorption [11], remodeling [44] and the resulted osteoporotic conditions [45] in OA patients has been reported in clinical studies over the years. Inhibition of subchondral bone resorption was investigated in animal experiments to study the effect of antiresorptive drugs on OA progression. By suppressing subchondral bone turnover, calcitonin markedly reduced the osteoporotic bone, cartilage, and synovium changes in the early stages of a canine anterior cruciate ligament transection (ACLT) model of OA [17, 18, 46].
Table 3:
Evaluation Groups Days post‐surgery
methods 10 20 30 90 180
Histology Untreated 0% 30% 70% 100% 100%
Aln treated 0% 0% 50% 80% 80%
BSE Untreated 0% 0% 0% 60% 80%
Aln treated 0% 0% 0% 50% 60%
Table 3. Alendronate inhibition of osteophyte formation in both tibias (evaluated by histology) and femurs (evaluated by BSE).
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Bisphosphonates, organic analogues of pyrophosphate, are potent inhibitors of osteoclast-mediated bone resorption [47]. Bisphosphonates are chemically simple compounds and exhibited exceptional affinity to bone. They have been used mainly for the treatment of osteoporosis, hypercalcaemia and osteolytic bone disease of malignancy, primary and secondary hyperparathyroidism. Treatment with bisphosphonates causes an early reduction in bone resorption followed by a later reduction in bone formation [48]. Bisphosphonates therefore play a major role in OA bone remodeling conditions that are characterized, at least partly, by an increased bone resorption.
Use of bisphosphonate in the treatment of OA has been explored in laboratory OA animal models and in the clinic. In the ACLT dog model, daily subcutaneous injections of bisphosphonate NE-10035 was used to effectively suppressed subchondral bone turnover in the OA joint and reduced proteoglycan degradation by 50% for 3 month [49]. Treatment with Zoledronate achieved 4-8 weeks of partial chondroprotection effect in an arthritis model where cartilage damage was induced by intraarticular chymopapain injection [50]. Clinically, clodronate, was reported to effect a significant decrease in spontaneous pain and pain on active movement, and this reduction of pain was correlated with the bisphosphonate-induced reduction of prostaglandin E2 (an inflammatory mediator) levels [51]. Carbone et al 2004 [52] reported that use of alendronate was associated with less severity of knee pain and with reduction of OA subchondral bone changes. These results suggested a constructive role of bisphosphonates in the treatment of osteoarthritis.
In this study, increased bone turnover was observed during the entire course of OA progression, with high resorption was leading in the early phase (within 30 days after surgery) and high formation taking the lead position afterwards. Cartilage biochemistry results in our experiment showed elevated levels of cartilage turnover in the arthritic joints; this finding is consistent with previous reports in osteoarthritis patients [53] and other animal models [54]. In this study, the changes of cartilage and bone were concurrent, and suggesting that they developed correspondingly during the disease progression.
It was shown in Cynomolgus Macaques OA that increased subchondral plate thickness was associated with increased severity of articular cartilage lesions, whereas low subchondral plate thickness did not result in cartilage lesion[55]. Our current study in guinea pig OA also showed a similar positive relationship between thicker subchondral plates and more severe articular cartilage lesions. The sensitivity of this experiment to the time-dependent progression bone remodeling picked up a phenomenon not previously reported in the bone remodeling response to experimental OA. A thinning in the subchondral plates was observed in the early phase of the untreated group (first 20 days) and did not result in a delay or prevention on the development of cartilage lesions.
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Closer examination of the data reveals that this thinning of the subchondral bone is due to a short resorption phase that is inherently part of the bone remodeling process that results in long-term bone thickening. As such, this event is concurrent with the formation of cartilage lesions.
High resorption occurred at not only the subchondral plate (Fig. 8), but also in the subchondral cancellous bone (Fig. 6), and affects the connectivity of the trabeculae in this area (Fig. 7). This change also reflected by decreased BMD in this period. The bone activation-resorption period takes about one month in human remodeling process [56]. After a quick reversal phase (few days), a period of bone formation (90 days) takes place, followed by a much longer mineralization period. The formation and mineralization period in our experiment is from 30 days to 180 days (when the experiment ended) post-surgery. We have confirmed an increased remodeling of subchondral bone in the partial medial meniscectomized OA model in guinea pigs, and this remodeling process followed the same pattern and time period of physiological remodeling procedure, albeit with a much shorter and intense progression. The greater-than-normal subchondral bone formation at later stages resulted in a thickening of the subchondral plate and consequent subchondral trabecular sclerosis. Due to the stronger effects and longer duration of bone formation period that starts after the resorption phase, most of clinic observations and OA animal model studies fall into this period. This observation has spurred the theory and discussion about the inverse relationship of osteoarthritis and osteoporosis for the past decade.
In conclusion, alendronate treatment effectively suppressed subchondral bone resorption, thereby achieving significant chondroprotective effect in this experiment. Our result explained the positive role of bisphosphonate in OA treatment. However, long-term benefits of bisphosphonate treatment for osteoarthritis cannot be concluded in this experiment. Further study need to be done in order to define an optimal treatment time and duration of OA with bisphosphonate.
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29. Jakobsen T, Kold S, Bechtold JE, Elmengaard B, Soballe K. Local alendronate increases fixation of implants inserted with bone compaction: 12-week canine study. J Orthop Res 2007; 25: 432-441.
30. Kim YJ, Sah RL, Doong JY, Grodzinsky AJ. Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem 1988; 174: 168-176.
31. Goldberg RL, Kolibas LM. An improved method for determining proteoglycans synthesized by chondrocytes in culture. Connect Tissue Res 1990; 24: 265-275.
32. Reddy GK, Enwemeka CS. A simplified method for the analysis of hydroxyproline in biological tissues. Clin Biochem 1996; 29: 225-229.
33. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, et al. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 1987; 2: 595-610.
34. Parisien M, Mellish RW, Silverberg SJ, Shane E, Lindsay R, Bilezikian JP, et al. Maintenance of cancellous bone connectivity in primary hyperparathyroidism: trabecular strut analysis. J Bone Miner Res 1992; 7: 913-919.
35. Bracci PM, Bull SB, Grynpas MD. Analysis of compositional bone density data using log ratio transformations. Biometrics 1998; 54: 337-349.
36. Baddeley AJ, Gundersen HJ, Cruz-Orive LM. Estimation of surface area from vertical sections. J Microsc 1986; 142: 259-276.
