phd thesis

Osteoinductive and Antibacterial

Biomaterials

for Bone Tissue Engineering

Dongyun Wang

The following institutions generously funded the printing of this thesis:

Academic Centre for Dentistry Amsterdam (ACTA)

VU University Amsterdam

Devsoon Technology Testing Co., Ltd, Weihai, China

Dent-Med Materials B.V. (Geistlich Bio-Oss® and Geistlich Bio-Gide

®)

Straumann B.V.

Cover design: Dongyun Wang & Qiushi Zhu

ISBN: 978-90-825654-0-9

Copyright © 2016 by Dongyun Wang. All Rights Reserved.

No part of this book may be reproduced, stored in a retrievable system, or transmitted in

any form or by any means, mechanical, photo-copying, recording or otherwise, without

the prior written permission of the holder of copyright.

VRIJE UNIVERSITEIT

Osteoinductive and Antibacterial Biomaterials

for Bone Tissue Engineering

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad Doctor aan

de Vrije Universiteit Amsterdam,

op gezag van de rector magnificus

prof.dr. V. Subramaniam,

in het openbaar te verdedigen

ten overstaan van de promotiecommissie

van de Faculteit der Tandheelkunde

op woensdag 21 september 2016 om 15.45 uur

in de aula van de universiteit,

De Boelelaan 1105

Door

Dongyun Wang

geboren te Taiyuan, China

promotor:

prof.dr. D. Wismeijer

copromotor: dr. Y. Liu

Contents

Chapter 1 General introduction 1

Chapter 2 Bone regeneration in critical-sized bone defect enhanced by

introducing osteoinductivity to biphasic calcium phosphate

granules

11

Chapter 3 A novel bone-defect-filling material with sequential

antibacterial and osteoinductive properties for repairing

infected bone defects

35

Chapter 4 Coatings for osseointegration of metallic biomaterials 57

Chapter 5 Accuracy of peri-implant bone thickness and validity of

assessing bone augmentation material

using cone beam computed tomography

77

93

Chapter 6 General discussion

Chapter 7 General summary 103

Samenvatting 109

Acknowledgements 113

Curriculum Vitae 119

Abbreviations

α-MEM Alpha-minimal essential medium

ACP Amorphous calcium phosphate

ALP Alkaline phosphatase

β-TCP β-tricalcium phosphate

BAM Bone augmentation materials

BCP Biphasic calcium phosphate

BIC Bone to implant contact

BMP2 Bone morphogenetic protein 2

BMP7 Bone morphogenetic protein 7

BMP2-cop.BioCaP BMP2-coprecipitated, layer-by-layer

assembled biomimetic calcium phosphate particle

BMP2-BioCaP BMP2-incorporated biomimetic calcium phosphate

CaP Calcium phosphate

CBCT Cone beam computered tomography

CFU Colony forming units

CLFM Laser scanning fluorescence microscopy

CSBC Critical-sized bone defect

ECM Extracellular matrix

ELISA Enzyme linked immunosorbent assay

FBS Fetal bovine serum

FDA Food and Drug Administration

HA Hydroxyapatite

HACC Hydroxypropyltrimethyl ammonium chloride chitosan

HU Hounsfield units

MIC Minimum inhibitory concentration

MBC Minimum bactericidal concentration

MRSA Methicillin-resistant staphylococcus aureus

OCN Osteocalcin

OCP Crystalline octacalcium phosphate

PCC Poly (methyl methacrylate)

P. gingvalis Porphyromonas gingivalis

PLLA poly L-lactic acid

PLGA poly D, L-lactide-co-gycolic acid

PMMA Pearson correlation coefficient

S. aureus Staphylococcus aureus

SEM Scanning electron microscopy

TRAP Tartrate resistant acid phosphatase

1

Chapter 1

General Introduction

Chapter 1

2

Critical-sized bone defect

Bone defects, resulting from trauma or tumor resections, are common clinical problems that

affect hundreds of thousands of patients worldwide. They are usually associated with pain,

stiffness of the surrounding joints and disability, which often prevents employment and

therefore imposes an economic burden on the patient and on society [1]. Thanks to self-

healing mechanism, humans and animals tend to spontaneously repair bone defects.

However, if the sizes of the defects are beyond the self-healing capacity, they cannot

repaire without medical intervention. Such defects are called critical-sized bone defects

(CSBD), which is defined as the smallest size intraosseous wound that will not

spontaneously heal completely with bone tissue, or the defects will heal by connective

tissue during the lifetime of the animal [2]. Although tremendous efforts have been made to

repair CSBD, current strategies encounter a variety of limitations. Further development of

effective repair of CSBD is therefore needed in the field of orthopedic, oral and

maxillofacial surgery.

Bone tissue engineering

Bone tissue engineering is an interdisciplinary field that combines the knowledge and

technology of material engineering and biological factors to regenerate damaged bone

tissues[3]. It is based on the understanding of bone structure, bone mechanics, and tissue

formation as it aims to induce new functional bone tissues. In other words, to successfully

regenerate or repair bone, knowledge of the process of bone defect healing is quite essential.

Bone defects heal by a process that recapitulates many of the events of both

intramembraneous and endochondral bone formation, and it uniquely heals without the

formation of scar tissue [4, 5]. Initially, hematoma formation is accompanied by an

inflammatory response and the recruitment of many of the signaling molecules involved in

the regulation of new bone formation (i.e., ILs, TNF-α, FGFs, BMPs, PDGF, VEGF, etc.).

At cortex and periosteum level, intramembranous bone formation immediately occurs. The

external soft tissues stabilize the fracture by the formation of a callus, which subsequently

undergoes chondrogenesis, and then a process highly similar to endochondral ossification.

More specifically, after the callus forms, chondrocyte proliferation decreases as the tissues

begin to mature (i.e., hypertrophy) and calcify the matrix. In-growing blood vessels carry

chondroclasts, which are responsible for resorbing the calcified cartilage and osteoblastic

progenitors, which begin the process of new bone formation. The mechanical continuity of

the cortex is achieved via subsequent remodeling of the newly formed bone. In the process

of repairing bone defects, several key factors are highlighted: (1) a biocompatible scaffold

that closely mimics the natural bone extracellular matrix niche, (2) osteogenic cells to lay

on the bone tissue matrix, (3) morphogenic signals that differentiate mesenchymal stem in

General introduction

3

cells into osteogenic cells, and (4) sufficient vascularization to meet the growing tissue

nutrient supply and clearance needs.

Bone grafts

Bone grafts must comply with some or all these factors to be able to facilitate bone

regeneration to various degrees. If the grafts have the ability to facilitate the migration and

proliferation of osteoblasts and progenitor cells [6], they are considered to be

osteoconductive material. A solo scaffold is usually an osteoconductive graft. When a

scaffold is constructed with osteogenic and/or vasculo-genic growth factors, they are

endowed with osteoinductivity─the ability to induce progenitor cells to differentiate down

osteogenic lineages [6]. Only when a biomaterial consists of a scaffold, osteogenic cells and

growth factors, it is considered to be osteogenic.

Autografts are osteogenic, because they can provide all the elements for bone regeneration

such as osteoconductive 3-dimensional scaffolds, osteogenic cells and osteoinductive

growth factors [7]. Autografts are therefore regarded as the “gold standard” for bone defect

repair. However, autografts need to be harvested from the iliac crest or other sites in

patients, and thus it requires a second operation at the site of the tissue harvest [8], which

makes patients in danger of significant donor site injury and morbidity, deformity and

scarring [9-11]. Another drawback of autografts is their uncontrollable and variable

spontaneous resorption, which may compromise the outcome of reconstructing the

curvature of the local sites [12].

Allografts represent the second most common bone-grafting technique. They are often from

a cadaver. Allogeneic bone is also biocompatible, and is available in various forms,

including demineralized bone matrix, morcellised and cancellous chips, cortico-cancellous

and cortical grafts, and whole-bone segments, depending on the host-site requirements. In

comparison to autografts, allografts are associated with risks of immunoreactions and

transmission of infections [13, 14]. Since donor grafts are devitalized via irradiation or

freeze-drying processing, they have reduced osteoinductive properties and no cellular

component [15, 16]. Furthermore, the bone grafting market is experiencing an unmet

supply and great demand; there is currently a shortage in allograft bone graft material [17].

Xenografts are composed of tissue taken from another species. The antigenic potential of

xenografts can be diminished or eliminated by chemical treatment. One of the most widely

used xenografts in clinical dentistry is deproteinized bovine bone (Bio-Oss®, Geistlich,

Switzerland). It is derived from a bovine source and is treated by a chemical extraction

process to remove all the organic components and pathogens [18].

Chapter 1

4

Thanks to technological evolution and better understanding of bone-healing mechanisms,

many synthetic calcium phosphate (CaP) grafts have been developed to mimic the content

and structure of natural bone for repairing bone defects. According to the chemical

compostition, CaP can be classified as either hydroxyapatite, beta-tricalcium phosphate,

biphasic calcium phosphate, carbonated apatite or calcium deficient hydroxyapatite [19].

Moreover, CaP can be fabricated into different application forms, such as pastes, granules,

blocks and composites, which can be classified as ceramic or cements. Ceramics are

defined as the inorganic, non-metallic solid materials prepared by sintering [20]. The

sintering process removes volatile chemicals and increases crystal size, resulting in a

porous and solid material. On the other hand, cements consist of a mixture of calcium

phosphates which can be applied as a paste and harden in situ due to a precipitation reaction.

Both xenograft and synthetic CaP have physical and chemical structure similar to that of

natural bone [21], which offers them excellent osteoconductivity. However, they lack

intrinsic osteoinductivity [22].

Acquiring and evaluating osteoinductivity

For most clinical cases, osteoconductive bone grafts can achieve excellent bone defect

reparation. However, there are still a significant number of eligible individuals with CSBD

suffering from diabetes, local osteoporosis and metabolic bone disorder which can

compromise bone healing. Bone grafts with enhanced ability to repair bone defects are

therefore needed. Although cell-based therapies are attracting increasing attention, they are

still in their infancy regarding the safety and efficacy for humans. In contrast, growth

factor-based therapy is more advantageous in safety, feasibility and practical potential for

clinical application in the immediate future. Therefore, we believe it is the safest and most

practical method to endow grafts with osteoinductivity by introducing growth factors. One

substantial group of growth factors to offer osteoinductivity to bone grafts is the bone

morphogenetic proteins (BMPs). BMPs have been found in the demineralized bone matrix

more than 40 years ago. They belong to the TGF-β superfamily and promote the

differentiation of osteoprogenic cells and induce osteogenesis [23]. The osteoinductive

properties of BMPs make them promising candidates to promote bone formation, which has

been confirmed in animal studies and clinical trials [24-26]. Some of them are therefore

approved by the food and drug administration (FDA) as a medical device [23]. These are

recombinant human bone morphogenetic protein-2 (rhBMP-2) and recombinant human

bone morphogenetic protein-7 (rhBMP-7).

To evaluate the osteoinductivity of biomaterials, we need to thoroughly understand

osteoinduction. It could be divided into 3 principles [27]：(1) mesenchymal cell

recruitment; (2) mesenchymal differentiation to bone-forming osteoblasts and (3) ectopic

bone formation in vivo. Based on this theory, the osteoinductivity in vitro can be


5

determined by evaluating the effect of biomaterials on the differentiation of mesenchymal

stem cells. Their osteoinductivity is confirmed only when mesenchymal stem cells are

induced to osteogenic differentiation, which can be characterized by alkaline phosphatase

(ALP), osteocalcin (OCN) expression and mineralization. ALP [28] and OCN [29] are

involved in the process osteoid formation and bone mineralization. OCN is considered a

specific marker of osteoblast function [30]. Alizarin red-S is usually used to stain calcite

dolomite to show the mineralization [31], which is the reliable signal for osteogenic

differentiation. In vivo the osteoinductivity of a material is usually demonstrated by bone

formation after being implanted in CSBC [32] or ectopic sites such as subcutaneous pockets

and intramuscular sites [27].

Antibiotic delivery vehicles for treatment of infected bone defects

Reparation of bone defects can be challenging not only because of their sizes, but also

when they are combined with local infections. For example, in bone defects caused by

trauma, up to 50 % infectious complications have been reported [33, 34] with the tibia

being most often affected [35]. Furthermore, the subsequent chronic osteomyelitis and/or

non-union represent a major source of disability and decreased quality of life for the

individual patient, and a socio-economic problem for public health systems.

Thorough elimination of local infection is the prerequisite of repairing bone defects[36] and

therefore much focus has been put on infection control. Systematical administration of

antibiotics is not preferable for local infection control, because it is difficult for

systemically administered antibiotics to cross bone tissue with relatively avascular bone

surrounding it before reaching the infected defect site. This not only diminishes its

effectiveness, but also increases the chances for the induction of bacterial resistance [37].

Logically, these two central shortcomings could be ameliorated with the use of locally

delivered antibiotics. Various delivery modes have been developed for infection control. At

the clinical level, the mainly applied antibiotic delivery vehicle is in the form of poly

(methyl methacrylate) (PMMA) beads. After they had been first clinically applied in the

early 1970s [38], they gradually established themselves as a standard option for the local

delivery of antibiotics to bone cavities and this trend continues to this very day. Although

PMMA beads loaded with hydrophilic antibiotics were successfully applied in the past [39-

41], numerous clinical limitations are associated with their use. These include: (1) their

non-biodegradable nature and the need for a secondary surgery to remove them [42]; (2) an

often insubstantial amount of the released antibiotic following the initial, burst release

phase [43], which has led to the promotion of pathogenic resistance to such therapies in the

past [44]; (3) proneness to biofilm formation, which hinders the antimicrobial action [45];

and (4) moderate toxicity resulting from the absorption of MMA monomers and the

carboxylesterase-mediated conversion of MMA to methacrylic acid [46]. On top of this, a

Chapter 1

6

comprehensive clinical study has yet to prove that PMMA beads are more effective than the

systemic antibiotic delivery in treating orthopedic infections [47]. The major clinically

available alternative capable of sustained release are calcium sulfate cements, which suffer

from other weaknesses, mainly their rapid degradation, which is faster than the bone

ingrowth rate and can lead to mechanical failure of the implant [48].

We therefore believe that the ideal antibiotic delivery vehicle to repair infected bone defects

should possess the following properties: (1) biodegradable and its degradation rate should

match with the ingrowth rate of new bone; (2) can be adjusted to carry proper amount of

antibiotics and deliver antibiotics in appropriate mode; (3) the vehicle itself is also a

functional bone substitute.

Outline of the thesis:

BMP2 is well-known as an effective osteoinductive agent. This thesis is about introducing

BMP2 into different carriers to develop various osteoinductive biomaterials with different

properties to repair CSBD and infected CSBDs.

To enhance bone regeneration in CSBD, most bone-defect-filling materials in clinics need

to be mixed with autografts to obtain osteoinductivity. Due to the obvious limitation in

using autografts, we therefore developed a BMP2-coprecipitated, layer-by-layer assembled

biomimetic calcium phosphate particle (BMP2-cop.BioCaP) as a potential osteoinducer. In

chapter 2, we combined BMP2-cop.BioCaP with clinically often used biphasic calcium

phosphate (BCP) to repair an 8mm rat cranial defect. Our hypothesis was that BMP2-

cop.BioCaP could introduce osteoinductivity to BCP and so function as effectively as

autografts for the repair of CSBD.

BMP2-cop.BioCaP cannot be applied as an independent bone-defect-filling material due to

its rapid degradation. We therefore developed a BMP2-incorporated biomimetic calcium

phosphate granule (BMP2-BioCaP). In chapter 3, we combined it with a strong

antibacterial agent ─ hydroxypropyltrimethyl ammonium chloride chitosan (HACC) to

fabricate an osteoinductive and antibacterial biomaterial─BMP2-BioCaP/HACC complex

for repairing infected CSBD. It was designed with a sequential release system: burst release

of HACC and followed by a controlled release of BMP2. We hypothesized that this BMP2-

BioCaP/HACC complex could rapidly eliminate residual bacteria and thereafter induce new

bone formation in subcutaneous pockets in rats.

Osteoinductivity is essential not only for bone-defect-filling materials to repair CSBD, but

also for coatings on metallic biomaterials to improve their osseointegration in patients with

comprised surrounding bone tissue. In chapter 4, we reviewed that introducing BMP2 to


7

calcium phosphate coatings endow metallic implant surfaces with osteoinductivity so as to

enhance and accelerate their osseointegration.

In clinical practice, unlike animal studies mentioned above, bone regeneration in defects

and osseointegration of implants can hardly be evaluated by histological analysis. However,

it is essential to have good validity and reliability of radiological evaluating system to

assess post-operative integration bone-defect-filling materials and osseointegration of

metallic implants. In chapter 5, we therefore evaluated the accuracy of cone beam

computed tomography (CBCT) by comparing the same measurements gained by CBCT

with those on their corresponding histological sections.

In chapter 6, the main conclusions of this thesis are discussed and placed in a broader

perspective. The limitations of this thesis are also addressed.

Objectives of the thesis

The aims of this thesis are as follows:

1. To introduce osteoinductivity to clinically used BCP by a novel osteoinducer and

evaluate if it can improve bone regeneration in CSBD. Furthermore, to explore the

mechanism of the osteoinducer enhancing bone regeneration.

2. To develop an antibacterial and osteoinductive biomaterial for treatment of

infected CSBDs and to evaluate its antibacterial activity and osteoinductivity in

vitro and in vivo.

3. To review the biological process of osseointegration and offer an overview of the

coatings designed for improving osseointegration of metallic biomaterials

4. To evaluate the accuracy of measuring bone thickness surrounding dental implants

and the reliability of assessing existence and completion of osseous integration of

augmentation material using CBCT.

Chapter 1

8

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9

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11

Chapter 2

Bone regeneration in critical-sized bone defect

enhanced by introducing osteoinductivity to

biphasic calcium phosphate granules

Dongyun Wang, Afsheen Tabassum, Gang Wu, Liquan Deng,

Daniel Wismeijer, Yuelian Liu.

Clinical Oral Implants Research, 2015, 00: 1-10

Chapter 2

12

Abstract

Objectives: Biphasic calcium phosphate (BCP) is frequently used as bone substitute and

often needs to be combined with autologous bone to gain an osteoinductive property for

guided bone regeneration in implant dentistry. Given the limitations of using autologous

bone, a bone morphogenetic protein-2 (BMP2)-coprecipitated, layer-by-layer assembled

biomimetic calcium phosphate particles (BMP2-cop.BioCaP) has been developed as a

potential osteoinducer. In this study, we hypothesized that BMP2-cop.BioCaP could

introduce osteoinductivity to BCP and so could function as effectively as autologous bone

for the repair of a critical-sized bone defect.