37. Johnsson LC. Joint remodeling as the basis for osteoarthritis. J Am Vet Med Assoc 1962; 141: 1237-1241.
38. Radin EL, Paul IL, Tolkoff MJ. Subchondral bone changes in patients with early degenerative joint disease. Arthritis Rheum 1970; 13: 400-405.
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39. Radin EL, Martin RB, Burr DB, Caterson B, Boyd RD, Goodwin C. Effects of mechanical loading on the tissues of the rabbit knee. J Orthop Res 1984; 2: 221-234.
40. Bobinac D, Spanjol J, Zoricic S, Maric I. Changes in articular cartilage and subchondral bone histomorphometry in osteoarthritic knee joints in humans. Bone 2003; 32: 284-290.
41. Anderson-MacKenzie JM, Quasnichka HL, Starr RL, Lewis EJ, Billingham ME, Bailey AJ. Fundamental subchondral bone changes in spontaneous knee osteoarthritis. Int J Biochem Cell Biol 2005; 37: 224-236.
42. Cantatore FP, Corrado A, Grano M, Quarta L, Colucci S, Melillo N. Osteocalcin synthesis by human osteoblasts from normal and osteoarthritic bone after vitamin D3 stimulation. Clin Rheumatol 2004; 23: 490-495.
43. Oegema TR, Jr., Carpenter RJ, Hofmeister F, Thompson RC, Jr. The interaction of the zone of calcified cartilage and subchondral bone in osteoarthritis. Microsc Res Tech 1997; 37: 324-332.
44. Lajeunesse D. The role of bone in the treatment of osteoarthritis. Osteoarthritis Cartilage 2004; 12 Suppl A: S34-38.
45. Del Puente A, Esposito A, Carpinelli A, Nutile G, Scognamiglio A, Savastano S, et al. [Longitudinal study on osteoarthritis and bone metabolism]. Reumatismo 2003; 55: 102-107.
46. Behets C, Williams JM, Chappard D, Devogelaer JP, Manicourt DH. Effects of calcitonin on subchondral trabecular bone changes and on osteoarthritic cartilage lesions after acute anterior cruciate ligament deficiency. J Bone Miner Res 2004; 19: 1821-1826.
47. Devogelaer JP. Treatment of bone diseases with bisphosphonates, excluding osteoporosis. Curr Opin Rheumatol 2000; 12: 331-335.
48. Vasikaran SD. Bisphosphonates: an overview with special reference to alendronate. Ann Clin Biochem 2001; 38: 608-623.
49. Myers SL, Brandt KD, Burr DB, O'Connor BL, Albrecht M. Effects of a bisphosphonate on bone histomorphometry and dynamics in the canine cruciate deficiency model of osteoarthritis. J Rheumatol 1999; 26: 2645-2653.
50. Muehleman C, Green J, Williams JM, Kuettner KE, Thonar EJ, Sumner DR. The effect of bone remodeling inhibition by zoledronic acid in an animal model of cartilage matrix damage. Osteoarthritis Cartilage 2002; 10: 226-233.
51. Cocco R, Tofi C, Fioravanti A, Nerucci F, Nannipieri F, Zampieri A, et al. Effects of clodronate on synovial fluid levels of some inflammatory mediators, after intra-articular administration to patients with synovitis secondary to knee osteoarthritis. Boll Soc Ital Biol Sper 1999; 75: 71-76.
52. Carbone LD, Nevitt MC, Wildy K, Barrow KD, Harris F, Felson D, et al. The relationship of antiresorptive drug use to structural findings and symptoms of knee osteoarthritis. Arthritis Rheum 2004; 50: 3516-3525.
53. Iwase T, Hasegawa Y, Ishiguro N, Ito T, Iwasada S, Kitamura S, et al. Synovial fluid cartilage metabolism marker concentrations in osteonecrosis of the femoral head compared with osteoarthrosis of the hip. J Rheumatol 1998; 25: 527-531.
54. van Valburg AA, van Roermund PM, Marijnissen AC, Wenting MJ, Verbout AJ, Lafeber FP, et al. Joint distraction in treatment of osteoarthritis (II): effects on cartilage in a canine model. Osteoarthritis Cartilage 2000; 8: 1-8.
55. Carlson CS, Loeser RF, Purser CB, Gardin JF, Jerome CP. Osteoarthritis in cynomolgus macaques. III: Effects of age, gender, and subchondral bone thickness on the severity of disease. J Bone Miner Res 1996; 11: 1209-1217.
56. Burr DB. Anatomy and physiology of the mineralized tissues: role in the pathogenesis of osteoarthrosis. Osteoarthritis Cartilage 2004; 12 Suppl A: S20-30.
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Chapter 9
Chapter 9 - Discussion and Conclusions
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1. General Discussion
Arthritis is one of the most prevalent disorders to strike the elderly population, causing disability, pain, and a general loss in the quality of life. Although arthritis includes a large group of diseases with many diverse causes and symptoms, the term is most often used to describe OA or RA, both of which have been the subject for many studies. While RA is known as an auto-immune disorder, the exact causes for OA remain relatively ambiguous, and despite the copious documentation on its pronounced and striking symptoms towards the end of its progression, its roots are still unknown.
It is widely recognized that the advancement of OA involves severe changes in both cartilage and bony tissues. However, early studies tend to primarily focus on cartilage in their investigations. This is probably due to cartilage’s notorious lack of intrinsic healing properties, while bone is known to have a high capacity for regeneration. Because of this difference in tissue biology, many early researchers assumed that the bottleneck for successful OA treatment lies in cartilage repair. However, as more information on OA progression becomes available, it became more obvious that focusing on cartilage alone is unlikely to yield a successful treatment. The high level of intimacy between bone and cartilage has made it apparent that the two tissues’ involvement in OA cannot be considered separately.
The close bond between the two tissues is evident from their roots during development. Bone and cartilage were originally one tissue, and jointly differentiated into the two distinct tissues to be found in the adult. It is generally accepted that the mutual feedback between the two tissues is required as they specialized and adapted to their unique roles and functions within the joint organ. These two tissues share numerous growth factors and cytokines together – bone morphogenetic proteins (BMPs), interleukins (ILs) [1], Transforming growth factor-beta1 (TGF-beta) [2], matrix metalloproteinases (MMPs) [3-5], and IGFs[6]. As such, bone and cartilage in the mature joint are interdependent, and are only capable of fulfilling their biomechanical functions with their mutual support from the macro-structural to the micro-molecular level. Furthermore, the intimate relationship between bone and cartilage involves multiple pathways of interaction, and many of which were shown to regulate the metabolic and remodeling mechanisms of the tissues. Consequently, repair attempts for one tissue without consideration for the other could cause disturbance to this delicate balance between the two tissues, potentially creating side effects that may, at the very least, undermine the self repair attempts or even accelerate the progression of the disease. Thus far, treatment efforts that focus on cartilage alone frequently were unsuccessful [7, 8].
Several of our studies show that in the OA disease model, bone and cartilage is linked in many ways. For example, a loss in cartilage integrity imposes increased mechanical stresses in the subchondral bone, which leads to its thickening over time and in turn,
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would inflict more damage unto the cartilage tissue. The background for OA development is filled with many such interrelated pathways, and this complexity only increases with time. As OA progresses, it recruits the involvement of additional tissues, further increasing the layers of complications. Because all changes to tissue physiology during OA progression are invariably connected, with the recruitment each additional component, the disease creates multiple new pathways that may serve to accelerate its development. Due to this self-feeding nature of OA, preventative or early stage treatments are far more likely to be successful. Instead of focusing on late-stage symptoms such as the loss of cartilage or gross joint deformation, more emphasis should be placed on stopping the disease at its initiation and earlier stage. As such, knowledge of key interactions between different tissues in the early stages of OA is critical in the development of successful treatments.
However, due to the complicated nature of OA progression, it is difficult to predict exactly when it is initiated. In fact, by the time it is diagnosed (usually with the radiographic evidence of reduced joint space), the disease is already in its late or advanced stages [9-11]. This coupled with the unavailability of early diagnostic tools made the characterization of early stage human OA progression almost unattainable. As a result, numerous animal models were developed and their use has become indispensable in this field of study. Many studies included in this thesis aimed to elucidate the early interactions between different tissues with the onset of OA and have successfully characterized, at least in animal models, many of the complex pathways responsible for OA progression.
To bring further clarity to the many unanswered questions concerning the role and importance of the subchondral bone in OA development, this thesis approached the problem from two primary directions. First, we examine the minute changes of subchondral bone and cartilage to elucidate their relationship and impact on OA progression. This was done by a study using three dimensional microCT analyses combined with stereological histology. By correlating three dimensional cartilage and bone changes, we have successfully established the connection between bone remodeling and cartilage destruction. We have concluded that bone has not only a biomechanical, but also a biochemical influence on all the tissues of the joint. As bone changes structurally and physiologically in response to OA progression, its impact on the entire joint organ is profound. Our studies using 3 dimensional analyses demonstrated that bone’s mechanical properties are combined products of both its structural and material properties. It was found that structural and material properties of bone, including its matrix volume, degree of mineralization, matrix content, the number, thickness, and orientations of its trabeculae, as well as their angle and level of the connectivity, are all affected, and in turn, affect all other components of the joint.
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The second direction taken by this thesis was to study the OA development in an slow progressive experimental OA in a time-sensitive manner. We believe that it is critical to examine the disease’s progression in detail over time on both surgical induced OA (mimic secondary OA) and spontaneous OA (mimic primary OA), especially in the early stage. This was accomplished in our time trial experiments using a guinea pig model. To our knowledge, we are the first group to study the progressive change of subchondral bone over a 6 month time period in an experimental OA guinea pig model. From the role of biomarkers to structural changes in the tissues, the study yielded essential insights into the early stages of OA development. It is now clear that subchondral bone change is an unavoidable step in the progression of OA and that the presence of cartilage lesion is always matched with significant bone remodeling directly below. This discovery has significant implications in both the understanding and treatment of OA.
As mentioned above, due to the nature of OA progression, achievement of successful treatment is much more likely in the early stages of the disease, before its spread and advancement throughout the entire joint organ. Thus, having dependable methods of early detection is essential for preventative treatment or early control of the disease. Without understanding the early events in the different tissues involved with OA, it is almost impossible to detect the disease in its beginning stages. Our time trial study showed that, at least in animal models, subchondral bone change can be a strong indicator of OA initiation and its early development. We believe that the monitoring of changes in bone metabolism (such as BMD, serum or urine levels of bone biomarkers and cytokines), can be a potentially powerful tool for the detection of OA for early treatment.
During the course of our study, we have also discovered that bone and cartilage change in OA is site-specific, with greater levels of remodeling found on the medial and anterior portions of the knee joint. This is consistent with previously reported data [12-14] and supports the theory that mechanical loading plays a significant role in the initiation and progression of OA [15-17]. This site-specificity of OA development indicates that there is a much higher risk of OA if the joint surface is unevenly loaded. This shows that uneven loading can be a potential cause of OA. From a treatment standpoint, one way to alleviate this problem is to help the joint avoid the development of high pressure areas. This may involve a combination of exercise and physical therapy to train and strength supportive muscle and to restore proper joint alignment. With this understanding, surgical procedures may be also developed to correct severe cases of joint misalignment (such as osteotomy) as a treatment against the onset of OA and alleviate syndromes [18-20].
Results from both of our model development studies have confirmed that the interaction of subchondral bone to the cartilage is one of the most important driving forces in OA
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progression. Bone, while it normally retains a higher capacity for healing than cartilage, do not necessarily respond in a manner that benefits the entire joint. In response to the higher stress levels, bone remodeling begins with an increase in bone metabolism, which initially decrease the level of mineralization in the tissue coupled with an increase in bone mass, followed by an increase in mineralization at later stages. The physiological change in the subchondral bone has a direct biomechanical impact on the cartilage tissue layer on top, perceived as an initial drop in stiffness and then a significant decrease in shock absorption. Other changes associated with bone remodel such as increase in vascularity and biochemical changes in the microenvironment may also have a significant affect on all tissue of the joint, in particular, perhaps playing a crucial role in the formation of osteophytes. We believe that the high bone turnover and subchondral bone changes, while they may not be solely responsible for its initiation, are critical and required steps in the progression of OA.
Building upon the results from our study models, we also investigated the potential of bone-affecting treatments to influence the progression of OA. In our study on the effects of alendronate on tissue, it was shown that alendronate treatment effectively suppressed subchondral bone resorption, thereby achieving significant chondroprotective effect in this experiment. This cross-tissue modifying effect is also found in our hyaluronan study, which demonstrated that the prevention of early subchondral bone changes in OA progression is an effective deterrent against the formation of cartilage lesions. These findings confirm the intimate and interdependent relationship between bone and cartilage and exemplify how effective future treatments should be designed to act with consideration for both bone and cartilage.