Materials and methods: We prepared BMP2-cop.BioCaP and monitored the loading and

release kinetics of BMP2 from it in vitro. Seven groups (n=6 animals/group) were

established: 1)Empty defect; 2)BCP; 3) BCP mixed with biomimetic calcium phosphate

particles (BioCaP); 4) BCP mixed with BMP2-cop.BioCaP; 5) BioCaP;6)

BMP2-cop.BioCaP; 7) BCP mixed with autologous bone. They were implanted into 8mm

diameter rat cranial critical-sized bone defects for an in vivo evaluation. Autologous bone

served as a positive control. The osteoinductive efficacy and degradability of materials

were evaluated using micro-CT, histology and histomorphometry.

Results: The combined application of BCP and BMP2-cop.BioCaP resulted in significantly

more new bone formation than BCP alone. The osteoinductive efficacy of

BMP2-cop.BioCaP was comparable with the golden standard use autologous bone.

Compared with BCP alone, significantly more BCP degradation was found when mixed

with BMP2-cop.BioCaP.

Conclusion: The combination of BCP and BMP2-cop.BioCaP showed a promising

potential for guided bone regeneration clinically in the future.

Keywords: Animal experiments; Biomaterials; Guided bone regeneration; Growth factor

Introduce osteoinductivity to BCP by a new osteoinducer

13

1. Introduction

The major concern in oral implantology is often the presence of insufficient alveolar bone

to facilitate the insertion of dental implants according to planned prosthetic position. Recent

development in guided bone regeneration has made it possible to install dental implants in

regions which were previously considered unsuitable due to presence of insufficient bone.

Various grafting materials, including autografts, allografts, xenografts, and synthetic

materials, have been used in implant dentistry for guided bone regeneration, however with

varying degree of success [1]. Although autografts are regarded as the “gold standards” for

bone regeneration [2, 3], their application is limited due to the low availability as well as

pain and morbidity from the donor site [4]. Synthetic calcium phosphate (CaP)-based

materials are therefore used clinically for the repair of bone defects [5, 6]. Biphasic calcium

phosphate (BCP), a mixture of hydroxyapatite and β tricalcium phosphate, is one of the

most commonly used synthetic CaP materials due to its excellent osteoconductivity [7-9]－

the ability to facilitate the migration and proliferation of osteoblasts and progenitor cells

[10]. In the progress of bone regeneration, growth factors are further needed to differentiate

progenitor cell into osteoblasts and subsequently to form new bone. Basing on this theory,

there is an increasing interest in combining osteogenic and/or vasculogenic growth factors

with osteoconductive materials to enhance bone regeneration. This kind of materials are

considered to possessing osteoinductivity－ the ability to induce progenitor cells to

differentiate down osteogenic lineages [10].

To endowing materials with osteoinductivity, bone morphogenetic protein-2 (BMP2) is an

often used osteogenic growth factor due to not only its powerful capacity of inducing

osteogenesis but also its saftey. Its application has been approved by the Food and Drug

Administration (FDA). The positive influence of BMP2 on bone regeneration has been

shown to induce bone formation in animal studies [11, 12] and clinical trials [13, 14].

However, the present way of delivering BMP2 clinically, the superficial adsorption of

BMP2 onto bone filling materials [15], causes burst release and consequently the transient

high local concentration of BMP2. This kind of delivery of BMP2 is often associated with

various potential side effects such as an excessive stimulation of bone resorption and the

induction of bone formation at unintentional sites [16]. To maximize its osteoinductivity,

BMP2 needs to be continuously delivered to target sites at a low concentration [17]. Our

research group has recently developed BMP2-coprecipitated layer-by-layer assembled

biomimetic calcium phosphate (BioCaP) particles (BMP2-cop.BioCaP). It could serve as an

independent osteoinducer when mixed with osteoconductive biomaterials [18]. BMP2 was

incorporated into the outermost layer of an inorganic crystalline latticework of

BMP2-cop.BioCaP. Very low doses of the incorporated BMP2 were released slowly and

steadily as the BMP2-cop.BioCaP particles underwent degradation at the site of the

implantation. These low doses (at μg level) were proved to induce bone formation

Chapter 2

14

efficiently in a pro-fibrotic environment and to suppress the foreign body reaction to a

clinically-used bone substitute in rat ectopic sites [18]. Compared with the high dose (at mg

level) of BMP2 used clinically (INFUSE® Bone Graft, 1.5mg BMP-2 per ml collagen

sponge), the BMP2-cop.BioCaP granules (about 150μg BMP2 per ml granule) achieved a

satisfactory bone regeneration with 10 times less BMP2. BMP2-cop.BioCaP was proved to

be not only an effective, but also an efficient method to deliver BMP2 for the repair of

critical-sized bone defects.

In the present study, we combined BMP2-cop.BioCaP with BCP to heal critical-sized

cranial defects in rats. The bone regeneration was evaluated 4 and 12 weeks after the

operation using micro-CT, histological and histomorphometrical analysis. Our hypothesis

was BMP2-cop.BioCaP could introduce osteoinductivity to BCP and it could function as

effectively as autologous bone for the repair of critical-sized bone defects.

2. Materials and Methods

2.1 Study design

Biomimetic calcium phosphate (BioCaP) and BMP2-coprecipitated layer-by-layer

assembled biomimetic calcium phosphate (BMP2-cop.BioCaP) granules were prepared and

evaluated both in vitro and in vivo. BioCaP and BMP2-cop.BioCaP were characterized in

vitro by using confocal laser scanning fluorescence microscopy (CLFM) and scanning

electron microscopy (SEM). The loading and release kinetics of BMP2 in each group was

also assessed. For the in vivo experiments, we adopted a rat critical-sized cranial defect

model for assessing the formation of bone tissue and degradation of the materials assessed

using micro-CT evaluation, histological and histomorphometrical analysis.

2.2. Fabrication of BioCaP and BMP2-cop.BioCaP granules

The BioCaP granules were prepared using the well-established protocol developed by our

group[18]. Briefly, 2000ml 5-fold-concentrated simulated body fluid (684mM NaCl,

12.5mM CaCl2·2H2O, 5mM Na2HPO4.2H2O, 21mM NaHCO3, 7.5mM MgCl2.6H2O

(Sigma, St. Louis, USA)) was incubated for 24 hours at 37°C to obtain amorphous calcium

phosphate (ACP) particles which served as a core of the particles. Subsequently, crystalline

octacalcium phosphate (OCP) was deposited on the core. This was produced by immersing

ACP granules into 1000 ml supersaturated calcium phosphate solution (40mM HCl, 4mM

CaCl2.2H2O, 2mM Na2HPO4·2H2O(Sigma, St. Louis, USA)), which was buffered to pH 7.4

with 50mM TRIS, for 48 hours at 37°C. ACP layer and OCP layer were obtained alternately

three times to obtain the final granules. To produce BMP2-cop.BioCaP, the final crystalline

layer was functionalized by the incorporation of BMP2 (INFUSE® Bone Graft, Medtronic,


15

Minneapolis, MN, USA) into this solution at a concentration of 2 μg/ml. The entire

procedure was conducted under sterile conditions.

2.3 Characterization of BioCaP and BMP2-cop.BioCaP granules

Structure characterization of BioCaP using CLFM

To investigate the inner structure of BioCaP granules, bovine serum albumin (BSA)

labelled with fuorescei-isothiocyanate (FITC-BSA, siga, St. Louis MO, USA) (0.1mg/ml)

was coprecipitated into ACP layer and Rhodamine B (0.1mg/ml) was co-precipitated into

OCP layer. After freeze drying, the granules were embedded in methyl methacrylate.

600μm-thick sections were prepared and affixed to Plexiglas holders. These sections were

then ground down to a thickness of 80 mm for an inspection in a fluorescence microscope.

Surface characterization of BioCaP and BMP2-cop.BioCaP using scanning SEM

The morphology of BMP2-cop.BioCaP granules was evaluated using a scanning electron

microscope (XL 30, Philips, the Netherlands). For this purpose, samples of the material

were mounted on aluminum stubs and sputtered with gold particles to a thickness of

10-15nm.

2.4 Quantification of the amount of the incorporated BMP2

The amount of incorporated BMP2 was determined using a commercially available

enzyme-linked immunosorbent assay (ELISA) kit (PeproTech, London, UK). 0.05g of

BMP2-cop.BioCaP (n=6) was dissolved in 1ml 0.5M EDTA (pH 8.0) and vortexed twice

for 5 minutes. The supernatants were withdrawn for analysis of the total loading of BMP2.

2.5 Release kinetics of BMP2 from BMP2-cop.BioCaP

To study the release kinetics of BMP2 from BMP2-cop.BioCaPgranules, FITC-BSA was

used as cost effective alternative for BMP2. Previous studies have indicated the similarity

between the release kinetics of BSA and BMP2 [19-21]. Six samples of FITC-BSA-cop

BioCaP were incubated in a sealed 10ml glass tubes containing 2ml of phosphate-buffered

0.9% saline (pH 7.4). The tubes were incubated for up to 35 days in a shaking waterbath at

37°C (60agitations/min). The supernatant of each sample was withdrawn for

spectrophotometric analysis in a fluorimeter (Spectrama M2, Molecular Devices, CA,

USA).The temporal release of FITC-BSA was expressed as a percentage of the total

amount that had been coprecipitated into the crystalline layer of the BioCaP.

2.6 Experimental groups for animal study

Chapter 2

16

A rat cranial defect (8 mm in diameter), which was considered as a critical-sized bone

defect [22] was used for the experimental animal model in this study. Seven groups (n=6)

were established for treating critical-sized bone defects (Table 1).

A) Empty defect (Blank control)

B) BioCaP alone

C) BCP alone (Negative control for the effects of BMP2-cop.BioCaP)

D) BMP2-cop.BioCaP alone

E) BCP mixed with BioCaP (Negative control for the effects of BMP2)

F) BCP mixed with BMP2-cop.BioCaP (Experimental groups)

G) BCP mixed with autologous bone (Positive control)

Straumann® BoneCeramic™ (Straumann, Basel, Switzerland) was applied as BCP. In

group C and D, volume ratio of BCP to BioCaP/BMP2-cop.BioCaP was 4:1. 0.28ml of

BCP (size: 0.25-1.00mm) and 0.07ml of BioCaP/BMP2-cop.BioCaP (size: 0.25-1.00mm)

per sample were placed into 1ml Eppendorf tube and homogenously vortexed. Autologous

bone in group G was harvested from the cranial defects in rats, ground to chips and then

mixed with BCP under sterile conditions. The amount of the materials in each group and

the corresponding volume ratio was determined as in our previous study [18].

Table 1. Composition and BMP2 loading dose of groups

2.7 Surgical procedure

Fourty two male Sprague-Dawley rats (12 weeks and weighing~500g) were randomly

divided into 7 groups mentioned above at each time point. In total, 84 rats were used for

two time points in the animal experiments, 4 or 12 weeks after surgery. The animal care

was performed in accordance with the guidelines of the Ethical Committee of Zhejiang

Chinese Medical University. All animal experiments were carried out according to the ethic

laws and regulations of China. Critical-sized cranial defects (8 mm in diameter) were

created in these rats [22]. Briefly, the rats were anaesthetized with an intraperitoneal

injection of pentobarbital (Nembutal 3.5 mg/100 g). A subcutaneous injection of 0.5 ml of 1%

lidocaine as a local anesthetic was given along the sagittal midline of the skull. Following

this, a sagittal incision was made over the scalp from the nasal bone to the middle sagittal

Groups Abbreviation Volume of BCP(ml) Volume of BioCaP with/without BMP2(ml) BMP2 loading dose (μg)

Empty defect Empty defect - - -

BioCaP alone BioCaP - 0.35 -

BCP alone BCP 0.35 - -

BCP mixed with BioCaP BCP+BioCaP 0.28 0.07 -

BMP2-cop.BioCaP alone BMP2-cop.BioCaP - 0.35 51.13 ± 9.68

BCP mixed with BMP2-cop.BioCaP BCP+ BMP2-cop.BioCaP 0.28 0.07 10.29 ± 1.94

BCP mixed with autologous bone BCP+autologous bone 0.28 0.07* -

* 0.07ml autologous bone, instead of BioCaP


17

crest and the periosteum was dissected (Fig. 1A). The 8mm defect was created using a

dental surgical drill with a trephine which was constantly cooled with sterile saline solution

(Fig. 1B and C). Subsequently, the calvarial disk was carefully removed to avoid tearing the

dura (Fig. 1D). After thoroughly rinsing with physiological saline to wash out any bone

fragments (Fig. 1E), samples from various groups were implanted randomly into these

defects (Fig.1F). Afterwards, a collagen membrane (Bio-Gide®, Geistlich Biomaterials,

Wolhusen, Switzerland) was used to cover the defect (Fig. 1G). The periosteum and the

scalp were closed in layers with interrupted 4-0 Vicryl resorbable sutures (Fig. 1H). The

rats were sacrificed with an overdose of Pentobarbital® (Merck, Darmstadt, Germany) 4

and 12 weeks after the operation, and samples with the surrounding tissues were taken.

Fig 1. Surgical procedure: 8mm cranial defect was created with intersection of sagittal suture and coronal suture as

center of round defect

2.8 Micro-CT evaluation

Samples with surrounding tissues were fixed chemically, and embedded in

methylmethacrylate (MMA) as previously reported [23]. After MMA got hardened, samples

were fixed in synthetic foam and placed vertically in a polyetherimide holder and scanned

at a 18μm isotropic voxel size, 70kV source voltage and 113μA current using a high

resolution micro-CT system (μCT 40, Scanco Medical AG, Bassersdorf, Switzerland). Grey

values, depending on radiopacity of the scanned material, were converted into the

corresponding degrees of mineralization by the analysis software (Scanco Medical AG)[24].

They were used to distinguish BCP, BioCaP/BMP2-cop.BioCaP and newly formed bone as

well as to measure their volumes.

2.9 Histological and histomorphometrical analysis

By applying a systematic random sampling strategy [25], samples were sawn vertically to

sagittal suture of rats into 6 or 7 slices of 600 μm thickness 1 mm apart. Slices of each

Chapter 2

18

sample were mounted on Plexiglas holders and polished. Subsequently, the surfaces of the

slices were stained with McNeal’s tetrachrome, basic fuchsine, and toluidine blue O.

Images were recorded in a stereo-microscope at a final magnification of 30 times and

printed in color for the histomorphometric analysis of various parameters. The volume of

the newly formed bone and the remaining material were determined stereologically from its

area density on tissue sections using the point counting technique [26]. The space within the

original bone defect was taken as the reference space [26]. The volume density of the newly

formed bone in each group was calculated as the new bone area in the defect divided by

corresponding reference area of each sample. There were two kinds of new bone observed

in the histological sections. The new bone found on the periphery of the bone defect in

contact with the host bone was named as osteoconductive bone, while the new bone found

in the center of the bone defect, without any contact with the host bone, was named as

osteoinductive bone (Fig. 2). Their volume density was again calculated as their area in the

defect divided by the corresponding reference area of each sample. The relative volume of

the remaining BCP granules was estimated by dividing the absolute volume of the

remaining BCP by the corresponding original volume of the BCP. The original volume of

BCP in group of pure BCP (0.35 ml) was considered as 1 unit, and the original volume of

BCP in other group (0.28ml) was considered as 0.8 unit.

Fig 2. Representative histological sections (A) of osteoconductive bone (Blue frame) and osteoinductive bone

(Yellow frame). The histological sections were stained with McNeal’s Tetrachrome, basic Fuchsine and Toluidine

Blue O. The schematic diagram is shown in B.

The volume density of the foreign body giant cell (FBGC) was determined to evaluate the

foreign body reaction to the BCP in the different groups. Due to small size of the FBGCs, a

higher magnification was needed for point counting strategy. About 20 images of each

sample were recorded at a final magnification of 320 times with a Leica DMRA microscope

(Leica, Wetzlar, Germany) and printed in color for the histomorphometric analysis.


19

Multinucleated giant cells on surface of the BCP were counted as FBGC and its volume

density was normalized to the corresponding volume density of BCP.

2.10 Statistical analysis

All data were presented as the mean values with the standard deviation (median ± standard

deviation). Data were compared using the Kruskal-Wallis one-way analysis of variance

with significance level 0.05. Post hoc comparisons were made using Dunn’s test.

3 Results

3.1 Preparation and characterization of BioCaP and BMP2-cop.BioCaP granules

In this study, we produced BioCaP granules using biomimetic layer-by-layer assembling

technique and incorporate BMP2 into the most outer layer of BioCaP granules to obtain

BMP2-cop.BioCaP granules. The CLFM images of cross sections of BioCaP granules

confirmed the layer-by-layer structure in that an amorphous CaP layer (Fig. 3A) and a

crystalline CaP layer (Fig. 3B) were deposited alternately onto each other three times to

form the final granules─BioCaP (Fig. 3C). BMP2-cop.BioCaP granules were seen with a

scanning electron microscope as irregular clusters of microspheres ranging from 100 to

1000μm (Fig. 3D). The outermost of BMP2-cop.BioCaP granules exhibited a uniform

crystalline surface (Fig. 3E). In BMP2-cop.BioCaP granules, the total loading of BMP2 in

0.35cm3 (size of each sample) was 51.13±9.68μg with a co-precipitation rate of 30.1±5.7%.

Protein coprecipitated into BioCaP was released gradually and at a steady rate from the 3rd

day until 35th

day [18].

Fig 3. Laser-scanning fluorescence micrographs of cross-sections BioCaP granules depicting that amorphous (A,

green signal) and crystalline (B, red signal) calcium phosphate were layer-by-layer assembled. The images in A

and B are merged in image C. Scanning electron micrographs depicting the morphology of BMP2-cop. BioCaP

granules (D) and crystalline outer layer of BMP2-cop. BioCaP granule (E).

Chapter 2

20

3.2 Clinical and micro-CT observations

All 84 rats remained healthy and all the surgical sites healed without any complications. No

visual sign of inflammation or adverse tissue reaction was observed. All specimens were

retrieved after implantation periods of 4 and 12 weeks. BCP could be recognized in the

micro-CT examination because its mineralization was significantly higher than in the rest

of the material or tissue. Newly formed bone and BioCaP/BMP2-cop.BioCaP can hardly be

distinguished from one another because their grey values were too close (Fig. 4).