OA and RA are both complex disorders that involve multiple interdependent tissues that interact throughout the subtle initiation and steady progression of the disease. The details of disease progression can be understood only if the relationship between bone and cartilage is fully appreciated. Throughout our studies, we have demonstrated and confirmed subchondral bone’s importance in OA progression. Since subchondral bone change is an essential step in OA progression, it maybe useful for the early detection of OA. It may also serve, as shown in our studies, as a possible avenue of treatment. The current studies have examined the role of bone in OA at the tissue level, and future studies may focus on cellular. It is believed that research in the area of cellular signaling between bone and cartilage would be extremely fruitful for the furthering of our understanding of OA [21-23]. Possible cell signaling factors of interest include BMP-2 and BMP-7, since they have been demonstrated to have an effect on both bone and cartilage. By treating subchondral bone with Alendronate and Hyaluronan we have effected partial cartilage protection. It is also shown that alternative treatments such as glucosamine do have structure-modifying effects on OA tissues, rather than the placebo only influence as suggested by some previous researchers [24-26]. Future studies may
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include testing different bone and cartilage anabolic and catabolic signaling pathways to alter the normal course of disease development.
2. Concluding Remarks:
This thesis evaluated and discussed the intimate relationship of bone and cartilage in great details. The close relationship of the two tissues’ involvement in OA cannot be considered separately. The research results presented in this thesis have confirmed that the interaction of subchondral bone to the cartilage is one of the most important driving forces in OA progression; understanding of interactions between different tissues in the early stages of arthritis is critical in the development of successful treatments. In addition, subchondral bone change can be a strong indicator of OA initiation and its early development. By suppressing subchondral bone turnover we have achieved chondroprotective effect in an animal model of OA; this result has not only confirmed the close relationship of cartilage and subchondral bone, but also indicated a new direction of OA treatment.
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2. Blaney Davidson EN, van der Kraan PM, van den Berg WB. TGF-beta and osteoarthritis. Osteoarthritis Cartilage 2007; 15: 597-604.
3. Flannelly J, Chambers MG, Dudhia J, Hembry RM, Murphy G, Mason RM, et al. Metalloproteinase and tissue inhibitor of metalloproteinase expression in the murine STR/ort model of osteoarthritis. Osteoarthritis Cartilage 2002; 10: 722-733.
4. Janusz MJ, Hookfin EB, Heitmeyer SA, Woessner JF, Freemont AJ, Hoyland JA, et al. Moderation of iodoacetate-induced experimental osteoarthritis in rats by matrix metalloproteinase inhibitors. Osteoarthritis Cartilage 2001; 9: 751-760.
5. Shibakawa A, Yudoh K, Masuko-Hongo K, Kato T, Nishioka K, Nakamura H. The role of subchondral bone resorption pits in osteoarthritis: MMP production by cells derived from bone marrow. Osteoarthritis Cartilage 2005; 13: 679-687.
6. Lajeunesse D. The role of bone in the treatment of osteoarthritis. Osteoarthritis Cartilage 2004; 12 Suppl A: S34-38.
7. Colao A, Pivonello R, Scarpa R, Vallone G, Ruosi C, Lombardi G. The acromegalic arthropathy. J Endocrinol Invest 2005; 28: 24-31.
8. Ike RW. The role of arthroscopy in the differential diagnosis of osteoarthritis of the knee. Rheum Dis Clin North Am 1993; 19: 673-696.
9. Messent EA, Ward RJ, Tonkin CJ, Buckland-Wright C. Cancellous bone differences between knees with early, definite and advanced joint space loss; a comparative quantitative macroradiographic study. Osteoarthritis Cartilage 2005; 13: 39-47.
10. Boniatis I, Cavouras D, Costaridou L, Kalatzis I, Panagiotopoulos E, Panayiotakis G. Computer-aided grading and quantification of hip osteoarthritis severity employing shape descriptors of radiographic hip joint space. Comput Biol Med 2007; 37: 1786-1795.
11. Botha-Scheepers S, Kloppenburg M, Kroon HM, Hellio Le Graverand MP, Breedveld FC, Ravaud P, et al. Fixed-flexion knee radiography: the sensitivity to detect knee joint space narrowing in osteoarthritis. Osteoarthritis Cartilage 2007; 15: 350-353.
12. Gurevitch O, Kurkalli BG, Prigozhina T, Kasir J, Gaft A, Slavin S. Reconstruction of cartilage, bone, and hematopoietic microenvironment with demineralized bone matrix and bone marrow cells. Stem Cells 2003; 21: 588-597.
13. Palmer JL, Bertone AL, Malemud CJ, Carter BG, Papay RS, Mansour J. Site-specific proteoglycan characteristics of third carpal articular cartilage in exercised and nonexercised horses. Am J Vet Res 1995; 56: 1570-1576.
14. van Osch GJ, van der Kraan PM, Blankevoort L, Huiskes R, van den Berg WB. Relation of ligament damage with site specific cartilage loss and osteophyte formation in collagenase induced osteoarthritis in mice. J Rheumatol 1996; 23: 1227-1232.
15. Arokoski JP, Jurvelin JS, Vaatainen U, Helminen HJ. Normal and pathological adaptations of articular cartilage to joint loading. Scand J Med Sci Sports 2000; 10: 186-198.
16. Buckwalter JA, Martin JA. Osteoarthritis. Adv Drug Deliv Rev 2006; 58: 150-167. 17. Oakley SP, Lassere MN, Portek I, Szomor Z, Ghosh P, Kirkham BW, et al. Biomechanical,
histologic and macroscopic assessment of articular cartilage in a sheep model of osteoarthritis. Osteoarthritis Cartilage 2004; 12: 667-679.
18. Freeman MA, Bargren J, Miller I. A comparison of osteotomy and joint replacement in the surgical treatment of the arthritic knee. Arch Orthop Unfallchir 1977; 88: 7-18.
19. Gunther KP. Surgical approaches for osteoarthritis. Best Pract Res Clin Rheumatol 2001; 15: 627-643.
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20. Saito S, Saito M, Nishina T, Ohzono K, Ono K. Long-term results of total hip arthroplasty for osteonecrosis of the femoral head. A comparison with osteoarthritis. Clin Orthop Relat Res 1989: 198-207.
21. Iannone F, Lapadula G. The pathophysiology of osteoarthritis. Aging Clin Exp Res 2003; 15: 364-372.
22. Kaiser M, Haag J, Soder S, Bau B, Aigner T. Bone morphogenetic protein and transforming growth factor beta inhibitory Smads 6 and 7 are expressed in human adult normal and osteoarthritic cartilage in vivo and are differentially regulated in vitro by interleukin-1beta. Arthritis Rheum 2004; 50: 3535-3540.
23. Loeser RF, Todd MD, Seely BL. Prolonged treatment of human osteoarthritic chondrocytes with insulin-like growth factor-I stimulates proteoglycan synthesis but not proteoglycan matrix accumulation in alginate cultures. J Rheumatol 2003; 30: 1565-1570.
24. Christgau S, Henrotin Y, Tanko LB, Rovati LC, Collette J, Bruyere O, et al. Osteoarthritic patients with high cartilage turnover show increased responsiveness to the cartilage protecting effects of glucosamine sulphate. Clin Exp Rheumatol 2004; 22: 36-42.
25. Largo R, Alvarez-Soria MA, Diez-Ortego I, Calvo E, Sanchez-Pernaute O, Egido J, et al. Glucosamine inhibits IL-1beta-induced NFkappaB activation in human osteoarthritic chondrocytes. Osteoarthritis Cartilage 2003; 11: 290-298.
26. Wang SX, Laverty S, Dumitriu M, Plaas A, Grynpas MD. The effects of glucosamine hydrochloride on subchondral bone changes in an animal model of osteoarthritis. Arthritis Rheum 2007; 56: 1537-1548.
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Samenvatting
Experimentele artritis: in vivo en in vitro modellen
Artritis of reuma, de belangrijkste oorzaak van bewegingsbeperking voor mensen ouder dan 45 jaar, heeft meer dan honderd verschijningsvormen. Artrose is het meest voorkomend, gevolgd door reumatoïde artritis. Artrose wordt gekenmerkt door progressief verlies van gewrichtskraakbeen, veranderingen in het subchondrale bot direct onder het kraakbeen, ontstaan van botuitsteeksels (osteofyten) en ontsteking van het omliggende weefsel. Er is nog veel onbekend over het ontstaan van artrose, maar het is wel duidelijk dat belasting, leeftijd en genetische factoren een belangrijke rol spelen.
Het ziektebeeld is complex omdat veel factoren van invloed zijn, maar onbekend is langs welk mechanisme ze het beloop beïnvloeden. Studies naar de pathogenese zijn moeilijk vanwege een relatief lange periode waarin er nauwelijks symptomen zijn, terwijl de kwaliteit van het bot en het kraakbeen al wel significant achteruitgaat. Voor betere behandeling is meer begrip van de etiologie nodig. Daarvoor zijn zowel in vivo als in vitro modellen nuttig, elk met hun eigen sterke en zwakke punten. Met in vivo modellen kan de complexe aandoening als geheel worden bestudeerd, inclusief alle factoren die het beloop beïnvloeden. In vitro modellen staan het toe om bepaalde aspecten separaat te bestuderen in snellere en goedkopere experimenten dan in vivo mogelijk is, en zonder dat variatie tussen dieren daarop van invloed is. De keus voor het te gebruiken model is belangrijk; elk model heeft unieke eigenschappen, geeft andere resultaten en biedt daardoor unieke inzichten in de pathologie. De informatie die wordt verkregen is daarom vaak additioneel. Het doel van dit proefschrift is om door de combinatie van een aantal in vivo en in vitro modellen meer inzicht te krijgen in het verloop van artrose, in het bijzonder in de interactie tussen botadaptatie en kraakbeendegradatie.
Hoofdstuk 1 geeft achtergrondinformatie over artritis en beschrijft modellen die worden gebruikt in onderzoek naar artrose en reumatoïde artritis. Hoofdstuk 2 geeft een overzicht van de structurele veranderingen die bij artrose en reumatoïde artritis optreden in subchondraal bot, gemeten met micro-computertomografie (micro-CT) in
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proefdieren. Hoofdstuk 3 beschrijft de ontwikkeling van artrose in een implantaat dat bestaat uit tissue engineered kraakbeen, gekweekt in een gesloten bioreactor met of zonder ondersteunend biodegradeerbaar en osteoconductief materiaal van verschillende samenstelling. De selectie van dit in vitro modelsysteem is gebaseerd op eerder onderzoek met celkweken in monolaag, meerdere lagen en pallets, inclusief studies waarin kraakbeencellen in co-cultuur werden gekweekt met botcellen en cellen uit het synovium.
Reumatoïde artritis is een systemische auto-immuun ziekte, gekarakteriseerd door chronische gewrichtsontsteking en wisselende mate van bot- en kraakbeenafbraak. Studies met proefdieren geven inzicht in de invloed van veranderingen in het bot op reumatoïde artritis. In hoofdstuk 4 wordt reumatoïde artritis geïnduceerd in ratten door een antigeen te injecteren dat aan de celwand bindt. Een deel van de ratten wordt behandeld met N-butyryl glucosamine (GlcNBu). De microstructurele veranderingen in het subchondrale bot worden gemeten. GlcNBu blijkt de ontsteking en zwelling in de enkel te verminderen, en verandering in botdichtheid en botstructuur tegen te gaan. Daarmee wordt botverlies in dit proefdier model voor chronische artritis voorkomen.
Hoofdstuk 5 focust op veranderingen in het subchondrale bot rondom het gewricht in een ander proefdier model. Bij konijnen wordt artrose geïnduceerd door de voorste kruisband door te snijden. Veranderingen in dichtheid, turnover, structuur en connectiviteit van trabeculair bot, en in dikte en mineralisatiegraad van de subchondrale plaat worden bestudeerd. Bij deze konijnen beschermt orale toediening van Glucosamine-HCl het subchondrale bot tegen veranderingen.