Fig 4. Micro-CT images and corresponding histological images of group containing only BioCaP (A, a) and group

containing BCP with BMP2-cop.BioCaP (B, b). In micro-CT image, BCP (white arrow) showed highest grey value

due to its strongest radiopacity; BioCaP(yellow arrow) and newly formed bone (blue arrow) showed quite similar

grey values which can hardly be distinguished the micro-CT machine.

3.3 Histological results

An empty defect was not completely healed even 12 weeks after implantation, which

proved that the 8mm diameter rat cranial defect was a critical-sized defect (Fig. 5 A, a).

In the groups with BCP alone or with BCP mixed with BioCaP, bone formation was found

along the borders of the bone defect, which was considered as osteoconductive bone. After

4 weeks, it showed as deep pink stained woven bone (Fig. 5B, 5C), while after 12 weeks,

the new bone had grown into lamellar bone and light pink stained. BCP granules were

distributed uniformly in the bone defect and no significant degradation of the granules was

seen after 12 weeks (Fig. 5b, 5c).

When BCP was mixed with BMP2-cop.BioCaP, a higher amount of new bone was

observed as compared to other groups. After 4 weeks, both osteoconductive and

osteoinductive bone were observed in the defect (Fig. 5D). After 12 weeks, these two kinds

of new bone had merged into bone bridge, which fulfilled most of the defect (Fig. 5d). In

the area of new bone, BCP granules were encapsulated by newly formed bone. The outline

of the embedded BCP granules was not as sharp as for the original granules. The granules


21

were gradually degraded and replaced by new bone.

In group of BioCaP alone, the amount and distribution of new bone formation was found to

be similar to that of the empty defect. After 4 weeks, BioCaP granules were distributed

uniformly in the defect (Fig. 5E). After 12 weeks, no BioCaP granules were observed any

more (Fig. 5e).

In group of BMP2-cop.BioCaP alone, osteoconductive and osteoinductive bone were both

found after 4 (Fig. 5F) and 12 weeks (Fig. 5f). They were similar to those found the group

of BCP mixed with BMP2-cop.BioCaP, but with a relatively smaller volume.

BMP2-cop.BioCaP particles cannot be found any more 4 weeks after the operation.

In the group of BCP mixed with autologous bone, obvious osteoconductive bone and a

relatively small piece of osteoinductive bone were observed at 4 weeks. Pieces of

autologous bone cannot be found anymore (Fig. 5G). After 12 weeks, osteoinductive bone

had merged into a bigger block of new bone. It decreased dramatically after 12 weeks

compared the amount of BCP on the 4 week histological sections (Fig. 5g).

In summary, in all the groups without BMP2 or autologous bone, only osteoconductive

bone was observed. The amount of osteoconductive bone did not increase with time, but the

structure of it became more mature. In the groups with BMP2 or autologous bone, not only

osteoconductive bone but also osteoinductive bone were found in the defects and more

newly formed bone could be expected in comparison with groups without BMP2.

Chapter 2

22

Fig 5. Representative histological images of each group after 4 and 12 weeks at a low magnification (30×).The

sections were stained with McNeal’s Tetrachrome, basic Fuchsine and Toluidine Blue O.

3.4 Histolomorphometric results

New bone formation

The descriptive measurements of the total new bone, including osteoconductive bone and

osteoinductive bone, 4 and 12 weeks after implantation was shown in table 2. Itrevealed

that the volume density of the newly formed bone was significantly higher when BCP was

mixed with BMP2-cop.BioCaP compared with BCP alone after 4 weeks and 12 weeks. The

volume density of newly formed bone was the same when BCP was mixed with

BMP2-cop.BioCaP or with autologous bone. The volume density of the newly formed bone

in the group with BMP2-cop.BioCaP was significantly less at both 4 and 12 weeks

compared to group of BCP mixed with BMP2-cop.BioCaP (Fig. 6).


23

Table 2. Descriptive measurements of volume density (mm3/mm3) of total newly formed bone, including

osteoconductive and osteoinductive bone, in all groups at 4 and 12 weeks after implantation (Median±Standard

deviation).

Fig 6. Histomorphometric measurements of volume density of total newly formed bone including osteoconductive

bone and osteoinductive bone at 4 (A) and 12 (B) weeks after implantation. Values are shown as median± standard

deviation. * p < 0.05, indicating significant difference between groups at the same time point; # p < 0.05,

indicating significant difference between 4 weeks and 12 weeks of the same group.

The amount of newly formed bone displayed different growth profiles with time. When the

bone defect was filled with BCP alone, the newly formed bone did not increase much from

4 weeks to 12 weeks. Whereas, BCP mixed with BMP2-cop.BioCaP induced a statistically

significant increase in bone formation after 12 weeks compared with after 4 weeks. A

similar phenomenon was also observed in positive control group of BCP mixed with

autologous bone. As mentioned above, the new bone was divided into two types namely

osteoconductive and osteoinductive bone. The amount of osteoconductive bone did not

increase with time (Fig. 7A). On the other hand, the amount of osteoinductive bone did

increase with time in the groups with BMP2-cop.BioCaP or with autologous bone (Fig. 7B).

To sum up, the amount of new bone in the group with BCP alone did not increase because

only osteoconductive bone formed, which did not show an obvious increase with time. In

contrast, when defects were implanted with BCP mixed with BMP2-copBioCaP, both

osteoconductive and osteoinductive bone were observed and a significant increase in

Osteoconductive bone Osteoinductive bone Total new bone Osteoconductive bone Osteoinductive bone Total new bone

Empty defect 0.04±0.01 - 0.04±0.01 0.04±0.01 - 0.04±0.01

BioCaP 0.05±0.02 - 0.05±0.02 0.06±0.02 - 0.06±0.02

BCP 0.23±0.04 - 0.23±0.04 0.25±0.05 - 0.25±0.05

BCP+BioCaP 0.24±0.04 - 0.24±0.04 0.27±0.06 - 0.27±0.06

BMP2-cop.BioCaP 0.18±0.03 0.08±0.02 0.26±0.07 0.19±0.03 0.15±0.06 0.38±0.04

BCP+BMP2-cop.BioCaP 0.30±0.02 0.17±0.03 0.43±0.04 0.30±0.04 0.28±0.04 0.58±0.05

BCP+autologous bone 0.30±0.02 0.15±0.03 0.44±0.04 0.30±0.04 0.26±0.04 0.59±0.02

4 weeks 12 weeks

Chapter 2

24

osteoinductive bone was observed from 4 weeks to12 weeks.

Fig 7. Histomorphometric measurements of volume density of osteoconductive bone (A) and osteoinductive bone

(B) at 4 and 12 weeks after implantation. Values are shown as median± standard deviation. * p <0.05.

Material degradation

The degradation of BCP was associated with the bone formation. BCP did not degrade

much when it was implanted alone or mixed with BioCaP. These were the groups with an

unfavorable new bone formation. When it was mixed with BMP2-cop.BioCaP or

autologous bone, more new bone formation and more BCP degradation were observed

simultaneously. This phenomenon was even more obvious after 12 weeks (Fig. 8A).

At 4 weeks, the volume density of BioCaP in the group containing BioCaP alone was

0.06±0.02 mm3/mm

3. Interestingly, BioCaP was hardly found in the group of BCP mixed

with BioCaP. Whereas, BMP2-cop.BioCaP degraded completely no matter if it was with or

without BCP. After 12 weeks, no BioCaP could be observed in groups with BioCaP alone or

with BCP mixed with BioCaP.

Foreign body reaction

The FBGCs at 12 weeks were too few to count. Therefore, only data at 4 weeks were

obtained (Fig. 8B). The volume density of FBGCs to BCP was significantly lower when

BCP was mixed with BMP2-cop.BioCaP than it was either alone or mixed with BioCaP. No

statistically significant differences were found between BCP mixed with


25

BMP2-cop.BioCaP and autologous bone.

Fig 8. Histomorphometric measurements of remaining BCP granules at 4 and 12 weeks after implantation (A) and

volume density of FBGC at 4 weeks after implantation (B). Values are shown as median± standard deviation.

* p <0.05.

4 Discussion

The aim of this study was to investigate if BMP2-cop.BioCaP could replace autologous

bone by functioning as an effective osteoinducer for BCP and enhancing bone regeneration

in a critical-sized bone defect. Our results supported the positive effect of

BMP2-cop.BioCaP on bone formation. BCP mixed with BMP2-cop.BioCaP induced more

new bone in rat cranial critical-sized defects than BCP alone and it was as effective as BCP

mixed with autologous bone. It indicated that BMP2-cop.BioCaP could be considered as an

effective method of introducing osteoinductivity into bone substitutes. Doctors and dentists

would not have to rely on autologous bone and the complications caused by autologous

bone transplantation could be avoided. What’s more, the outstanding benefit of this

osteoinducer was that it could be mixed with any commercially available bone substitute

granules without altering their original property. Thus BMP2-cop.BioCaP could be made

into an off-the-shelf product. Compare to other approaches of conferring bone substitutes

with osteoinductivity, adding BMP2-cop.BioCaP to clinically available bone substitutes

should be easier for clinical application.

BMP2-cop.BioCaP is a kind of biomimetic calcium phosphate granule on which an

amorphous calcium phosphate layer and a crystalline calcium phosphate layer are deposited

alternately on the granule. When these two kinds of calcium phosphate layers were stained

with different fluorescent dyes, their emerged confocal images showed the two overlapping

Chapter 2

26

fluorescent colors (Fig. 3C), which confirmed the layer-by-layer structure of

BMP2-cop.BioCaP granules.

Rat cranial critical size defect (8mm in diameter) [22] was used as an implantation model to

evaluate the osteoinductive efficacy of materials. This kind of preclinical testing has been

widely used for large skeletally mature animals including dogs, goats, sheep and pigs.

Compared to rodents, these animals were considered to have more similar structure and

composition of bone tissue as humans. However, there was no substantiation that one

model was proved to be better than any other when it was used to address clinical

translation of assayed therapeutic approaches [27]. What is more, rats are inexpensive, easy

to house and to manipulate. In addition, the reduced lifespan of rats avoids the influence of

animals’ ageing on the bone metabolism and regeneration processes.

BMP2-cop.BioCaP improved bone formation because it could induce osteoinductive bone

that was new bone formed in the center of bone defect. The formation of osteoinductive

bone produced a new bone regenerating center besides the one at the periphery of host bone.

According to the understanding of bone defect healing process in animals, bone

regeneration starts from the borders of the defect, moves centripetally to fill the defect and

so tends to heal the defects finally [28]. This does not happen for critical sized bone defects

and the growth to the center stops at a certain point. This phenomenon could be observed in

the empty defect in our study. To some extent, BCP filled in the defect enhanced the healing

but it did not change the conventional healing pattern. Whereas, the addition of

BMP2-cop.BioCaP altered the healing by introducing a new center of regenerating

bone—osteoinductive bone. Consequently, the two centers of bone regeneration accelerated

and enhanced the healing of the defect.

The efficacy of BMP2-cop.BioCaP in improving bone formation was comparable with the

most often used osteoinducer in clinical practice─autologous bone. Although the

distribution, composition and the amount of new bone in these two groups were similar as

the histological sections showed, the mechanism of osteoinductive bone formation in these

two groups was quite different. Autologous bone is osteogenic, including hydroxyapatite

functioning as scaffold, osteogenic cells and a bunch of bioactive agents, which could form

new bone by itself. Therefore it can be applied to almost all bone defects independently or

combined with other bone grafting materials. Whereas, BMP2-cop.BioCaP needed an extra

resource of osteogenic cells and scaffold to heal the critical-sized bone defect. Therefore,

although our results support the use of BMP2-cop.BioCaP for replacing autologous bone,

further studies with other animal models and human beings are still needed to evaluate this

hypothesis.

Osteoinductive bone was able to grow on its own even in the absence of BMP2. According


27

to our results, BMP2-cop.BioCaP stimulated new bone formation within 4 weeks. After 4

weeks, the BMP2-cop.BioCaP particles were completely degraded. Theoretically it could

not then stimulate bone formation any more. However, we found osteoinductive bone

growing bigger from 4 weeks to 12 weeks, indicating the ability of the osteoinductive bone

to grow by itself. But it could not heal the complete bone defect even after 12 weeks, which

contradicted other studies using the same animal model [29]. There could be two possible

explanations for the unhealed bone defects in the present study: one was that osteoinducer

(BMP2-cop.BioCaP) only needed to form osteoinductive bone at the early stage of bone

regeneration to stimulate bone regeneration. Even after the BMP2-cop.BioCaP was

degraded, the existence of osteoinductive bone was enough to completely heal the defect

since it could grow on its own. The incomplete healing was only because the observing

period was not long enough; the other one was that during the entire healing period the

existence of osteoinducer was needed, the BMP2-cop.BioCaP particles degraded too fast to

ensure a complete healing of the defect. To find the real reason we will need to study extra

groups of BMP2-cop.BioCaP with a slower degradation profile or groups of BioCaP loaded

with more BMP2 and to measure for a longer time to assess if the results will be different

from the current ones.

The sustained release of BMP2 guarantees that BMP2-cop.BioCaP functions as an effective

osteoinducer [30]. It has been proved in vitro that protein coprecipitated into BioCaP was

released gradually and at a steady rate after the 3rd

day until the 35th day, at which juncture

the initial depot had been depleted by no more than 50.1% [18]. Although there are no

direct in-vivo supporting experiments, it is logic to speculate that the release of BMP2 from

BMP2-cop.BioCaP in vivo would be slow and sustained, because BMP2-cop.BioCaP is

derived from biomimetic calcium phosphate coating and they used the same technique to

carry BMP2－incorporating BMP2 into the inorganic lattice network work of crystalline

calcium phosphate. Our previous experiment proved that calcium phosphate coating

degraded slowly in vivo and BMP2 was liberated as the crystalline calcium phosphate

degrades [23]. Titanium alloy discs with a biomimetic BMP2-incorporated calcium

phosphate coating were implanted subcutaneously in rats and retrieved for

histomorphomtrical analysis at 7 day intervals over a period of 5 weeks. From the first to

the fifth week, volume of coating remaining was 9.22, 9.21, 5.78, 5.54, 2.04 mm3. It proved

directly that the coating degraded slowly and supported indirectly the in vivo slow release

of BMP2 from BMP2-cop.BioCaP. Compared with the degradation profile in vitro, the

degradation of BMP2-cop.BioCaP in vivo was much faster due to the acceleration by many

types of cells (e.g. fibroblasts, monocytes/macrophages) through phagocytotic mechanism

[31].

A bone substitute should have proper degradation profile to match the bone growth rate

[32]. It cannot degraded too fast since it has to function as a scaffold for the osteoblast to

Chapter 2

28

attach, proliferate, differentiate and finally form new bone. Also it cannot degrade too

slowly since the newly formed bone needs finally to replace it. As a bone substitute, BCP

does not degrade easily. It was reported that 26.6% of BCP granules were still not degraded

after 6 to 8 months in a treated maxillary sinus in a clinical trial [33], which needed to be

accelerated. The present study showed that in the group with BCP alone, the volume of

BCP did not change much even after 12 weeks. Significantly more BCP granules degraded

in the groups containing BCP mixed with BMP2-cop.BioCaP or autologous bone than in

the group with BCP alone. This indicated that BMP2-cop.BioCaP stimulated the

degradation of BCP because in the process of osteogenesis, BCP served as a scaffold and a

resource of calcium and phosphate ions for the new bone formation. With osteogenesis

going on, BCP would be gradually degraded and replaced by new bone. The more actively

the bone regenerated, the more BCP granules were degraded [34].

One major concern of biomaterials is their biocompatibility. Unfavorable biocompatibility

is often characterized by a foreign body reaction to biomaterials [35, 36], which

histologically is seen by the local accumulation of macrophages, their fusion to form

FBGCs, and the deposition of dense fibrous connective tissue. Therefore, the FBGCs shown

in histological sections could be a reliable parameter to evaluate the foreign body reaction.

Some studies [23, 37] applied tartrate resistant acid phosphatase (TRAP) reaction to

recognize osteoclasts (TRAP-positive) and subtracted them from the total of multinucleated

giant cells found on the histological images to calculate the number of FBGCs. The

undecalcified sections we used in this study were not optimal for the histochemical

demonstration of TRAP reactivity. The poor staining reactivity could potentially raise the

possibility that the population of TRAP-negative cells included not only FBGCs but also

osteoclasts registering falsely negative. Therefore, TRAP staining was not applied for

evaluating foreign body reaction in the present study. Instead we counted multinucleated

giant cells on the surface of BCP as FBGC to evaluate the foreign body reaction caused by

different materials. This was based on the theory that FGBC should appear on the surface of

foreign material and that the other multinucleated giant cells on the newly formed bone are

osteoclasts for bone remodeling. In the present study, we recognized that the volume ratio

of FBGCs to BCP was significantly lower in the group with BCP mixed with

BMP2-cop.BioCaP as compared to that in the group of either BCP alone or BCP with

BioCaP. It showed that BMP2-cop.BioCaP was not only able to induce bone formation

efficiently but it could also inhibit the host foreign body reaction to BCP. The inhibition to

foreign body reaction was probably caused by the extensive osteogenesis [38]. During bone

regeneration the level of osteopontin increases. Furthermore, osteopontin was previously

shown to suppress the fusion of macrophages into FBGCs both in vitro and in vivo [39].

Therefore, it was reasonable to speculate that the extensive osteogenesis, stimulated by

BMP2-cop.BioCaP, helped to inhibit foreign body reaction.