Tijdens artrose is er altijd een wisselwerking tussen de veranderingen die plaatsvinden in kraakbeen, onderliggend bot en omliggende weefsels. Hierdoor is het moeilijk om exact te bepalen waar en hoe artrose begint. Verschillende diermodellen zijn ontwikkeld met als doel inzicht te krijgen in deze wisselwerking. Twee benaderingen worden uitgewerkt in dit proefschrift. Ten eerste worden de veranderingen in het subchondrale bot en het kraakbeen bij proefdieren in detail beschreven, om de samenhang tussen deze veranderingen tijdens het ontstaan van artrose duidelijk te maken. In hoofdstuk 6 worden driedimensionale micro-CT analyses gecombineerd met stereologische histologie van kraakbeen om veranderingen te registreren tijdens de ontwikkeling van spontane artrose in de knie bij twee soorten cavia’s. Met deze methode wordt een verband aangetoond tussen botadaptatie en kraakbeendegradatie. De tweede aanpak in dit proefschrift is om ontwikkeling van artrose in de tijd te volgen in een langzaam progressief diermodel voor artrose. Hoofdstuk 7 vergelijkt de eerste stadia van artrose in cavia’s waarin artrose chirurgisch wordt geïnduceerd (vergelijkbaar met secundaire artrose) met cavia’s die spontaan artrose ontwikkelen (vergelijkbaar met primaire artrose). De progressieve veranderingen in het subchondrale bot, die gedurende zes maanden in detail zijn vastgelegd, tonen aan dat toegenomen turnover van het
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subchondrale bot een essentiële stap is bij artrose ontwikkeling. Bovendien wordt bewezen dat kraakbeenschade altijd samengaat met significante adaptatie van het onderliggende bot. Deze ontdekking heeft belangrijke implicaties voor zowel het begrip als de behandeling van artrose.
Op basis van deze inzichten in de wisselwerking tussen subchondraal bot en artrose, vormen we de hypothese dat kraakbeendegradatie kan worden vertraagd door de turnover van subchondraal bot te voorkomen. In hoofdstuk 8 worden cavia’s die artrose ontwikkelen behandeld met Alendronate, een medicijn dat botresorptie tegengaat. Deze behandeling heeft inderdaad een positief effect op de structuur en mineralisatie van het subchondrale bot, op de kwaliteit van het kraakbeenoppervlak, en het beschermt het kraakbeen tegen verlies van collageen en proteoglycanen. De conclusie is dat medicatie tegen botresorptie een positief effect heeft op zowel kraakbeen- als botdegradatie.
Samengevat laat dit proefschrift zien dat veranderingen in kraakbeen en bot sterk aan elkaar gekoppeld zijn en samen moeten worden bestudeerd. De samenhang tussen deze weefsels kan niet onafhankelijk gezien worden van de ontwikkeling van artritis; de wisselwerking tussen subchondraal bot en kraakbeen lijkt een van de belangrijkste factoren voor de ontwikkeling van artrose of reumatoïde artritis. Door het onderdrukken van de veranderingen in subchondraal bot is het mogelijk gebleken om kraakbeendegradatie te vertragen in cavia’s die artrose ontwikkelen. Dit bewijst dat verhoogde turnover van subchondraal bot een oorzakelijke factor is bij de ontwikkeling van artrose. De combinatie van in vitro en in vivo modellen in dit proefschrift heeft bijgedragen aan dit betere begrip van de etiologie. In het bijzonder zijn in vitro modellen op basis van tissue engineered kraakbeen van belang geweest voor het bestuderen van veranderingen aan het kraakbeen oppervlak, en voor het screenen van potentiële medicijnen. Voor het bestuderen van de progressie van artrose op de lange termijn is het cavia model zeer bruikbaar. Dit model simuleert veel aspecten van de normale ontwikkeling van artrose bij mensen en kan gebruikt worden om behandelingen.
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Acknowledgement
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Acknowledgement First and for most, I would like to thank my promoter prof. Rik Huiskes for giving me the opportunity to write this thesis, for his supervision, guidance and advice, and especially for his faith in me to manage a (series of) high demanding full time job(s) and write this thesis at the same time.
I owe a debt of gratitude to my co-promoter dr. René van Donkelaar for his guidance, deep involvement, and constant support throughout the thesis work. He is the one who reviewed many times this manuscript from early to final versions. He always goes the extra mile to help me. His patience, dedication, and devotion revealed during his supervision presented his excellence as a scientist and as an educator.
I would like to express my immeasurable gratitude to my lifelong mentor and best friend prof. Ernst Hunziker who introduced me to tissue engineering and arthritis research ten years ago – since then, I have thoroughly enjoyed exploring this fascinating field. Prof. Hunziker is a pioneer in this field of research and I have significantly profited from his constructive suggestions, intellectual discussions and legitimate directions. His mentoring to me started from the day I stepped into his institute, but continues on today – many years after I graduate from his lab.
I am profoundly grateful for the wonderful opportunity to work with professors Marc Grynpas and Pierre Mainil. In Dr. Mainil’s laboratory I had the chance to combine my previous clinical experience with my newly acquired cell biology knowledge for the design of experimental cell therapy for the treatment of arthritis. In his lab, I have acquired many useful skills in designing and developing animal models of human disease. This experience made my career transition from surgery to cell biology and then to preclinical (translational) research smooth and meaningful. By the time I moved to prof. Grynpas lab, I was ready to explore the fascinating field of musculoskeletal research. Prof. Grynpas is an internationally renowned scientist in bone biology, working in his lab I was indisputably trained with outstanding bone research practice. Combining the bone biology and cartilage biology knowledge I worked on several preclinical arthritis models. Prof. Grynpas has giving me constructive instructions in designing the experiment and constant support in carrying out experiments. I was very lucky to work with Prof. Grynpas and had fruitful results - most of the papers presented in this thesis are resulted from the work I did at his lab.
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I sincerely thank the committee members; prof. Keita Ito, prof. Ruud Geesink and dr.ir. Bert van Rietbergen for their valuable comments on this thesis.
My gratefulness to my family and beloved ones is beyond words. Many thanks for all your endless love and support.
Curriculum Vitae
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Curriculum Vitae Xuanhui Wang graduated from China Medical University, Shenyang, China in 1987. She completed her residency in the Fifth Hospital of Shenyang, China, and became a vascular surgeon in the Second Hospital of Shenyang Medical Collage, China. Her passion for science led her to graduate school and began three years of cardiovascular research, for which she received a M.Sc degree in 1997. Her M.Sc thesis title is “Antiphospholipid Antibodies in Thrombangiitis Obliterans and Deep Vein Thrombosis”
She then continued her research in the field of cartilage cell biology at the University of Bern, Switzerland and was awarded a doctor’s degree in Human Medicine, in May 2000. Her doctor’s degree’s thesis is titled "Chondrocyte Biosynthesis and Metabolic Activity in Alginate Culture”. After that, she completed her first term of postdoctoral research at osteoarthritic research laboratory, Pathology Institute, University of Bern, Switzerland, in the field of cartilage repair and tissue engineering. Her second term of postdoctoral research was in the bone biology laboratory at the Department of Laboratory Medicine and Pathobiology, University of Toronto; Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Canada. Her research there was primarily focused on preclinical arthritis models. Having trained extensively in several academia labs she was recruited by a biotech company to conduct chondroprotective drug screening using in vitro and in vivo preclinical models in 2005. Afterwards, she worked at two other biotech/pharmaceutical companies in the field of arthritis and orthopedic research. Currently, she is the Technical Director of Orthopaedics and Tissue Engineering, MDS Pharma Services, WA. USA.
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Bibliography
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Bibliography
Book chapter:
1. Wang X. Treatment of common peripheral vascular disease. In: Lee B. ed. “Brief Therapeutic Solutions for Common Cardiovascular Disease”. Press of Shanghai Medical University. Oct. 1995: 55‐67
2. Wang SX, Subchondral bone 3D microarchitecture changes in animal models of osteoarthritis quantified by micro‐CT. (Book chapter) In. Qin Ling, Harry Genant ed. “Advanced Bioimaging Technologies in Assessment of the Quality of Bone and Scaffold Materials”. Springer‐Verlag Inc. (Berlin, New York, Tokyo) 2007
Peer reviewed articles:
1. Wang X. et al,. Thrombectomy using Fogarty embolectomy catheter for non‐traumatic acute lower limb ischaemia. Journal of Shenyang Medical College 1993(4):273‐5
2. Wang X. et al,. Advanced therapies for patients with reflux veins in the lower extremities. Journal of Shenyang Medical College 1994(1):55‐8
3. Wang X. et al,. Antiphospholipid antibodies and vascular disease. Chinese Journal of Surgery 1997;1(2):129‐35
4. Wang X. et al,. Plasma levels of antiphospholipid antibodies are associated with disease progression in Thrombangiitis obliterans. Shanghai Medical Journal 1997;5(20):112‐7
5. Wang X. et al. Lupus anticoagulants in Buergers disease and deep vein thrombosis. Chinese Journal of Experimental Surgery 1997;5(14):1703‐9
6. Wong M, Siegrist M, Wang X, Hunziker E. Development of mechanically stable alginate/chondrocyte constructs: effects of guluronic acid content and matrix synthesis. J Orthop Res 2001 May;19(3):493‐9.
7. Ciolfi VJ, Pilliar R, McCulloch C, Wang SX, Grynpas MD, Kandel RA. Chondrocyte interactions with porous titanium alloy and calcium polyphosphate substrates. Biomaterials 2003 Nov;24(26):4761‐70.
8. Wang X, Grogan SP, Rieser F, Winkelmann V, Maquet V, Berge ML, Mainil‐Varlet P. Tissue engineering of biphasic cartilage constructs using various biodegradable scaffolds: An in vitro study. Biomaterials 2004 Aug;25(17):3681‐8
9. Suh WK, Wang SX, Jheon AH, Moreno L, Yoshinaga SK, Ganss B, Sodek J, Grynpas MD, Mak TW. The immune regulatory protein B7‐H3 promotes
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osteoblast differentiation and bone mineralization. Proc Natl Acad Sci U S A 2004 Aug;101(35):12969‐73.
10. Wang SX. Cherian A, Dumitriu M, Grynpas MD, Carran J, Wainman D, Anastassiades T. Disease modifying effects of N‐butyryl glucosamine in a streptococcal cell wall induced arthritis model in rats. J Rheumatol. 2007 Apr;34(4):712‐20.
11. Wang SX, Laverty S, Plass A, White L, Grynpas MD. The effect of glucosamine hydrochloride on subchondral bone changes in an animal model of OA. Arthritis and Rheumatism, 2007 May;56(5):1537‐48.
12. Ng AHM, Wang SX, Turner CH, Beamer WG, Grynpas MD. Bone quality in BXH recombinant inbred mice. Calcif Tissue Int. 2007 Sep;81(3):215‐23.
13. Wang SX, Dumitriu M, Tam RH, Kandel RA, Reulund R, Marc D. Grynpas. Progressive Change of Cartilage and Subchondral Bone in Partial Medial Menisectomy and Accelerated Spontaneous OA Models ‐ A 6 Month Study with Dunkin Hartley Guinea Pigs (to be submitted)
14. Wang SX, Dumitriu M, Tam RH, Renlund R, Kandel R, Grynpas MD. The Effects of Alendronate on the Progression of OA in Cartilage and Subchondral Bone Tissues using a Partial Medial Menisectomy Guinea Pig Model (to be submitted)
15. Wang SX, Eyre D, Tam RH, Arsenault L, Hunziker EB. Stereological study of cartilage and subchondral bone changes in spontaneous knee osteoarthritis: results from two strains of guinea pig. (to be submitted)
Conference proceedings:
1. Wong M, Siegrist M, Wang X, Hunziker E. The effect of GAG and collagen on the compressive properties of alginate constructs. 13th Congress of the International Society of Bioengineering and Skin, Israel, March, 2000.
2. Whiteside RA, Wang X, Jakob R, Wyss UP, Mainil‐Varlet P. Impact loading of articular cartilage during autologous osteochondral transplantation. The International Cartilage Repair Society (ICRS), 4th Symposium, Toronto, June 15‐18, 2002.
3. Wang X, Grogan SP, Winkelmann V, Schittny J, Rieser F, Bittmann P, Jakob R, Mainil‐Varlet P. Tissue engineering biphasic implants using biodegradable scaffolds. The International Cartilage Repair Society (ICRS), 4th Symposium, Toronto, June 15‐18, 2002. (podium presentation)
4. Whiteside RA, Wang X, Jakob R, Monin D, Wyss UP, Mainil‐Varlet P. The effect of impact energy on articular cartilage during autologous osteochondral
Bibliography
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transplantation. The International Cartilage Repair Society (ICRS), 4th Symposium, Toronto, June 15‐18, 2002.
5. Wang SX, Coulomb J, Laverty S, Grynpas MD. The effects of glucosamine on bone in a rabbit model of osteoarthritis. Canadian Arthritis Network 2002 scientific symposium, Calgary, CA. Sept 26‐28, 2002
6. Grynpas MD, Wang SX, Chahine CJ, Sodek J. The role of CD44 in bone remodeling: a study in CD44‐knockout mice. Functional Analysis of the Mouse Genome Symposium, Toronto, Dec 12, 2002
7. Wang X, Grogan SP, Rieser F, Bittmann P, Jakob R, Mainil‐Varlet P. Tissue engineering biphasic cartilage implants using biodegradable scaffolds. 49th Orthopaedic Research Society (ORS), New Orleans, US, Feb 2‐5, 2003.