29

BMP2-cop.BioCaP could function as an effective osteoinducer to introduce

osteoinductivity to BCP, however it could hardly be applied alone as a bone substitute

material. The result of new bone formation in group with BMP2-cop.BioCaP confirmed this

speculation. This was because if the CaP-based materials degraded too quickly, it could not

function as scaffold long enough for the osteoblasts to form new bone. Apparently, 4 weeks

was too short for BMP2-cop.BioCaP to heal critical-sized bone defect. To optimize the

degradation rate of BMP2-cop.BioCaP, it may be helpful to understand the possible factors

involved: Firstly, the incorporation of BMP2 played a role in the on degradation of BioCaP

granules. We found that the degradation rate of BMP2-cop.BioCaP increased significantly

in comparison with BioCaP. Interestingly, the suppression of FBGCs to CaP coatings in the

presence of coprecipitated BMP2 could be found from 2 to 3 weeks [23]. Therefore, the

degradation of BMP2-cop.BioCaP could hardly be attributed to phagocytic activity. In

contrast, mineralization mediated by osteoblasts may possibly affect its degradation. This

mineralization process generated many protons [40]. Therefore, an extra cellular buffering

system was needed to neutralize them so as to prevent their accumulation, otherwise it

would influence the activity of osteoblasts negatively. Calcium and phosphate ions

dissolved from BMP2-cop.BioCaP and BioCaP granules could directly neutralized the

protons. The more osteogenesis there was, the more the protons were generated and the

more calcium and phosphate ions were needed to neutralize them. As we learnt from results,

BMP2-cop.BioCaP showed more osteogenic efficacy than BioCaP. It degraded faster to

release more calcium and phosphate ions to neutralize protons. Secondly, the mixture with

BCP did not affect significantly the degradation rate of either BioCaP or BMP2-cop.BioCaP,

which suggested that the degradation of BMP2-cop.BioCaP was not impacted by the

materials used for bone grafting. Thirdly, the impact of the environment on the degradation

rate of BMP2-cop.BioCaP and BioCaP granules should also considered. In the present

study, the BMP2-cop.BioCaP completely degraded after 4weeks in a rat cranial bone defect.

A previous study showed that BMP2-cop.BioCaP had not been completely degraded during

a 5 week period at an ectopic site[18]. The granules may result in a faster degradation in the

orthotopic site compared with the ectopic site. The possible reasons for this could be that

compared with a connective tissue environment, the osteogenic environment was more

suitable for bone formation. Enhanced bone formation needed more calcium and phosphate

ions from BioCaP degradation.

BMP2-cop.BioCaP was based on the technique of producing lay-by-layer assembled

calcium phosphate carrier loaded with BMP2. These BioCaP particles could also be

incorporated with other proteins to serve as a carrier for the slow release of those proteins.

Given this valuable property, we can add many specific functions by coprecipitating with

different agents, such as those against inflammation and cancer.

4 Conclusion

Chapter 2

30

With the limitation of the animal model and the timeframe used in this study, we conclude

that BMP2-cop.BioCaP successfully introduced osteoinductivity to BCP and function as

effective as the golden standard－autologous bone. Our findings further showed that the

excellent osteoinductivity of BMP2-cop.BioCaP resulted from its slow and sustained

delivery of BMP2, BCP combined with BMP2-cop.BioCaP has shown a promising

potential for guided bone regeneration clinically in the future.

5 Acknowledgements

We would like to thank Prof. Tony Hearn for his scientific input and English editing as a

native speaker for this publication. This project was supported by ITI Research Grant (No.

836_2012).


31

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Chapter 2

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35

Chapter 3

A novel bone-defect-filling material with

sequential antibacterial and osteoinductive

properties for repairing infected bone defects

Dongyun Wang, Yuelian Liu, Yi Liu, Liquan Deng, Sebastian A.J. Zaat,

Daniel Wismeijer, Gang Wu

In preparation

Chapter 3

36

Abstract

Objectives: The repair of infected critical-sized bone defects is hindered by both active

infection of residual bacteria and compromised bone regenerative capacity. We recently

develop biomimetic calcium phosphate (BioCaP) granules that can internationally

incorporate and slowly release osteoinductive bone morphogenetic protein-2 (BMP2) to

efficiently induce new bone formation. To provide a viable treatment option for infected

bone defects, we hereby develop a novel bone-defect-filling material with a sequential

release system: burst release of a powerful antibacterial agent-

Hydroxypropyltrimethylammonium chloride chitosan (HACC) and followed by a

controlled release of BMP2. We hypothesize that BMP2-BioCaP/HACC complex can

rapidly kill residual bacteria and thereafter induce new bone formation so as to repair

infected bone defects.

Materials and methods: The minimum bactericidal concentration (MBC) of HACC against

infection-associated bacteria and cytotoxicity of HACC to pre-osteoblasts were both

evaluated to determine the optimal concentration of HACC. The influence of HACC at the

optimal concentration on the BMP2-induced differentiation of pre-osteoblasts was

evaluated by assessing alkaline phosphatase (ALP) activity, osteocalcin (OCN) expression

and mineral nodule formation. Corresponding amount of was then adsorbed on BMP2-

BioCaP granules and air-dried to obtain BMP2-BioCaP/HACC complex. The release

kinetics of HACC and BMP2 were determined using quantitative spectrophotometric

analysis. To evaluate osteoinductivity of BMP2-BioCaP/HACC complex and optimize the

HACC concentration in vivo, the following groups (n=6 samples, 100uL per sample) were

randomly implanted into subcutaneous pockets in rats: 1) BMP2-BioCaP/HACC complex

(BMP2-BioCaP/HACC complex carrying 20, 4 or 0.8μg HACC were respectively named

as BMP2-BioCaP/HACC-20, BMP2-BioCaP/HACC-4, BMP2-BioCaP/HACC-0.8); 2)

BMP2-BioCaP; 3) BioCaP/HACC; 4) BioCaP. Five weeks after implantation, samples were

retrieved for histological and histomorphometric analysis.

Results: The optimal HACC concentration is 40μg/mL, at which HACC can kill bacteria

without harming pre-osteoblasts. 40μg/mL HACC didn’t significantly influence the BMP2-

induced ALP, OCN and mineralization. The in-vitro release profile showed that HACC was

completely exhausted after 3 days, while BMP2 was gradually and slowly released with

about 20% depletion after 30 days. In the in-vivo pro-fibrotic environment (subcutaneous

sites), bone formation was observed only in the BMP2-containing groups. Furthermore, in

comparison with the positive control group (BMP2-BioCaP), BMP2-BioCaP/HACC-4 and

BMP2-BioCaP/HACC-0.8 complex resulted in similar amount of new bone formation,

while BMP2-BioCaP/HACC-20 was associated with significantly less new bone formation.

A novel osteoinductive and antibacterial material

37

Conclusion: BMP2-BioCaP/HACC complex could rapidly eliminate antibiotic-resistant

bacteria and efficiently promote new bone formation both in vitro and in vivo, which

conferred this novel material a promising application potential to repair infected bone

defects.

Keywords: Bone morphogenic protein-2, Hydroxypropyltrimethylammonium chloride

chitosan, Antibacterial activity, Osteoinductivity, Bone repair

1. Introduction

Achieving satisfactory bone regeneration in infected bone defects is a great challenge in the

field of orthopaedic, oral and maxillofacial surgery. The infected bone defects are often

caused by trauma combined with direct contamination. Such acute bone infections

sometimes progress to chronic infections (clinically referred to as osteomyelitis). Routine

systemic antimicrobial therapy is typically sufficient to clear an acute bone infection; but

chronic osteomyelitis can be extremely difficult to treat and requires radical surgical

debridement of the necrotic and infected tissues, followed by extensive systemic

application of antibiotics [1, 2]. This radical debridement process often results in a large

bone and soft tissue defect, where the dead space must be effectively managed to gain

tissue regeneration and so as to reduce the chance of reinfection [3, 4]. If the bone defect is

too large and beyond the intrinsic self-healing capacity, which is considered as critical-

sized bone defects, bone grafting is needed to facilitate bone regeneration. Although

autografts are regarded as “gold standards” for repairing bone defects [5, 6], their

application is limited because of its low availability and donor-site pain and morbidity [7].

Synthetic calcium phosphate (CaP)-based materials are therefore widely adopted to heal

bone defects clinically [8, 9]. This is due to its excellent osteoconductivity [10-12]－the

ability to facilitate the migration and proliferation of osteoblasts and progenitor cells [13].

However, they lack osteoinductivity－the ability to induce progenitor cells to differentiate

down osteogenic lineages [13], which is essential for satisfactory bone regeneration in

critical-sized bone defects. One approach to solving this problem is to introduce

osteoinductive agents into CaP materials, such as bone morphogenetic protein 2 (BMP2).

To maximize its osteoinductivity, BMP2 needs to be delivered to target sites at low

concentration in a sustained manner [14]. Our research group has recently developed

BMP2-incorporated biomimetic calcium phosphate (BMP2-BioCaP) which could slowly

and continuously deliver BMP2. The BMP2-BioCaP granules were proved to be an

osteoinductive bone substitute, which can induce bone formation efficiently in

subcutaneous pocket model in rats [15].

Despite the aggressive tissue debridement, the spatial heterogeneity of bacterial

Chapter 3

38

colonization in the bone and the surrounding tissue makes it impossible to ensure complete

elimination [16]. The residual bacteria release various inflammatory mediators and tissue-

destructive enzymes, which can compromise bone regeneration [17]. Therefore, to gain

favorable bone reconstruction, it is necessary to locally use bactericidal or bacteriostatic

antibiotics to stop bacteria from secreting inflammatory mediators. The most often used

antibiotics are vancomycin, gentamicin and tobramycin. However, their application is more

and more limited due to the emergence of resistance in bacteria in hospitals worldwide by

use and misuse of these conventional antibiotic [18]. On the other hand, a novel water-

soluble chitosan derivative (hydroxypropyltrimethyl ammonium chloride chitosan, HACC)

has attracted much attention due to its strong antibacterial activity, broad spectrum [19, 20]

and very few reported bacterial resistance [21], which made it a promising candidate

antibiotics for treatment of infected bone defects [22].

To optimize the local delivery of antibiotics, much focus has been put on developing

antibiotics-loaded biomaterial system to help eradicate an established bone infection.

Poly(methyl methacrylate) (PMMA) bone cement is the gold standard biomaterial for local

antibiotic therapy in orthopaedics and has been used for over 35 years for both prophylaxis

and therapy. Seminal studies conducted by Buchholz and Engelbrecht in 1970 [23] and

Klemm in 1979 [24] described the use of antibiotic-loaden PMMA to prevent infection and

treat chronic bone infections, respectively. However, PMMA lacks degradability, which is

troublesome due to unfavorable release patterns of the antibiotic as well as necessity of a

second surgery to remove it and to implant bone grafts. The ideal approach to solve this

problem is to develop a biodegradable local biomaterial system, functioning as both carrier

for antibiotics and bone grafts, to not only clear the infection but to also contribute to the

subsequent bone regeneration process [16].

In the present study, to develop a favorable local biomaterial system for the treatment of

infected critical-sized bone defects, we used our previously developed osteoinductive bone

substitute, BMP2-BioCaP granules, as antibiotic carrier. HACC solution was then adsorbed

on them and air-dried to obtain an antibacterial and osteoinductive biomaterial

system─BMP2-BioCaP/HACC complex. It can achieve sequential release of antibiotic and

osteoinductive agent－ a burst release of HACC and following slow and sustained release

of BMP2. We hypothesized that the BMP2-BioCaP/HACC complex could rapidly eliminate

residual bacteria and thereafter induce new bone formation so as to repair infected critical-

sized bone defects.

2. Materials and methods

2.1 Antibacterial assay of HACC


39

Minimum bactericidal concentration (MBC) against methicillin-resistant staphylococcus

aureus (MRSA) were evaluated. MBC of HACC were determined by standardized broth

microdilution techniques with inoculums of 5×105

CFU/mL according to Clinical

Laboratory Standards Institute guidelines and incubated at 35 °C for 24 h [25].

2.2 Cytotoxicity testing of HACC

MC3T3-E1 cells were cultured in alpha-minimal essential medium (α-MEM, Invitrogen

Co. CA) containing 10% fetal bovine serum (FBS) at 37°C in a CO2 incubator (5%

CO2/95% air). MC3T3-E1 cells were sub-cultured at approximately 80% confluence in

every 3 days in 75cm2 flasks.

The cytotoxicity of HACC to MC3T3-E1 cells was assessed using Alamar Blue Assay

(Invitrogen, CA, USA). MC3T3-E1 cells were seeded at 6×104 cells/well in 12-well plates.

After 24 hours of incubation, the culture medium was changed into α-MEM culture

medium containing 1000, 200, 40μg/mL of HACC. Alamar Blue was also added to each

well in an amount equal to 5% (v/v) of the culture medium volume. Cells treated with a-

MEM without HACC served as negative control. The supernatants of each well were

extracted after 6, 24, 48 and 72 hours, and their fluorescence was measured with excitation

at 530 nm and emission at 590 nm.

2.3 Effect of HACC on BMP2-induced differentiation of pre-osteoblasts

To investigate the influence of HACC on BMP2-induced differentiation of MC3T3-E1

cells, they were seeded at 1×105 cells/well in 12-well plates. The cells were firstly treated

with or without 40μg/mL HACC for three days. Subsequently, culture medium was

changed to a-MEM containing 1% FBS with or without 50ng/mL BMP2. Accordingly, four

groups were established as follows:

Alkaline phosphatase (ALP) activity and Osteocalcin (OCN) expression was assessed after

BMP2 treatment for 4 and 7 days. At each time point, supernatants of each well were

harvest to detect OCN level using the OCN enzyme linked immunosorbent assay (ELISA)

Chapter 3

40

kits (Cloud-clone, Houston, TX, USA). Subsequently, cells were rinsed three times with

PBS. 400μL of distilled water was then added to each well to obtain cell debris for ALP

activity by using a p-nitrophenyl-phosphate colorimetric assay. The total protein was

determined with a bicinchoninic acid assay (BCA assay) protein assay reagent kit (Pierce,

Rockford, IL, USA).

To assess mineralized modules formation, alizarin red S staining was used after BMP2

treatment for 28days. The substrates were washed three times with PBS, fixed in 4%

formaldehyde for 15 min and stained with 0.5 mL/well Alizarin Red Stain Solution for 20

min at room temperature. The cell monolayers were then washed with distilled water until

the runoff ran clear. The images were produced using an optical microscope.

2.4 Fabrication of BioCaP and BMP2-BioCaP granules

BioCaP was fabricated by refining a well-established biomimetic mineralization approach

[15]. A CaP solution (200 mM HCl, 20 mM CaCl2·2H2O, 680 mM NaCl, and 10 mM

Na2HPO4) buffered by TRIS (250 mM) to a pH of 7.4. The whole solution was incubated in

a shaking water bath (50agitations/min) at 37°C for 24 hours. Thereafter all precipitations

were retrieved and gently washed by PBS, strongly filtered and compressed to a block

using a vacuum exhaust filtering method with a vacuum filter (0.22-μm pore, Corning, NY,

USA) and an air pump. After drying in air circulation at room temperature overnight, the

hardened block was ground and filtered with metallic mesh filters to obtain granules with a

size of 0.25-0.5mm. The calcium phosphate solution was filtered with the vacuum filter

(0.22μm pore) before buffering for sterilization. All the following procedures were

performed under aseptic conditions.

BMP2 (INFUSE® Bone Graft, Medtronic, USA) can be introduced into the calcium

phosphate solution at a final concentration of 2μg/ml before buffering as mentioned above

and thereafter was co-precipitated into the interior of BioCaP to form BMP2-BioCaP.

2.5 Fabrication and characterization of BMP2-BioCaP/HACC complex

HACC powder was purchased from Lvshen Bioengineering Co., Ltd. (Jiangsu, China) and

sterilized by gamma rays from a cobalt-60 source (Synergy Health, Ede, the Netherlands).

HACC powder was dissolved into distilled water to prepare HACC solution, 30μL of which

was thoroughly adsorbed on the surface of 100uL BioCaP-BMP2 to obtain one sample of

the BMP2-BioCaP/HACC complex (60μg BMP2 for per sample). The BMP2-

BioCaP/HACC complex carrying various amounts of HACC was fabricated to optimize the

HACC content of each sample. The BMP2-BioCaP/HACC complex carrying 20μg, 4μg or

0.8μg was respectively named as BMP2-BioCaP/HACC-20, BMP2-BioCaP/HACC-4,


41

BMP2-BioCaP/HACC-0.8.

The surface characterization of the BMP2-BioCaP and BMP2-BioCaP/HACC complex was

evaluated by scanning electron microscope (SEM) (XL30, Philips, the Netherlands). For

this purpose, samples of the material were mounted on aluminum stubs and sputtered with

gold particles to a thickness of 10-15nm.

2.6 Release kinetics of HACC and BMP2 from the BMP2-BioCaP/HACC complex

To monitor the release kinetics, six samples of fluorescence-labeled BMP2-BioCaP/HACC

complex (100uL/sample) were incubated in 1mL 0.9% phosphate-buffered saline (PBS) at

37°C, pH 7.4 for up to 30 days with mild shaking (50 agitations/min). At each time point

(hour 3, 6, day1, 2, 3, 5, 10, 15, 20, 25, 30), triplicate 100μl aliquots of the medium

(containing released FITC-BSA and rhodamine B) were collected and replaced with fresh

PBS. Fluorescence densities of FITC-BSA and rhodamine B were measured in a

spectrophometer (Spectramax M2 Molecular Devices, CA, USA) (FITC-BSA: excitation

wavelength at 485 nm and emission wavelength at 528 nm; rhodamine B: excitation

wavelength at 530 nm and emission wavelength at 590 nm). Fluorescence readings were

converted to amounts of protein using a standard curve, which was generated by preparing

a dilution series of FITC-BSA or rhodamine B in PBS. The temporal release of FITC-BSA

was expressed as a percentage of the total amount that had been incorporated into BioCaP

granules. Likewise, the temporal release of rhodamine B was expressed as a percentage of

the total amount that had been adsorbed on the surfaces of BioCaP granules.

2.7 Animal experiment

To evaluate osteoinductivity of BMP2-BioCaP/HACC complex and optimize the HACC

concentration in vivo, six groups were established (n=6 samples per group): 1) BMP2-

BioCaP; 2) BioCaP/HACC; 3) BioCaP; 4) BMP2-BioCaP/HACC-20; 5) BMP2-

BioCaP/HACC-4; 6) BMP2-BioCaP/HACC-0.8. Each sample contains 100uL BMP2-

BioCaP/HACC complex. We used dorsal subcutaneous pockets in Sprague-Dawley rats (12

weeks and weighing~500g) as the animal model. Two samples per rat were randomly

implanted into (one on the left side and one on the right) the subcutaneous pockets and be

trapped therein by suturing the incision site. The animal study adhered to the ethic laws and

regulations of China and was approved by the Ethical Committee of Zhejiang Chinese

Medical University.

2.8 Histological preparation and histolomorphometrical analysis

After 5 weeks, the samples with surrounding tissues were retrieved and fixed chemically,

Chapter 3

42

and embedded in methylmethacrylate (MMA) as previously reported [26]. By applying a

systematic random sampling strategy [27], samples were sawn into 6 or 7 slices of 600μm

thickness 400μm apart. Slices of each sample were mounted on Plexiglas holders and

polished. They were then stained with McNeal’s tetrachrome, basic fuchsine, and toluidine

blue O. Images of 10 random areas from each slide were recorded under a microscope

(Nikon-Eclipse) at final magnification of 200 times for histomorphometric analysis. The

volume density of tissues and materials were determined stereologically using the point

counting technique [28]. The volume density of newly formed bone was calculated as their

volume divided by the corresponding reference volume of each sample. The space under

the fibrous capsule that embraced the whole block of implants (subcupsullar space) was

taken as the reference volume. The volume density of the remaining BioCaP granules was

calculated as the absolute volume of the remaining BioCaP granules divided by the original

volume of the BioCaP granules (100μL).


All data were presented as the mean values with the standard deviation (mean ± standard

deviation). Data were compared using a one-way variance analysis (ANOVA) of with the

significance level being set at p < 0.05. Post hoc comparisons were made using Bonferroni

corrections.

3. Results

3.1 Determination of the optimal concentration of HACC

To determine the ideal point of balance between cellular biocompatibility an antibacterial

performance of HACC, the influence of HACC concentration on viability of MC3T3-E1

cells was evaluated. The results (Fig.1) revealed that HACC at 200μg/mL and above could

significantly suppress cell viability, whereas 40μg/mL HACC did not exhibited cytotoxicity

to cells. What’s more, the MBC of HACC against MRSA was 40μg/mL. 40μg/mL HACC

was therefore considered as the optimal concentration of HACC to kill bacteria without

harming osteoblasts, which was used for the following experiments.


43

Fig 1. Cell viability of MC3T3-E1 after 6, 24, 48, 72h exposure to HACC at concentrations of 1000, 200, 40 and

0μg/mL. Mean values are presented together with the standard deviation. *p<0.05.

3.2 The influence of HACC on BMP2-induced cell differentiation

According to our pilot study, HACC was resorbed within 3 days in vivo. To mimic the in-

vivo situation, MC3T3-E1 cells were firstly treated with HACC at its optimal

concentration-40μg/mL for 3 days. After removal of HACC, cells were then exposed to

BMP2. ALP and OCN, markers of osteoid formation and bone mineralization, were used to

examine the influence of HACC on BMP2-induced cell differentiation. The ALP assay (Fig.

2A) and OCN assay (Fig. 2B) showed that when cells were not pre-treated with HACC,

BMP2 significantly improved ALP and OCN expression of cells at 4 days and 7 days.

When cells were pretreated with 40μg/mL HACC, the ALP and OCN expression of them

was also improved with exposure to BMP2. Moreover, the ALP and OCN expression

showed no significant difference between group +HACC+BMP2 and –HACC+BMP2.

Besides, mineralized modules formation was found in group +HACC+BMP2 and –

HACC+BMP2. These results indicate that the BMP2-induced cell differentiation were not

influence by pretreatment of HACC or not.

Chapter 3

44

Fig 2. The ALP activity (A) and OCN concentration (B) of MC3T3-E1 cells after treatment of HACC and/or

BMP2 for 4 and 7 days. Mean values are presented together with the standard deviation (* p<0.5; # p>0.5).

Alizarin red staining (C) on MC3T3-E1 cells with 3-days pre-treatment of HACC and following treatment of

BMP2 for 28 days. 100× magnification.

3.3 Fabrication and characterization of the BMP2-BioCaP/HACC complex

As 40μg/mL has been confirmed as the optimal concentration of HACC, we therefore need

to assure the in-vivo functioning concentration of HACC should be 40μg/mL. We assumed

that in vivo HACC would release from the BMP2-BioCaP/HACC complex and functioned

locally in a niche around the BMP2-BioCaP/HACC complex where HACC concentration

was 40μg/mL. Given that the volume of each BMP2-BioCaP/HACC complex is 100μL, we

speculated the niche around the BMP2-BioCaP/HACC complex should be around 100μL.

500, 100, 20μL were therefore taken. Accordingly, the HACC amount carried by each

BMP2-BioCaP/HACC complex sample was 20, 4 and 0.8μg, which were respectively

named as BMP2-BioCaP/HACC-20, BMP2-BioCaP/HACC-4, BMP2-BioCaP/HACC-0.8.

The size of BMP2-BioCaP granules ranged from 0.25- 0.50 mm. The SEM images of


45

BMP2-BioCaP/HACC showed that BMP2-BioCaP granules were embedded into the

HACC network.

Fig 3. SEM micrographs of BMP2-BioCaP (A) and BMP2-BioCaP/HACC complex (B).

3.4 Release kinetics of HACC and BMP2 from the BMP2-BioCaP/HACC complex

The in-vitro release profile (Fig. 3) showed that HACC was therefore released rapidly and

completely exhausted after 3 days, while BMP2 revealed a low burst release and

subsequently a sustained release from the internally-incorporated depot in BioCaP granules.

About 10-14% of BMP2 released from BioCaP granules in an initial burst release stage

(within in the first 24 hours). It was then gradually released at a steady rate until 30 days.

Fig 4. Graph depicting the cumulative release kinetics of rhodamine B (HACC substitute) and FITC-BSA (BMP2

substitute) from BMP2-BioCaP/HACC complex. Mean values are presented together with the standard deviation.

Chapter 3

46

3.5 Ectopic bone formation of BMP2-BioCaP/HACC complex

After initial optimization on HACC content of each the BMP2-BioCaP/HACC complex

sample in vitro, the ectopic bone formation of the BMP2-BioCaP/HACC complex in dorsal

subcutaneous pockets of rats were used to evaluate the osteoinductivity of the BMP2-

BioCaP/HACC complex in vivo. The light microscope of representative histological

sections of each group at 5 weeks (Fig. 5) showed that newly formed bone was deposited

directly upon the surface of BioCaP granules, and was only found in BMP2-containing

groups. According to histomorphometrical analysis (Fig. 6), in comparison with the positive

control group (BMP2-BioCaP), BMP2-BioCaP/HACC-4 and BMP2-BioCaP/HACC-0.8

complex resulted in similar amount of new bone formation, while BMP2-BioCaP/HACC-

20 was found significantly less new bone formation and less mature bone structure (Fig.

5D). In group of BMP2-BioCaP/HACC-4, we observed that compact bone area in an active

phase with osteoblasts (Fig. 5E). On the other hand in groups without BMP2, no bone

tissue but fibrous capsular tissue was found around embracing the BioCaP granules (Fig.

5B&C).

HACC could not be observed on histological sections any more after 5 weeks (Fig. 5).

The degradation of BioCaP with or without BMP2 did not exhibit significant difference

among groups, indicating the existence of HACC did not influence BioCaP degradation in a

pro-fibrotic environment (Fig. 6B).

Fig 5. Light microscope of representative histological sections of each group at 5 weeks after subcutaneous

implantation in rats: A) BMP2-BioCaP; B) BioCaP/HACC; C) BioCaP; D) BMP2-BioCaP/HACC-20; E) BMP2-


47

BioCaP/HACC-4; F) BMP2-BioCaP/HACC -0.8. The sections were stained with McNeal’s Tetrachrome, basic

Fushine and Toludine Blue O. Newly formed bone (white arrow head)was deposited directly upon the surface of

BioCaP granules (asterisks), and was only found in BMP2-containing groups (A,D, E and F). New bone tissue in

group D exhibited less mature structure and smaller quantities that in A. Compact bone area in an active phase

with osteoblasts (black arrow) was observed in groups E. In groups without BMP2 (B&C), no bone tissue but

fibrous capsular tissue (white arrow) was found around embracing the BioCaP granules. Scale bar=100μm.

Fig 6. Graphs depicting the volume of newly formed bone (A) and the volume of remaining BMP2-

BioCaP/BioCaP granules (B) 5 weeks after implantation. Mean values are presented together with the standard

deviation. *p<0.05.

4. Discussion

Treatment of infected critical-sized bone defects remains a great challenge of orthopedic

surgery, oral and maxillofacial surgery because of the local residual bacteria and beyond-

self-healing bone defects. To overcome these two difficulties and consequently repair the

infected critical-sized bone defects, we believe the ideal local biomaterial system should

meet the following requirements: 1) The biomaterial system can function as both antibiotic

carrier and bone substitute, to not only clear the infection but to also contribute to the

subsequent bone regeneration process; 2) The antibiotics used for local delivery should

have a broad spectrum of activity and a low rate of bacteria resistance; 3) The antibiotic

should also be delivered to its optimal concentration, at which it reaches the balance

between cellular toxicity and antibacterial activity; 4) Osteoinductive bone grafts are more

favorable for improving bone regeneration; 5) The release kinetics of antibiotic and

osteoinductive agents should meet their optimal delivery mode respectively.

Firstly, the application of current available local biomaterial system is limited due to their

Chapter 3

48

intrinsic drawbacks. PMMA bone cement is considered as the gold standard biomaterial for

local antibiotic therapy. PMMA bone cements containing either gentamicin or tobramycin

at low doses (0.5-1 g per 40 g cement), are sufficient products for prophylaxis, but not for

therapeutic applications in established osteomyelitis [29]. In addition to the limitations on

antibiotic choice and the elution kinetics and efficiency, the other major shortcoming of

PMMA is that it is non-biodegradable. Consequently, it must be removed after infection

management as it could impair healing of the debrided bone defect. To preclude the issue of

biodegradation, calcium sulfate is developed as the primary resorbable material that has

been used clinically for local antibiotic delivery. On average, calcium sulfate pellets take

approximately 2-3 months to radiographically resorb [30]. These calcium-based antibiotic-

delivery systems have advantages over PMMA in that they can carry a wider range of

antibiotics and do not need a second surgery to remove them. However, for critical-sized

bone defect cases, after the removal or resorption of these local antibiotic-delivery systems,

a second surgery is still needed for bone grafting. The drawback of this two-step surgical

approach, however, is that it requires considerable time and additional surgeries, which

increase treatment costs and patient burden. Therefore, development of novel local

biomaterial system has primarily focused on biodegradable materials which can function as

both antibiotic carrier and bone substitute. Based on this theory, we used our previously

developed osteoinductive bone substitute, BMP2-BioCaP granules, also as antibiotic carrier

to develop an antibacterial and osteoinductive biomaterial─the BMP2-BioCaP/HACC

complex. It could also rapidly kill site-infection associated bacteria, and then induce bone

formation in profibrous environment (Fig. 5), which is as challenging as in critical-sized

bone defects. The complex was observed being incorporated into newly formed bone and

showing the trend of being degraded eventually. Compared to two-step surgical approach,

the benefit of using the BMP2-BioCaP/HACC for treating infected critical-sized bone

defects is that it only needs one surgery, which can dramatically decrease treatment costs

and patient burden.

Secondly, the antibiotics used in the local biomaterial system plays a key role for the

infection control. Since a wide of Gram-positive (e.g. S. aureus and S. epidermidis) and

Gram-negative (e.g. Pseudomona aeruginosa and Escherichia coli) bacteria are found in

the infected defects [18], the biomaterial system should have a broad-spectrum antibiotic.

Vancomycin, one of the most commonly used antibiotics in local delivery system, is only

active against Gram-positive bacteria such as Staphylococci (including MRSA),

Streptococci and Enterococci [31]. It is therefore not an ideal choice for local infection

control. In contrast, another most commonly used antibiotic─gentamicin has a broad-

spectrum of activity, rapid concentration-dependent bactericidal effect and low cost [32]. It

is available for intramuscular and intravenous injection, in antibiotic impregnated PMMA

beads, in sponge-like collagen implants and as antibiotic component in a coating on

intramedullary nails for tibial fracture fixation [18]. However, it cannot be the perfect


49

antibiotic for the local biomaterial system due to the more and more severe bacterial

resistance. As a result of indiscriminate use of these conventional antimicrobials, the spread

of antimicrobial resistance is now a global problem. The emergence of resistant strains has

increased the morbidity and mortality associated with wound infections [33]. Therefore,

research has also focused on encapsulating other novel antimicrobial compounds into

biomaterials, such as modified antibiotics, silver or antimicrobial peptides. HACC, a new

water-soluble quaternary ammonium salts from the reaction of chitosan with glycidyl

trimethylammonium chloride, have been reported as a chitosan derivative with a broad

spectrum of antibacterial ability [34]. The mechanism of antibacterial action of HACC may

explain its broad spectrum. Despite the distinction between Gram-positive and Gram-

negative bacterial cell walls, antibacterial modes both begin with interactions at the cell

surface and compromise the cell wall first. Gram-positive bacteria, lipoteichoic acids may

provide a molecular linkage for HACC at the cell surface, allowing it to disturb membrane

functions [35]. Lipopolysaccharides and proteins in the Gram-negative bacteria outer

membrane are held together by electrostatic interactions with divalent cations that are

required to stabilize the outer membrane. Polycations may compete with divalent metals,

such as Mg2+

and Ca2+

ions present in the cell wall, which will disrupt the integrity of the

cell wall or influence the activity of degradative enzymes [36]. Therefore, HACC shows

antibacterial activity to both Gram-positive and Gram-negative bacteria.

Furthermore, there is very few report of the bacteria resistance of HACC, which is perhaps

because it can inhibit biofilm formation. The presence of bacteria in a biofilm drastically

reduces their susceptibility to antimicrobial drugs and host defense cells. For this reason, it

is better to prevent biofilm formation rather than trying to kill bacteria after they are

shielded by biofilm. The first step of biofilm formation is bacterial adherence to orthopedic

implant surfaces [37]. Its adherence to the implanted surface is followed by an

accumulation process and the production of extracellular substances. Researchers found

that HACC could inhibit bacteria in expressing the intercellular adhesin, a kind of

extracellular substance, so as to inhibit biofilm formation.

Thirdly, many antibiotics are naturally associated with the issue of their cytotoxicity [38,

39], it was therefore of great importance to find the optimal HACC concentration to

effectively kill bacteria without harming cells and tissues so as to guarantee the two equally

important properties of the BMP2-BioCaP/HACC complex─antibacterial activity and

osteoinductivity in vitro and in vivo. In the present study, the proliferation of rat pre-

osteoblast MC3T3-E1 cells and antibacterial efficacy for different concentrations of HACC

were evaluated to strike a balance between cellular cytotoxicity and antibacterial efficacy

(Fig. 1). As 40μg/mL has been confirmed as the optimal concentration of HACC, we need

to assure the in-vivo functioning concentration of HACC is also 40μg/mL. We assumed that

in vivo HACC would release from the BMP2-BioCaP/HACC complex and functioned

Chapter 3

50

locally in a niche around it where HACC concentration was 40μg/mL. Given that the

volume of each BMP2-BioCaP/HACC complex is 100μL, we speculated the niche around

the BMP2-BioCaP/HACC complex should be around 100μL. Volume around 100μL, such

as 500, 100, 20μL, were therefore taken to verify how wide HACC can spread in vivo.

Accordingly, the HACC amount carried by each BMP2-BioCaP/HACC complex sample

was 20, 4 and 0.8μg, which were respectively named as BMP2-BioCaP/HACC-20, BMP2-

BioCaP/HACC-4, BMP2-BioCaP/HACC-0.8. The in-vivo results of osteoinductivity (Fig.

6A) revealed that in comparison with the positive control group (BMP2-BioCaP), BMP2-

BioCaP/HACC-4 and BMP2-BioCaP/HACC-0.8 complex resulted in similar amount of

new bone formation, while BMP2-BioCaP/HACC-20 was found significantly less newly

formed bone and less mature bone structure. It indicated that in the niche around

BioCaP/HACC-20, the concentration of HACC was higher than 40μg/mL, and

consequently showed certain bone formation suppression. This phenomenon suggested

HACC would not spread as widely as 5 times wider than its own volume and confirmed our

assumption that HACC released from the BMP2-BioCaP/HACC complex and functioned

locally in a niche around it.

Fourthly, in the progress of bone regeneration, growth factors are needed to differentiate

progenitor cell into osteoblasts and subsequently to form new bone. Based on this theory,

there is an increasing interest in combining osteogenic and/or vasculogenic growth factors

with osteoconductive materials to enhance bone regeneration. This kind of materials is

considered to possessing osteoinductivity, which is defined as the ability to induce

progenitor cells to differentiate down osteogenic lineages [13]. The golden proof of

osteoinductivity of certain biomaterial is that it form bone tissue in ectopic site, such as

subcutaneous pockets of rats [40]. As shown in figure 5 and 6A, compared to BMP2-free

BioCaP granules, the BMP2-BioCaP/HACC complex could form bone tissue in the ectopic

sites, which confirmed its osteoinductivity and its capacity of enhance new bone formation.

BMP2 is an effective, but expensive osteoinductive agent. An ideal BMP2-carrier should

consume BMP2 efficiently. In current study, 100μL BMP2-BioCaP/HACC complex,

carrying only 60μg BMP2, successfully gain bone tissue in a profibrous environment.

Compared to milligram of BMP2 used in previous study [22], only 60μg BMP2 can achieve

bone formation in the profibrous environment, which indicated that the BMP2-

BioCaP/HACC complex delivered BMP2 efficiently. Moreover, BMP2 is internally

incorporated into the BMP2-BioCaP/HACC complex. This could facilitate the retention of

BMP2 at the site of implantation for a prolonged period and protect the integrity of BMP-2

against interstitial proteases [41], eventually enhancing the osteoinductive capacity of the

complex.

Fifthly, sequential release of antibiotic and osteoinductive agent also guarantees the success


51

of the BMP2-BioCaP/HACC complex in repairing infected bone defects. As shown in in-

vitro release kinetic assay (Fig. 4), the burst delivery of HACC was followed by slow and

sustained release of BMP2 at its low concentration, which was in line with the optimal

delivery mode of both HACC and BMP2 as mentioned above.

The burst release of HACC was designed to rapidly kill residual bacteria in infected bone

defects without resulting in bacterial resistance. Data emerging from in-vitro and in-vivo

studies suggested that inappropriately low antibiotic dosing may be contributing to the

increasing rate of antibiotic resistance [42]. We speculated that long-term, low-dosage of

antibiotics offered enough time for resistance strains being selected and significantly

proliferated, therefore it is reasonable to avoid resistance by burst release a strong antibiotic

to kill bacteria rapidly without giving bacteria time to proliferate. Since HACC, the strong

antibiotics, was absorbed on the surface of BMP2-BioCaP, once BMP2-BioCaP/HACC

complex was implant in vivo, it could rapidly dissolve into body fluid to achieve burst

release so as to kill bacteria. Moreover, as mentioned above, the ability of HACC in

inhibiting biofilm formation also contributed to avoid resistance.