8. Grynpas M D, Jurisicova A, Wang SX, Moreno L, Taniuchi L, Canning J, Perez G, Tilly J. The relationship between bone quality and ovarian senescence in aging mice model. 30th European Symposium on Calcified Tissues, Rome, Italy, May 8‐12, 2003.
9. Wang SX, Laverty S, Stuart K, White L, Plaas A, Grynpas MD. The effects of glucosamine on bone in a rabbit model of osteoarthritis. 8th World Congress of the Osteoarthritis Research Society International (OARSI), Berlin, Germany, Oct 12‐15, 2003.
10. Wang SX, Stuart K, Kandel R, Grynpas MD. The effects of bisphosphonate on the development of bone change in the guinea pig OA model. 8th World Congress of the Osteoarthritis Research Society International (OARSI), Berlin, Germany, Oct 12‐15, 2003.
11. Wang SX, Kandel R, Grynpas MD. Effect of bisphosphonate treatment on the progression of osteoarthritis. Canadian Arthritis Network 2003 scientific symposium, Montreal, Nov 13‐15, 2003.
12. Wang SX, Coulomb J, Tiraloche G, Laverty S, Grynpas MD. Glucosamine for osteoarthritis: study: bone change in an experimental arthritis. Canadian Arthritis Network 2003 scientific symposium, Montreal, Nov 13‐15, 2003.
13. Wang SX, Laverty S, Plass A, Grynpas MD. The effects of glucosamine on bone in a rabbit model of osteoarthritis. 50th Othopaedic Research Society (ORS), San Francisco, March 7‐10, 2004.
14. Chahine CJ, Wang SX, Sodek J, Grynpas MD. Effects of CD44 on bone quality: a study in knock out mice. 50th Orthopaedic Research Society (ORS), San Francisco, March 7‐10, 2004.
15. Wang SX, Kandel R, Grynpas MD. Evaluation of OA in an animal model: when and where to intervene. The International Cartilage Repair Society (ICRS), 5th Symposium, Gent/Belgium, May 26‐29, 2004. (podium presentation)
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16. Formosa R, Wang SX, Kandel R, Grynpas MD. Characterization of Bone and Cartilage change in a guinea pig model of OA, A long‐term (6‐month) Study. 10th Canadian Connective Tissue Conference, Toronto, May 23‐26, 2004.
17. Andrieu MC, Wang SX, Formosa R, Grynpas MD. Bone Material Property Change in the Progression of Osteoarthritis. 10th Canadian Connective Tissue Conference, Toronto, May 23‐26, 2004.
18. Anastassiades TP, Grynpas M, Wang SX, Carran JR. Wainmain DS. Orally Administered N‐Butyrylated Glucosamine (GlcNBu) Decreases Inflammation in the Streptococcal Cell Wall Rat Arthritis Model and Increases the Bone Mineral Density and Content of the Subchondral Regions of the Femur and Tibia. The American Society for Bone and Mineral Research (ASBMR), 2004 Annual Meeting, Seattle, Washington, Oct 1‐5 2004.
19. Wang SX, Grynpas MD. Changes in subchondral bone mineralization during the progression of osteoarthritis, 8th International Conference on the Chemistry and Biology of Mineralized Tissues (ICCBMT), Banff, Alberta, Canada, Oct 17‐22, 2004.
20. Wang SX, Kandel R, Grynpas MD. The course of osteoarthritis is accelerated by partial meniscectomy in the guinea pig model. Canadian Arthritis Network 2004 scientific symposium, Vancouver, Nov11‐13, 2004.
21. Wang SX, Phan A, Kandel RA, Grynpas. Alendronate reduce bone and cartilage changes in a model of osteoarthritis. Canadian Arthritis Network 2004 scientific symposium, Vancouver, Nov11‐13, 2004. (podium presentation)
22. Wang SX, Kandel R, Grynpas MD. Characterization of the meniscectomized guinea pig model of osteoarthritis ‐ a long term study. Osteoarthritis Research Society International (OARSI), 9th world congress, Chicago, IL. Dec 2‐5, 2004.
23. Wang SX, Kandel R, Grynpas MD. The long term effect of alendronate on the development of the guinea pig accelerated spontaneous osteoarthritis model. Osteoarthritis Research Society International (OARSI), 9th world congress, Chicago, IL. Dec 2‐5, 2004. (podium presentation)
24. Wang SX, Kandel R, Grynpas MD. Characterization of subchondral bone changes during the progression of osteoarthritis: a 6‐month study in the partial medial meniscectomized guinea pig. 51st Annual Meeting of the Orthopaedic Research Society (ORS), Washington, DC, Feb 20‐23, 2005.
25. Vogel WF, Friese‐Hamim M, Wang, SX, Alves F, Missbach J. Function of the DDR collagen receptors in bone development. 11th Canadian Connective Tissue Conference, Mountreal, May 26‐28, 2005
26. Wang SX, Laverty S, Grynpas MD. Quantifying joint changes in an OA model using 3D and 2D imaging techniques. 2005 Symposium on Bone & Biomaterial
Bibliography
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Evaluation by mCT, pQCT&MRI, HongKong Oct 16‐18, 2005 (podium presentation)
27. Wang SX, Arsenault L, Hunziker EB. Alterations of calcified cartilage and subchondral bone observed in osteoarthritic joints: A Stereological Study in two strains of Guinea Pig. Osteoarthritis Research Society International (OARSI), 10th world congress, Boston, MA. Dec 8‐11, 2005
28. Wang SX. Osteoarthritic animal models for the study of cartilage repair. The International Cartilage Repair Society (ICRS), 6th Symposium, San Diego, Jan 8‐11, 2006. (podium presentation)
29. Wang SX. Eyre D, Arsenault L, Nüssli S, Hunziker EB. 3‐D subchondral bone evaluation of guinea pig spontaneous OA and the effects of hyaluronan in this model. 53rd Annual Meeting of the Orthopaedic Research Society (ORS), San Diego, CA, Feb 11‐14, 2007 (podium presentation)
30. Guelakis M, Zhang X, Wang SX., Rendon H, Ramsay H, Wang S, Abramson S, Labazzo K, Hariri R, Hofgartner W, Heidaran M. Placenta‐Derived Adherent Cells (PDACs) for Orthopedic Applications. 8th World Biomaterials Congress (WBC), Amsterdam, The Netherland, May 28‐June 1st 2008 (podium presentation)