To function as an effective osteoinductive agent, BMP2 needs to be released slowly and

continuously at its low concentration [14]. Based on this theory, we developed BMP2-

BioCaP granules [15] where BMP2 was internally incorporated into the BioCaP granules.

BMP2 was therefore released slowly and continuously with the undergoing degradation of

BioCaP granules in vivo. This has been indirectly proved by our previous study

(Unpublished data) revealing the parallel release kinetics of BMP2 and Ca2+

from BMP2-

BioCaP. The bone formation (Fig. 5) observed in the BMP2-BioCaP/HACC complex also

supports that it reached the optimal delivery mode of BMP2.

In the present study, the feasibility of the BMP2-BioCaP/HACC complex applied in bone

tissue engineering was assessed. The current results suggested that the BMP2-

BioCaP/HACC complex is particularly suitable to repair infected bone regeneration due to

rapid killing of residual bacteria and effective promotion of osteogenesis. However, the

present study has notable limitations. Because the pathogenesis of infections in infected

bone defects is complex process and involves interactions between the pathogen,

biomaterial, and host, the in-vitro assays discussed here do not account for host defense and

some other in-vivo factors. Moreover, although ectopic bone formation is the gold standard

to confirm the osteoinductivity of a material, the animal model used in the present study

cannot fully represent bone-defect model. In our ongoing studies, in-vivo infected-bone-

defect models will be utilized to evaluate the antibacterial efficacy of BMP2-

BioCaP/HACC complex.

Chapter 3

52

5. Conclusion

In this study, we developed a novel antibacterial and osteoinductive BMP2-BioCaP/HACC

complex with sequential release of antibiotic and osteoinductive agent－short-term delivery

of HACC and following slow and sustained release of BMP2. It could rapidly eliminate

antibiotic-resistant bacteria and efficiently promote new bone formation both in vitro and in

vivo, which conferred this novel material a promising application potential to repair

infected critical-sized bone defects. In our ongoing studies, in-vivo infected-bone-defect

models will be utilized to evaluate the antibacterial efficacy of BMP2-BioCaP/HACC

complex.


53

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32. Begg, E.J. and M.L. Barclay, Aminoglycosides--50 years on. Br J Clin Pharmacol,

1995. 39(6): p. 597-603.

33. Goswami, N.N., et al., Antibiotic sensitivity profile of bacterial pathogens in

postoperative wound infections at a tertiary care hospital in Gujarat, India. J

Pharmacol Pharmacother, 2011. 2(3): p. 158-64.

34. Qin, C., et al., Calorimetric studies of the action of chitosan-N-2-hydroxypropyl

trimethyl ammonium chloride on the growth of microorganisms. Int J Biol Macromol,

2004. 34(1-2): p. 121-6.

35. Tan, H., et al., Quaternized chitosan as an antimicrobial agent: antimicrobial activity,

mechanism of action and biomedical applications in orthopedics. Int J Mol Sci, 2013.

14(1): p. 1854-69.

36. Kong, M., et al., Antimicrobial properties of chitosan and mode of action: a state of

the art review. Int J Food Microbiol, 2010. 144(1): p. 51-63.

37. Ribeiro, M., F.J. Monteiro, and M.P. Ferraz, Infection of orthopedic implants with

emphasis on bacterial adhesion process and techniques used in studying bacterial-

material interactions. Biomatter, 2012. 2(4): p. 176-94.

38. Duewelhenke, N., O. Krut, and P. Eysel, Influence on mitochondria and cytotoxicity of

different antibiotics administered in high concentrations on primary human

osteoblasts and cell lines. Antimicrob Agents Chemother, 2007. 51(1): p. 54-63.

39. Ayaki, M., A. Iwasawa, and Y. Niwano, In vitro assessment of the cytotoxicity of six

topical antibiotics to four cultured ocular surface cell lines. Biocontrol Sci, 2012.

17(2): p. 93-9.

40. Miron, R.J. and Y.F. Zhang, Osteoinduction: a review of old concepts with new

standards. J Dent Res, 2012. 91(8): p. 736-44.

41. Mastrogiacomo, M., et al., Engineering of bone using bone marrow stromal cells and

a silicon-stabilized tricalcium phosphate bioceramic: evidence for a coupling between

bone formation and scaffold resorption. Biomaterials, 2007. 28(7): p. 1376-84.

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42. Roberts, J.A., et al., Antibiotic resistance--what's dosing got to do with it? Crit Care

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57

Chapter 4

Coatings for osseointegration of

metallic biomaterials

Dongyun Wang, Gang Wu, Xingnan Lin, Yuelian Liu

Coatings for osseointegration of metallic biomaterials.in: Cuie Wen. Surface coating and

modification of metallic biomaterials. Elsevier. 2015, 11: 345-358

Chapter 4

58

Abstract:

This chapter discusses the application of coatings for improving the osseointegration of

metallic implant materials. Firstly, the definition of osseointegration and the methods for

evaluating the osteointegaration are described. The chapter then reviews the biological

process of osseointegration; Secondly, the definition, preparation methods and mechanisms

to promote osseointegration of the coatings are discussed. At the end, the current clinical

application and the future trends of these coatings are reviewed.

Keywords：Osseointegration; coatings; metallic biomaterials; bone; biomimetic coating

Coatings for osseointegration of metallic biomaterials

59

Contents

1 Clinical background

2 Osseointegration

2.1 Definition of osseointegration

2.2 Biological process of osseointegration

2.3 Methods of evaluating osseointegration

3 Overview of coatings for osseointegration of metallic biomaterials

3.1 Improve recognition and adhesion of pre-osteoblasts

3.2 Enhance proliferation and differentiation of pre-osteoblasts

3.3 Promote mineralization of bone matrix

3.4 Regulate bone remodeling by inhibiting osteoclast function

4 Clinical application and future trends

4.1 Current clinical application

4.2 Potential modification of coatings for clinical use

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60

1. Clinical background

Medical metallic implants, such as dental implants as well as hip implants, have been

widely used in the past decades. Diseases or complications that are caused by disordered

bone tissue (such as trauma and inflammation) affect millions of patients every year [1].

The affected bone tissues often appear as a bone defect and its biological function is

impaired. They need to be repaired by the implantation of metallic biomaterials (dental

implants or hip implants) [2, 3]. Therefore, the function of surrounding tissues is regained

and the life quality of patients is improved.

The metallic implants that are currently used in clinic can achieve satisfactory efficacy for

most clinical cases. However, it is still a great challenge to gain proper osseointegration in

some circumstances, such as a low bone density. Patients with medical conditions, such as

diabetes, radiation treatment for cancer, xerostomia and osteoporosis, often exhibit

compromised bone conditions for placing metallic implants [4]. These conditions may

significantly decrease the survival rates of metallic implants. This situation has led

continuous efforts for improving the surface of implants in order to achieve proper

osseointegration. In addition, immediate and early loading of implants is needed to decrease

the waiting time for the functioning of implants. This needs an acceleration of

osseointegration process. Therefore various methods have been developed to prepare

coatings on the surface of implants to enhance and accelerate osseointegration [5].

2. Osseointegration

2.1 Definition of osseointegration

In 1952, Per-Ingvar Brånemark accidently found that the titanium chamber could

completely integrate into surrounding bone in rabbit. He named the phenomenon as

"osseointegration" and saw the possibilities for human use. Brånemark (1985) defined

osseointegration as "the direct structural and functional connection between the ordered,

living bone and the surface of a load-carrying implant." With the development of modern

medicine, the definition of osseointegration has been developed. The most widely accepted

definition of osseointegration is that it is the formation of a direct structural and functional

connection between the implant and surrounding bone tissues without any intervening

connective tissue [6]. This is a definition based on a histological view. In addition, a more

clinically oriented definition was advised for osseointegration as a process in which

clinically asymptomatic rigid fixation of alloplastic materials is achieved and maintained in

bone during functional loading [7].

2.2 Biological process of osseointegration

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61

Biomaterials can trigger a biological reaction from the host after being implanted. Different

biological response happens according to the surface properties of metallic biomaterials. An

inappropriate metallic biomaterial surface can lead to an encapsulation of the material with

dense connective tissues and a consequent isolation from body fluid [8]. On the contrary,

the well-designed implant surface would form an interlock with the surrounding bone and

achieve osseointegration, which indicates the biocompatibility of the metallic biomaterial

[9].

The first step of body’s reaction to a biomaterial is the adsorption of the molecules onto the

surface of biomaterials from the surrounding fluid [10]. This is essential for the subsequent

cell adhesion since the protein film bridges the biomaterial and the cells to assure cell

adhesion. The cells are mainly considered to be mesenchymal stem cells [11]. The second

step is the proliferation of mesenchymal stem cells, which are in need of various growth

factors. In the third stage, the multipotent mesenchymal stem cells would differentiate into

osteoblasts [12] and produce cross-linked collagen, and several additional specialized

proteins in much smaller quantities (such as osteocalcin and osteopontin) to form the

organic matrix of bone. The organic matrix is subsequently calcified to form a mineralized

bone matrix [13]. Thereafter follows the remodeling process of the newly formed woven

bone [14]. In the remodeling phase, the woven bone is transformed into lamellar bone with

the trabecular being formed along the pressure trajectories. This remodeling phase

represents the most active period of osteogenesis with high levels of osteoblast and

osteoclast activity. Osteoblasts synthesize very dense and cross-linked collagen to form an

organic matrix of bone. Osteoclasts are responsible for remodeling newly formed bone

along press trajectories by demineralizing the matrix and degrading the organic components

with proteinases [15]. The outcome of the bone remodeling stage is the establishment of the

osseointegration of metallic implants. The construction of osseointegration is the biological

foundation of the secondary stability of dental implants.

2.3 Methods of evaluating osseointegration

The in-vitro parameters that are used to evaluate the osseointegration are the adhesion,

proliferation, differentiation and mineralization of osteogenic cells on the surface of an

implant.

Histomorphometric parameters and biomechanical testing are recommended as direct

measurable indicators for assessing osseointegration in vivo. The biomechanical tests

usually measure the force that is needed to break the the bone-implant interface. Bone to

implant contact (BIC) is the most widely used parameter in in-vivo studies to assess

osseointegration quantitatively (Fig. 1).

http://en.wikipedia.org/wiki/Collagen

http://en.wikipedia.org/wiki/Osteocalcin

http://en.wikipedia.org/wiki/Osteopontin

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62

Fig 1. Histological micrograph showing BIC. Dental implant implanted in a sheep femur (after 12 weeks

postoperative), retrieved together with the surrounding tissue and stained with McNeil’s Tetrachrome, basic

Fushine and Toluidine Blue O.

3 Overview of coatings for osseointegration of metallic biomaterials

As above mentioned the biological process of osseointegration of biocompatible implants

can be divided roughly into four phases: First, recognition and adhesion; Second,

proliferation and differentiation; Third, synthesis and mineralization of bone matrix; Fourth,

bone remodeling.

Various kinds of coatings on implant surfaces have been designed to regulate these four

phases either by improving the function of osteoblast lineage or by adjusting the function of

osteoclasts so as to promote osseointegration. The coatings mentioned in this chapter refer

to an additional layer that was biological functionalized on the surface of implant.

3.1 Improve recognition and adhesion of cells

Once implants are placed, the molecules from the surrounding environment adsorb on the

surface of biomaterials. These molecules are critical for the adhesion of cells, because they

recognize their favorable implant surfaces by detecting and binding to molecules adsorbed

on implant surface.

These molecules are mainly considered as extracellular matrix (ECM). The process of

recognition and adhesion is initiated by cell-to-ECM contact which is achieved by cells


63

binding to ECM through adhesion receptors on cells membranes, e.g. integrins [16]. ECM

consists of a variety of proteins, such as collagen type I, fibronectin, vitronectin,

osteopontin, and bone sialoprotein. These proteins all have the specific amino acid

sequences, Arg-Gly-Asp (Arginine-glycine-aspartic acid), which is known as RGD [17].

RGD has a high affinity for some proteins from the integrin family and it is of great

importance for the cell-to-ECM contacts. This has led to developing the coatings of RGD

peptides or proteins with RGD amino acid sequence for improving the osseointegration of

metallic implantss [18]. While the effect of RGD to improve osseointegration is still

controversial. On one hand, RGD peptides were found to significantly increase the

adhesion of MC3T3-E1 mouse osteoblasts to titanium surfaces [19]. In the same line of

research, RGD peptides were also found to improve the properties of their coatings on

biomaterials such as hydroxyapatite (HA) [17]. On the other hand, another in-vitro study

showed that the presence of the RGD sequence did not improve the adhesion onto titanium

[20]. These findings were in line with other in vivo animal studies that had been unable to

confirm any increased bone regeneration or BIC around titanium transplants treated with

RGD peptides [17, 21]. Another study suggested that the effects of RGD peptiedes can be

related to checking time points. In general, significantly different results between implants

coated with RGD or ECM and implants carrying no peptides are observed mainly in the

early stage, whereas in the later stage almost no difference can be detected any more.

Coating of titanium pins with collagen I showed that the cellular reaction on the implants

appeared more intense in the early stages of bone healing in a rat tibia model compared

with uncoated pins. Four days after implantation, Cathepsin D, osteopontin and

osteonectin-positive cells were detected in higher numbers around the collagen I-coated Ti

pins compared with the uncoated Ti pins. The early appearance of these proteins around

collagen I-coated Ti pins indicates an earlier onset of the bone remodeling process

compared with uncoated Ti pins. After 28 days, both the direct BIC and the amount of

newly formed bone showed no statistically significant influence in collagen I-coated pins

compared with Ti pins. A similar phenomenon has been observed in a different animal

model. In a sheep tibia model, the coating of Ti implants with collagen I under loaded

conditions (external fixator pins) was investigated [22]. The extraction torque of the pins

fixed externally was not altered by the collagen I coating 6 weeks after implantation.

However, a significantly increased activity of osteoblasts around pins suggested an

increased remodeling of the bone around the collagen I-coated implants. All the data

indicated that the faster remodeling by collagen I-coating affects primarily the earlier stages

of bone healing without altering the mechanical stability.

Not only do the positive results of the early stage lack credibility but also the results of the

later stage need further discussion. In some instances, RGD peptides either had no effect on

the synthesis of new bone [23], or they were actually detrimental [24]. Hennessy et al [25]

proposed that this can be explained by a competitive inhibition between the endogenous

Chapter 4

64

adhesive agents and the coated RGD. They found that coated RGD could disturb the

adsorption of fibronectin, vitronectin and fibrinogen from blood onto the implant surface.

Unlike RGD, which binds to integrins through RGD-dependent mechanisms, there are other

multiple domains within fibronectin, vitronectin and fibrinogen that bind to integrins and

stimulate either synergistically or additively the integrin signaling [26]. Hence, the RGD

sequence by itself elicits weaker integrin activation than the full-length adhesion proteins.

This results in poor cell adhesion and thus osseointegration. [25].

3.2 Enhance cell proliferation and differentiation

Adhesion of osteogenic cells on biomaterial surface is necessary for osseointegration, but it

is not sufficient to ensure osseointegration. More signals are needed for the proliferation

and differentiation of osteogenic cells.

Bone morphogenetic protein-2 (BMP-2) is the most intensively studied for this purpose.

BMP-2 has shown considerable potency for stimulating bone formation both in ectopic

sites [27] and in orthotopic sites in different species [28]. As BMP-2 possesses a high

osteoinductive potential, it was considered to be an interesting candidate for a growth factor

to coat titanium implants [18]. Over the past decade, recombinant human bone

morphogenetic protein-2 (rhBMP-2) has been studied as a bone-modulating agent for

applications in dentistry, oral and maxillofacial surgery, and other biomedical settings [29].

Animal model studies have corroborated its capacity in inducing bone formation [30]. The

induced bone has demonstrated a long term stability after the placement of loaded implants.

It was found that the success of BMP-2 functionalized coatings was strongly dependent on

the concentration [31] and delivery mode of BMP-2 [32].

In the critical-size, supra alveolar, peri-implant defect dog model, Wikesjö et al examined

the ability of rhBMP-2 coated onto a titanium porous oxide implant surface to stimulate the

formation of local bone including osseointegration and vertical augmentation of the

alveolar ridge [31]. Animals received either implants coated with 0.75, 1.5 or 3mg/ml

rhBMP-2, or uncoated control implants. After 8 weeks of healing, cortex formation was

observed in sites receiving implants coated with rhBMP-2 at 0.75 or 1.5 mg/ml. Sites

receiving implants coated with rhBMP-2 at 3.0 mg/ml exhibited more immature trabecular

bone formation, seroma formation, peri-implant bone remodeling and an undesirable

displacement of the implant. The authors concluded that higher concentrations of rhBMP-2

were associated with problematic effects. Similar dose-dependent bone remodeling and

seroma formation have been observed in the previous studies evaluating implants coated

with rhBMP-2 at 0.2 and 4.0mg/ml in the alveolar bone in dogs [33]. The mechanisms

accounting for the side effects of overdosed BMP-2 can be that BMPs stimulate the

recruitment, proliferation and differentiation of osteoclasts [34]. Hence, they may promote

http://www.researchgate.net/profile/Ulf_Wikesjoe/


65

the resorption of newly formed bone almost as soon as it has been laid down onto a

titanium implant surface [35]. Another possible mechanism is that the overdosed BMPs can

trigger the production of intrinsic BMP inhibitors, such as noggin [36]. Therefore, the

applied dose of BMP-2 is critical in gaining optimal bone regeneration. In contrast to the

enhancement desired of bone regeneration at the bone–implant interface, an excessive dose

of BMP-2 may possibly impair the osteoconductivity of the implant surface [32].

The delivery mode of BMP-2 can also have a dramatic influence on osseointegration. Liu et

al. assessed the effects of BMP-2 and its mode of delivery on the osteoconductivity of

dental implants in the maxillae of miniature pigs [32]. Six different types of surfaces were

evaluated: uncoated titanium surfaces (Ti), BMP-2 adsorbed to Ti (Ti/BMP-2 ads),

biomimetic calcium phosphate-coated surfaces (CaP), BMP-2 adsorbed to CaP

(CaP/BMP-2 ads), BMP-2 incorporated into CaP (CaP/BMP-2 inc), as well as BMP- 2

adsorbed to and incorporated into CaP (CaP/BMP-2 ads & inc). After 3 weeks, implants

bearing only adsorbed BMP-2 displayed the lowest volume of bone deposited within the

osteoconductive space and lowest bone-interface coverage among all the groups. The

authors concluded that the osteoconductivity of functionalized implant surfaces was

drastically impaired when BMP-2 was present as a superficially adsorbed depot upon

CaP-coated or uncoated surfaces. The phenomenon might be due to the burst release of

adsorbed BMP-2 to the sites of bone defect, which caused an excessive application of

BMP-2 and subsequently resulted in side effects of stimulating the degradation of the bone.

Therefore, the sustained delivery of a moderate amount of BMP-2 is essential for

maximizing its osteoinductivity. A biomimetic CaP coating can be a suitable carrier for the

sustained release of BMP-2. The composition of CaP is quite similar to the inorganic phase

of bone tissue. CaP coating is also widely considered as an osteogenic drug carrier. The

preparation of a CaP coating with a biomimetic method enables the co-precipitation

osteogenic drugs into the latticework of CaP coatings. Biomimetic deposition is a method

whereby a biologically active bone-like apatite layer is formed on a substrate surface by

immersing the substrate in simulated body fluid under physiological conditions of

temperature (37 °C) and pH (7.4) [37] instead of the exceedingly high temperatures that can

make osteogenic drugs inactive.

Liu et al [38] developed a biomimetic two layer CaP coating on a titanium alloy plate (Fig.

2). The first layer is the initial “Rainbow” coating made of a layer of calcium phosphate.

The second layer is then biomimetically prepared by co-precipitating protein and calcium

phosphate upon the first layer. Scanning electron microscope images suggested that protein

has been incorporated into the mineral latticework instead of being deposited upon its

surface. In order to assess the delivery mode of CaP coating as a carrier system, the coated

titanium alloy plates were dissolved in a simulated physiological solution to observe the

Chapter 4

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release kinetics of the incorporated

Fig 2. Illustration of the biomimetic CaP coating technique. The coating was co-precipitated together with bone

growth factor of BMP-2 on the surface of titanium implant. The growth factor was incorporated into the crystal

layer of CaP without losing their bioactivities protein. Preformed calcium phosphate coatings that had been later

immersed in solutions containing protein lost about 3% of their associated protein during the first 2 h of incubation

at pH 7.3. The dissociation process was complete within 6h. This burst release is highly characteristic of

superficially adsorbed material, but coatings prepared by the co-precipitation of calcium phosphate and protein

released only 0.3% of their associated protein during the first 2 h of incubation and no more than 0.85% after 6

days. The small burst release component almost certainly represents superficially adsorbed protein. That a mere

0.85% of the protein was released after 6 days demonstrates that this protein formed an integral part of the mineral

matrix.

The biological property of the implant that are coated with BMP-2 was studied in vitro [39].

Titanium alloy implants were coated biomimetically with a layer of calcium phosphate in

the presence of different concentrations of rhBMP-2 (0.1–10 ug/mL). Protein blot staining

showed that rhBMP-2 was successfully incorporated into the crystal latticework. The

absorption of rhBMP-2 by the calcium phosphate coatings was dependent on the dose, as

determined by ELISA. Rat bone marrow stromal cells were grown directly on these

coatings for 8 days. Their osteogenicity was then assessed quantitatively by monitoring the

alkaline phosphatase activity, which increased as a function of rhBMP-2 concentrations

within the coating medium. The rhBMP-2 incorporated into calcium phosphate coatings

was more potent in stimulating the alkaline phosphatase activity of the adhering cell layer

compared with the stimulation of the cell layers grown on a plastic substratum using the

freely suspended drug. The osteoinductivity of BMP-2eincorporated implant coating was

also evaluated in an ectopic rat model. Bone formation along implants, which was used to

describe osteoinductivity of implant materials, was first observed 2 weeks after

implantation and thereafter continued unabated until 5 weeks. By the end of the fifth week,

implants with BMP-2eincorporated CaP coating were almost completely encapsulated by

woven bone (Fig. 3).


67

Fig 3. Light micrograph of titanium-alloy disc bearing a co-precipitated layer of calcium phosphate and BMP-2,

(subcutaneous place in rat) retrieved together with the surrounding tissue 5 weeks after implantation and stained

with McNeil’s Tetrachrome, basic Fushine and Toluidine Blue O.

3.3 Promoting mineralization of bone matrix

The organic matrix produced by osteoblasts needs to calcify to form the mineralized bone

matrix [13]. This can be modulated by drugs such as fluorine. The implants that are treated

with fluorine are currently available for the clinician to use. The mechanism relies upon the

property of fluoride, which interacts with HA that presents in bone tissue or teeth and

generates fluorapatite. This has better crystallinity and a lower dissolution rate compared

with HA [40]. Hence, a tight junction between bone and the implant was noted. The

incorporation of fluoride into CaP coating on implants is supposed to improve the bone

formation along implant surface.

Farley et al studied the effect of fluoride on osteoblasts in an in vitro model of avian cells

[41]. They reported that fluoride increases the proliferation and alkaline phosphatase

activity of osteoblasts. Based on the biomechanical and histomorphometric data,

the titanium implants modified with fluoride demonstrated a firmer bone anchorage than for

the unmodified titanium implants. These implants achieved a greater osseointegration after

a shorter healing time than unmodified titanium implants [42]. These findings were later

confirmed by others [43, 44]. Results from these studies suggest the application of fluoride

can facilitate a very specific stimulus on osteoblasts leading to an increase in the bone

apposition rate in the early phases of osteogenesis.

3.4 Regulation of bone remodeling by inhibiting the osteoclast function

A current clinical challenge in the field of orthopedics is to obtain a stably fixed implant in

weak osteoporotic bones. The fixation of orthopaedic implants in bone relies strongly upon

the initial stability of the implant [45]. For osteoporotic patients this early phase is

particularly delicate as the bone is already weak before the surgery. The resorption of a

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small amount of bone near the implant may induce a dramatic decrease in early fixation,

accelerating failure [45]. Bisphosphonates are a class of drugs that prevent the loss of bone

mass by inhibiting the osteoclast activity used to treat osteoporosis and similar

diseases. Therefore, bisphosphonates can be used to reduce periprosthetic osteolysis, which

allows orthopaedic implants to achieve a stronger primary fixation[46].

There are two classes of bisphosphonates: the N-containing and non-N-containing

bisphosphonates. The two types of bisphosphonates work differently in inhibiting

osteoclast cells. Zoledronate, one of N-containing bisphosphonates, was studied as a

coating agent for osteoporotic bone [45]. Four implants coated with zoledronate and two

control implants were inserted in the femoral condyle of ovariectomized sheep for 4 weeks.

The bone at the implant surface was 50% higher in the zoledronate-group than in the

control group. This was significant up to a distance of 400μm from the implant surface. The

results are similar to those observed in the osteoporotic rat model. This finding suggests

that the concept of releasing zoledronate locally from the implant to increase the implant

fixation is not species-specific. The results of this trial support the claim that local

zoledronate could increase the fixation of an implant in weak bone [45].

The mechanism of bisphosphonates for improving osseointegration can be explained by the

following study [47] where screws used for fracture fixation was coated with

bisphosphonate. Histological results showed that a shell of new bone had formed around

the screw, inside the marrow cavity. This was not seen in the control group [48, 49]. This

does not mean that bisphosphonates induce new bone formation in the way as BMPs. The

explanation appears to be that very scant, patchy loose bone or osteoid also formed around

the control screws after 1 week, but then disappeared when resorption was not suppressed.

When protected by the bisphosphonate, this early, scant bone served as a scaffold for

continued bone growth leading to the formation of a bony shell. It proves that

bisphosphonate coatings take effects by the inhibiting of bone resorption rather than the

promotion of bone formation.

4. Clinical application and future trends

4.1 Current clinical application

Although a variety of coatings have been developed for improving osseointegration in

preclinical studies, only a few of them have been evaluated in clinical applications, such as

HA and bisphosphonate coatings [50].

HA coating has been most extensively studied in clinical applications which are applied

mainly on hip implants and dental implants and in rare cases on knee prosthesis [51]. HA

http://en.wikipedia.org/wiki/Osteoporosis

http://en.wikipedia.org/wiki/Nitrogen

http://en.wikipedia.org/wiki/Nitrogen


69

coating could dramatically enhance the osseointegration and survival rate of implants when

placed in a compromised bone condition. Interestingly, HA-coated implants showed hardly

any statistically significant difference compared with non-coated implants as long as the

surrounding bone tissue exhibited sufficient quantity and quality of the bone to achieve

initial stability. The phenomenon was observed in dental [2, 52], hip [3, 53, 54] and knee

implants[51].

According to the classification of Lekholm and Zarb, bone was divided into four types (I, II,

III and IV). The bone quality is more and more challenging for the achievement of the

initial stability going from types I to type IV. Saadoun and LeGall [55] studied 673

Steri-Oss (Denar) implants (titanium screws, HA titanium screws, and HA-titanium

cylinders) during a 5-year period. They recommended that the choice of design should be

based on implant lengths, bone quality, and anatomic region. They recommended that the

closer the bone is to type IV, the greater the indication is for HA-titanium cylinders. The

coatings’ effect on the survival rate as a function of bone quality has been studied [56].

HA-coated implants had an overall failure rate of 3.9% over 36 months for all bone

qualities combined, while non-coated implants had a 13.4% failure rate for the same

parameters. The highest failure rates for non-coated implants were in bone qualities III and

IV (19.1% and 25.5%, respectively). Therefore, HA-coated implants are more appropriate

for patients with a compromised bone quality, whereas HA-coating implants should not be

recommended for cases with an acceptable bone quality.

Caution should also be taken to apply HA-coated to patients since HA coating was found to

have negative effects on bone formation. Reports in the literature [57, 58] have suggested

that HA coatings are unstable, more susceptible to bacterial infection, may be predisposed

to rapid osseous breakdown, and do not demonstrate any significant advantages over

titanium implants [56]. Furthermore it was discovered the HA is not able to improve the

long-term outcome of bipolar hemiarthroplasty. This is because in the initially well-fixed

HA-coated prostheses the sealing effect of a HA coating creates high concentrations of

polyethylene in the limited joint space resulting in massive proximal femoral osteolysis.

Consequently, a HA coating introduces a new failure mechanism and HA-coated implants

should only be recommended for patients with compromised bone quality.

Bisphosphonates-coated dental implants are also studied in clinical trials. Apart from the

conventional therapy for patients suffering from osteoporosis using systemic treatment with

bisphosphonates, bisphosphonates-coated dental implants were developed to release

bisphosphonate locally [50]. The promising results may lead to new possibilities for

orthopedic surgery in osteoporotic bone and for dental implants.

4.2 Potential modification of coatings for clinical use

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70

A variety of implants that are surface-treated with bioceramics or drugs, are commercially

available. They have been shown to accelerate and enhance osseointegration formation.

Nevertheless, take BMP-2 for example, there are other promising bioactive agents whose

true potential for application as biomimetic agents on implant coatings has yet to be

established [17]. RhBMP-2 has been approved by United States Food and Drug

Administration (FDA) to be used for spinal fusion and has been shown to induce bone

formation in animal studies and clinical trials [1, 59]. Moreover, CaP coatings have proved

to be an efficient carrier of BMP-2 so as to stimulate differentiation of rat bone marrow

stromal cells in vitro. It would be, therefore, worthwhile to perform clinical trials to

evaluate the osseointegration of implants bearing CaP coatings incorporated with BMP-2.

http://en.wikipedia.org/wiki/Food_and_Drug_Administration

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5. References

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fractures: a prospective, controlled, randomized study of four hundred and fifty patients. J Bone Joint Surg

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2. Geurs, N.C., et al., Influence of implant geometry and surface characteristics on progressive

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3. Landor, I., et al., Hydroxyapatite porous coating and the osteointegration of the total hip replacement. Arch

Orthop Trauma Surg, 2007. 127(2): p. 81-9.

4. Diz, P., C. Scully, and M. Sanz, Dental implants in the medically compromised patient. J Dent, 2013. 41(3):

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6. Guo, C.Y., J.P. Matinlinna, and A.T. Tang, Effects of surface charges on dental implants: past, present, and

future. Int J Biomater, 2012. 2012: p. 381535.

7. Zarb, G.A. and A. Schmitt, Osseointegration and the edentulous predicament. The 10-year-old Toronto

study. Br Dent J, 1991. 170(12): p. 439-44.

8. Allan, B., Closer to nature: new biomaterials and tissue engineering in ophthalmology. Br J Ophthalmol,

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77

Chapter 5

Accuracy of peri-implant bone thickness and

validity of assessing bone augmentation material

using cone beam computed tomography

Dongyun Wang, Andreas Künzel, Vladimir Golubovic, Ilya Mihatovic, Gordon John,

Zhuofan Chen, Jüngen Becker, Frank Schwartz.

Clinical Oral Investigations, 2013, 17: 1601-1609

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Abstract

Objectives: The aim of this study was to evaluate the accuracy of measuring bone

thickness surrounding dental implants and the reliability of assessing existence and

completion of osseous integration of augmentation material using a cone beam computed

tomography (CBCT) system.

Materials and methods: In jaws of foxhounds, artificial defects were regenerated by

guided bone regeneration and then dental implants were placed. After putting down the

dogs, the jaws were separated from the bodies and exposed in a CBCT system. The bone

thickness was measured on both buccal and oral sides of the implants at different levels.

Every examiner evaluated existence and integration of bone augmentation materials (BAM)

and the completeness of marginal implant covering. The same measurements and

evaluations were performed at digital images of the corresponding histological

sections.

Results: The mean and the standard deviation of the differences between radiological

and histological measurements of peri-implant bone thickness were −0.22 mm and

0.77 mm, respectively. Sensitivity and specificity were 0.77 and 0.60 for existence of

BAM, 0.59 and 0.74 for completed integration, and 0.39 and 0.71 for full covering of the

implant surface.

Conclusions: The present study indicates that the PaX Duo3D® CBCT system

allows measurements of peri-implant bone thickness at an accuracy of half a millimeter,

and—within limits—assessing the existence and integration of BAM. It is not possible to

evaluate whether the implant is covered completely by hard tissue.

Clinical relevance: Peri-implant bone thickness is a key factor for obtaining initial

implant stability. The accuracy of its measurement has clinical impact. Radiological

assessment of existence and integration of BAM would be of great benefit to the evaluation

of augmentation procedures.

Accuracy evaluation of CBCT

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1. Introduction

The development of oral implantology partially depends on the improvement of oral

radiological techniques. Regardless of the pre-operative assessment of bone quantity and

quality in the region of interest, or postoperative evaluation of integration of bone

augmentation material (BAM), good validity and reliability of radiological evaluation are

essential. Cone beam computed tomography (CBCT) has been widely used in clinical

practice of oral and maxillofacial surgery, demonstrating the advantages of high resolution,

easy handling, easy accessibility, reduced costs, lower radiation dose, and possibly less

disturbance from metal artifacts [1] compared to CT imaging. Research has concentrated

around the accuracy of measurements in CBCT images, but only a few studies focus on the

accuracy of measurements of the peri-implant bone thickness next to implants [2–5]. This

topic is, however, of great clinical importance since obtaining initial stability is the key to

the overall success of dental implant surgery. Besides implant design and placement

technique, sufficient bone thickness is also an essential factor for assurance of initial

stability. Braut et al. investigated the radiological evaluation of the facial bone wall prior to

extraction [6]. In a finite element study of orthodontic mini-implants, Dalstra et al.

demonstrated that increasing cortical bone thickness drastically reduced the peak strain

development in the peri-implant bone tissue [7]. An inverse relationship between cortical

bone thickness and peak strain development suggests that cortical bone thickness is one of

the key determinants of initial stability, although two cortical bones could have the same

thickness but completely different bone mineral densities and different initial stability.

Razavi et al. [2] placed dental implants in bovine ribs and exposed these specimens in two

different CBCT scanners (i-CAT NG, Accuitomo 3D60 FPD). They found the accuracy of

measuring bone thickness near implants was different depending on the CBCT scanner

used and concluded the different scanner resolutions being the reason. Fienitz et al. [3] used

another CBCT (Galileos) to scan the jaws of dogs being treated with dental implants and

bone augmentation procedures. Fienitz concluded that the evaluation of peri-implant bone

defect regeneration by means of CBCT is not accurate for sites providing a bone width

below 0.5 mm and that a safe assessment of the success of the guided bone regeneration

technique is not possible after the application of a radiopaque bone substitute material.

Corpas et al. used the Accuitomo 3D (Morita, Kyoto, Japan) to investigate implants in

minipigs. They found the CBCT deviating 1.20 mm from the histology regarding bone

defects [5]. The present study was designed to reevaluate the accuracy of linear

measurements of the peri-implant bone thickness and the radiological evaluation of

boneaugmentation procedures using a different CBCT scanner.

In addition, assessment of BAM integration might be also a point of interest of CBCT

application in oral implantology. In cases of bone augmentation before implant placement,

integration of BAM is a prerequisite to implant surgery. Similarly, in cases of endosseous

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implants with simultaneous bone augmentation, radiological assessment of BAM

integration would be helpful for the evaluation of therapeutical outcome. An animal

experiment is supposed to be a very accurate method to investigate the accuracy of such a

radiological diagnostic procedure, although few directly related papers have been published

[3]. Hence, this study aims to assess the reliability of estimation of BAM existence and

integration using a CBCT system.

2. Materials and methods

2.1 Animal study

In 2009, Schwarz et al. and Mihatovic et al. made a foxhound experiment to investigate

bone augmentation procedures prior to implant surgery [8, 9]. The experiment was

approved by the Animal Care and Use Committee of the Heinrich Heine University and the

local government of Düsseldorf. The biopsies obtained at the end of the experimental

phases were used for a supplementary radiological assessment and therefore served as a

basis for the present study.

The animal study included a total of six foxhound dogs (age 18–22 months, weight 32–42

kg). It was performed in three surgical phases. In the first phase, the mandibular and

maxillary first, second, third, and fourth premolars as well as the first and second molars

(P1–M2) were extracted. After a healing period of 10 weeks, a total of 48 standardized

saddle-type defects (mesio-distal width=10 mm; height=8 mm) were randomly prepared in

both the upper and the lower jaw of each dog. The defects were filled using natural bone

mineral or biphasic calcium phosphate, halfway with added autogenous bone and with

random assignment of these treatment procedures to anterior and posterior sites,

respectively. Subsequently, the treated defects were randomly allocated in a split-mouth

design to the use of either a polyethylene glycol membrane or a collagen membrane. At 8

weeks, modSLA titanium implants were inserted at the respective treated defect sites and

left to heal in a submerged position for 2 weeks before the dogs were put down. All surgical

autopsy procedures were performed by two experienced operators (F.S. and I.M.). For

details, see Mihatovic et al. and Schwarz et al. [8, 9].

2.2 CBCT radiography

The complete oral tissues of the put down dogs were perfused with formalin; both maxilla

and the mandible including soft tissues were dissected from the bodies and packed tightly

in airtight bags. All of the jaws were scanned using a CBCT system (PaX Duo3D®; Vatech,

Seoul, Korea) within a few hours after dissection. A wooden scaffold served to place the

jaws in a reproducible position for scanning in the CBCT machine. The laser orientation


81

beam was used to adjust the jaws accurately to the scanning volume. The exposure settings

were adjusted to 85 kV and 4.9 mA. The scanning parameters were set to a field of view

of 50-mm diameter and 50 mm height at a voxel size and a slice thickness of 0.08 mm

both. Following the exposures, the image data was saved to DICOM files by the Byzz

program version 5.7.2 (copyright 2009; Orangedental GmbH & Co. KG, Biberach,

Germany).

2.3 Image processing

The viewing software utilized to analyze the CBCT images was Ez3D 2009 Professional

(version 1.2.1.0; Vatech). For every single implant, the image display was standardized to

adjust the implant vertically and transversal to the alveolar bone in the multiplanar

reconstruction view at a slice thickness of 80 μm and at an image zoom of 250 %.

Following, these standardized views were saved as “projects” separately for every implant.

Two examiners with different levels of experience in 3D imaging and CBCT usage

participated in the first part of the study. One examiner was an oral surgeon (A.K.) having

many years’ experience in CBCT imaging. The other examiner was a dentist (D.W.). Both

of them were trained thoroughly on operating the CBCT machine used.

The examiners were instructed by a detailed step-by-step image processing protocol. Both

were given two individually randomized viewing lists to reload the saved projects and

carry out the measurements on identical slices twice in differently randomized orders to

produce the data for interand intra-examiner reproducibility. The interval between first

and second reading was 6 weeks at minimum.

In order to facilitate the measurement and save examiner time, a transparent plastic sheet

was printed which showed five black horizontal lines and one vertical line, following a

suggested method of Razavi et al. [2]. The distance between the first and last line was

exactly the whole length of the implants on the computer display from interface edge to

apex. These lines represent 0 %, 25 %, 50 %, 75 %, and 100 % of the implant length,

respectively. The sheet was fixed by adhesive tape on the display directly over the

radiographic images to indicate the levels where measurements were required from the

examiners and was readjusted for every image. The peri-implant bone thickness on both the

buccal and oral side of the implant was measured at the levels 0 %, 25 %, 50 %, and 75 %

(Fig. 1).

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Fig. 1 Schematic diagram: Measurement of the bone thickness at four levels

Three more observers joined to the second part of the study to provide a total of five

examiners. One of them was an examined oral surgeon and two of them dentists advanced

in postgraduate education of oral surgery, all of them trained and licensed for the use of

cone beam tomography. The assessment of integration of BAM was accomplished by the

examiners answering the three following questions:

– Is there any augmentation material?

– If yes, is the augmentation material completely integrated into the bone?

– Is the implant completely covered with bone and/or augmentation material?

The five observers repeated their readings after at least 4 weeks. In the end, there were 10

evaluations per implant of existence, completed BAM integration and completed bone

covering.

2.4 Histological reference

After exposure of CBCT, the specimens were processed in hard-tissue histology according

to Donath [10] with some modifications. The jaws were cut at every implant site in the

bucco-oral direction resulting in sections of approximately 40-μm thickness, as close as

possible to the implant axis. All sections were stained with toluidine blue. For details, see

Schwarz et al. [8, 9].

The histomorphometrical analysis was performed twice by both of the investigators (D.W.

and A.K.) in randomized order. For image acquisition, a color CCD camera (Color View III;

Olympus, Hamburg, Germany) was mounted on a binocular stereomicroscope (SZ61;

Olympus, Tokyo, Japan). The optical zoom was calibrated to a magnification factor of ×1.0.

The histological slices were repositioned for every microscopic photo.

The histological images were checked for conformity with the corresponding radiological

images. Seven out of the 48 implants were eliminated from the study due to nonconformity

of histological and radiological image planes leaving 41 implants for this investigation.


83

The histological section measurements, corresponding to the levels examined on the CBCT

images, were then carried out using a microscopic imaging program (Cell D® v3.1;

Olympus Soft Imaging Solutions GmbH, Münster, Germany). The evaluators answered the

same questions regarding the augmentation materials as before in the radiological

evaluation. Complete integration of BAM was defined as completed osseous inclusion of

BAM granules with direct contact to living bone. To ensure a reliable reference, a number

of borderline cases were excluded from the second part of the study if the amount of BAM

was very small (e.g., only a few granules) or if the integration of the BAM was nearly but

not fully completed. Complete covering of the implant was rated if the top level of bone or

BAM being in contact to the implant was at or above the marginal edge of the interface at

both sides of the implant. Concluding 30 specimens with identical results in all repetitions

of the histological evaluations were selected for the evaluation of recognition and

integration of BAM and complete covering of the implants.


Statistical analysis was performed using a commercially available software program

(SPSS Statistics 20.0; SPSS Inc., Chicago, IL, USA). To provide a reliable reference, at

first the differences between the repeated histological readings were calculated. Wherever

the difference between two histological readings at the same site was above 0.5 mm—

usually because the bone surface was uneven or oblique to the measurement line—this

measurement point was eliminated from the investigation. Finally, the statistics were

based on 279 points measured eight times (twice radiology and twice histology by two

observers each) making a total of 1,116 radiological and of 1,116 histological

measurements both. The histological measurements were averaged for every site. The

differences between every radiographic measurement and the mean of the histological

measurements at the corresponding point were calculated. The correlation between

radiology and histology was calculated by using the Pearson correlation coefficient (PCC).

Then both intra-observer reliability and inter-observer reliability of the radiological

measurements were examined by PCC.

3. Results

The PCC for intra-observer correlation of the radiological readings was 0.937 (observer

D.W.) and 0.972 (observer A.K.), respectively. Figure 2 visualizes the correlation between

the first and second measurements of observer A.K. The PCC for the inter-observer

correlation was 0.936 (Fig. 3). Both demonstrated good reproducibility within and between

examiners. The Pearson correlation between radiological and histological readings was

0.912 (Fig. 4).

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Fig 2. Intra-observer correlation for CBCT

measurement (first and second measurement of

observer A.K.)

Fig 3. Inter-observer correlation for CBCT

measurement (first and second measurement of

observer A.K. to observer D.W.)

Fig 4. Correlation of radiological and

histological measurements


85

The mean value and the standard deviation of the differences between radiological and

histological measurements at all measurement points were −0.22 mm and 0.77 mm,

respectively (complete table of measurements in Online Resource 1). The largest

underestimation of bone thickness was −3.31 mm for observer D.W. and −3.11 mm for

observer A.K; the largest overestimations were 3.47 mm and 2.47 mm, respectively.

According to the histological sections, BAM existed in 23 of 30 cases (76.7 %) at the time

of CBCT exposure (Table 1). Six out of the 23 cases with existing BAM (26.1 %) were

considered as completely integrated BAM. In 18 of the 30 specimens (60.0 %), the top

level of bone or BAM was at or above the marginal edge of the interface at both sides of the

implant, thus classifying these cases as full coverings of the implant surface.

Table 1 Qualitative assessment of peri-implant bone and BAM in CBCT and in histology

BAM existence BAM integration Implant covered

Histology 23/30(76,7%) 6/23(26.1%) 18/30 (60%)

CBCT 205/300 (68.3%) 60/177 (33.9%) 106/300 (35.3%)

CBCT sensitivity 0.77 0.59 0.39

CBCT specificity 0.60 0.74 0.71

In the CBCT images, the existence of BAM was diagnosed in 205 of 300 readings (68.3 %).

In 177 cases in which the existence of BAM was diagnosed correctly in radiology, the

observers rated 60 times (33.9 %) for completed integration. In 106 of 300 readings

(35.3 %), the viewers rated the implant being fully covered by bone or BAM (complete

table of measurements in Online Resources 2).

Sensitivity and specificity were 0.77 and 0.60 for evaluating the existence of BAM, 0.59

and 0.74 for evaluating its completed integration, and 0.39 and 0.71 for evaluating full

covering of the implant surface. Checking the differences with chi-square test did not show

statistical significance for any of the diagnostic questions (α=0.05).

Examples of partially integrated BAM on the oral side (left side of the figure) and non-

integrated bone on the buccal side in a CBCT image and in a histological section are shown

in Figure 5. The BAM which is almost completely integrated shows homogeneity in the

CBCT image whereas loose BAM material looks granular. In CBCT, new bone (dark blue

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in histology) looks fainter compared to elder bone. The implants appear thickened with an

increased diameter.

Figure 6 shows CBCT and histological images without BAM. One should note that it is

possible to distinguish the band of new woven bone from the compact bone at the lingual

side (right side of the figure) and to identify some but not all of the gaps between implant

and bone in CBCT, which show in histology. However, it is difficult to identify correctly in

CBCT the contour of the bone at the buccal side of the implant.

Fig 5. Histological and corresponding CBCT image: measurement of the bone thickness at four levels. The

radiopacity at the buccal side of the implant is granular and proves as non-ossified augmentation material in

histology.

Fig 6. Histological and corresponding CBCT image: a double contour line, a lingual band of new, not yet fully

mineralized bone (dark blue in histology) shows at the lingual side both in histology and CBCT. The BIC is less

than expected in the CBCT image. The implant contour is thickened in CBCT


87

4. Discussion

Many papers related to the accuracy of linear measurements have been published [2, 11–

24]. Usually dry skulls [2, 11, 12, 14–16, 18–20, 22, 23, 25] were used as study model,

sometimes artificial specimens [17, 21]. Often measurement data obtained from these dry

skulls or mandibles by using a caliper were considered as objective standard. This is

sufficient to examine superficial anatomical sites and distances; however, measurements in

the sagittal plane are difficult and prone to bias in this way. Moreover, due to the lack of

soft tissue, dry skulls and mandibles show less blurring, compared to typical imaging

results in clinical cases, and therefore might show different results.

To overcome the limitations mentioned above, Suomalainen et al. exposed the mandible

immersed in sucrose solution isointense with soft tissue [20]. Razavi et al. used bovine ribs

embedded in a poly-ethylene mold with laboratory putty with implants planted in each rib

to measure the cortical bone thickness surrounding implants using CBCT [2]. Subsequently,

histological sections were prepared in buccal–oral direction along the long axis of the

implants, allowing an evaluation of the accuracy of measuring the cortical bone thickness

adjacent to the implants. Even though in vitro bovine rib is feasible to assess the accuracy of

linear measurements, this study does not give information to measurements of bone with

BAM. Fienitz [3] and Corpas [5] used animal experiments to evaluate the accuracy of CBCT

measurements at the peri-implant tissues with bone defects, and Fienitz included BAM.

An ongoing beagle study designed for the investigation of peri-implant bone augmentation

procedures gave chance to add a radiological investigation allowing to evaluate the

diagnostic accuracy of peri-implant bone and implant measurements and of the evaluation of

peri-implant bone regeneration in CBCT.

Compared to the golden standard of histological measurements, the linear measurement of

bone using CBCT provides values at an accuracy in a sub-millimeter range [16]. The

differences between CBCT and histology measured in this study seem similar to the results

of Fienitz and below the results of Corpas.

Even though the results demonstrate sufficient accuracy of linear measurements using this

CBCT system, it has to be kept in mind that the accuracy of metric measurements might be

limited by several factors. First of all, in a preliminary test some distortion was found in the

images obtained from the PaXDuo3D® CBCT system, which may result in different

magnifications at different positions in the volume. This might be a reason for the

limitations of the diagnostic accuracy found in this investigation. Furthermore, in this

investigation the voxel size setting of the CBCT machine was set to 80 μm. Any distance

measurement might contain an unavoidable error of at least this voxel size. In addition,

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usually low contrast range and low resolution of radiological images are charged as

potential sources for error, limiting the precision of measurements by blurring the

boundaries between bone and soft tissue and bone-to-implant interface [16]. It should be

noticed that high resolution images of perfect technical quality might be related to similar

measurement inaccuracies if the measured objects have irregular borderlines. The placing

of measurement lines on such borderlines gets a stochastic component giving it the property

of a more or less statistical sample. This might be a reason for the limits of intraand inter-

observer reliability seen in the histological measurements. The blurring in radiological

images compared to histological images seems to have a smoothing effect on the surfaces

under inspection, thus smoothing the stochastic variations in placing the measurement lines.

The existence of radiodense objects like implants causes beam hardening artifacts [26] and

might complicate the visualization of the bone–implant interface [2]. Although larger flat

panel detectors used in current CBCT scanners possibly lead to less beam hardening

artifacts [1], they might be affected by more scattered radiation because of an enlarged field

of view [27].

It might be of interest whether BAM could cause beam hardening artifacts. Our preliminary

tests showed the radiopacities of the BAM used in this investigation being only slightly or

not above the radiopacities of human bone or dog bone. This is no surprise as the BAM

used is of bone origin or is composed of the same chemical elements as bone. Therefore,

significant beam hardening artifacts by the BAM used in this investigation are unlikely.

The integration state of BAM on histological sections served as an objective standard in the

present study. Compared to it, the existence of BAM was seen less often in CBCT than in

histology. Vice versa, the observers more often stated a completed integration of existing

BAM in CBCT than in histology. However, rating existence and completed integration of

BAM in radiological images did not show a significant difference to the histology.

Sensitivity and specificity to evaluate the existence and integration of BAM with the CBCT

system at the experimental settings used are below expectations for a diagnostic procedure.

In clinical practice, the PaX Duo3D® CBCT system seems to provide some clue, at least,

whether BAM is completely integrated or not.

It would be of clinical impact to answer the diagnostic question whether the implant is

completely covered by bone or BAM. BAM shows as high-density radiopaque granule

structures. If these were in a homogenous contact to the adjacent bone, they were supposed

to represent BAM completely integrated into living bone in the present study. Often there is

some doubt or error because it is difficult to distinguish newly formed bone and bone

substitute granules in CBCT images. One should keep in mind that the implants were

placed 8 weeks after guided bone regeneration procedure. Unfortunately, the sensitivity of


89

the CBCT system to answer such a question is poor and the specificity moderate, at least at

the exposure settings used.

For successful implantology, both bone quantity and bone quality are essential. This study

focused on the accuracy of assessment bone quantity by measuring peri-implant bone

thickness in CBCT. This study did not assess bone quality, which is mainly manifested as

bone density. However, a systematic review of the literature [1] demonstrated that present

CBCT machines can hardly be used for the estimation of bone density because of

inconstant gray levels not representing the Hounsfield units (HU). HU are CT values in the

measurement of CT images used to quantitatively describe tissue density. In other words, it

meant that scanned regions of the same density in the skull can have different gray scale

values in the reconstructed CBCT dataset. However, to the authors’ knowledge, no paper

has focused on the feasibility and validity of bone density measurements using the PaX

Duo3D® CBCT system, which might be a future study.

5. Conclusion

The present study indicates that the PaX Duo3D® CBCT system allows measurements of

peri-implant bone thickness at an accuracy of half a millimeter, and─within some

limits─assessing the existence of BAM and its integration into the bone, but not the

evaluation of complete hard-tissue covering of the implant surface.

6. Acknowledgments

The authors appreciate the contribution of Orange Dental, Biberach, Germany, for providing

the PaX Duo3D® CBCT system for the present study. Dongyun Wang was kindly

supported by grant number CF 51001 of the Camlog Foundation, Basel, Switzerland for her

research fellowship at the Department of Oral Surgery at the Heinrich Heine University

Düsseldorf.

Chapter 5

90

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2. Razavi T, Palmer RM, Davies J, Wilson R, Palmer PJ (2010) Accuracy of measuring the cortical bone

thickness adjacent to dental implants using cone beam computed tomography. Clin Oral Implants Res

21(7):718–725.

3. Fienitz T, Schwarz F, Ritter L, Dreiseidler T, Becker J, Rothamel D (2012) Accuracy of cone beam computed

tomography in assessing peri-implant bone defect regeneration: a histologically controlled study in dogs. Clin

Oral Implants Res 23(7):882-887

4. Miyamoto Y, Obama T (2011) Dental cone beam computed tomography analyses of postoperative labial bone

thickness in maxillary anterior implants: comparing immediate and delayed implant placement. Int J

Periodontics Restorative Dent 31(3):215–225

5. Corpas Ldos S, Jacobs R, Quirynen M, Huang Y, Naert I, Duyck J (2011) Peri-implant bone tissue assessment

by comparing the outcome of intra-oral radiograph and cone beam computed tomography analyses to the

histological standard. Clin Oral Implants Res 22(5):492–499

6. Braut V, Bornstein MM, Belser U, Buser D (2011) Thickness of the anterior maxillary facial bone wall—a

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31(2):125–131

7. Dalstra M, Cattaneo PM, Melsen B (2004) Load transfer of miniscrews for orthodontic anchorage. J Orthod

1:53–62

8. Mihatovic I, Golubovic V, Hegewald A, Becker J, Schwarz F (2012) Influence of two barrier membranes on

staged guided bone regeneration and osseointegration of titanium implants in dogs. Part 2: augmentation using

bone graft substitutes. Clin Oral Implants Res 23:308–315

9. Schwarz F, Mihatovic I, Golubovic V, Hegewald A, Becker J (2012) Influence of two barrier membranes on

staged guided bone regeneration and osseointegration of titanium implants in dogs: part 1. Augmentation

using bone graft substitutes and autogenous bone. Clin Oral Implants Res 23(1):83-89

10. Donath K (1985) The diagnostic value of the new method for the study of undecalcified bones and teeth with

attached soft tissue (Sage–Schliff (sawing and grinding) technique). Pathol Res Pract 179(6):631–633

11. Berco M, Rigali PH, Jr., Miner RM, DeLuca S, Anderson NK, Will LA (2009) Accuracy and reliability of

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(1):16 e11–16

13. Fatemitabar SA, Nikgoo A (2010) Multichannel computed tomography versus cone-beam computed

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14. Kobayashi K, Shimoda S, Nakagawa Y, Yamamoto A (2004) Accuracy in measurement of distance using

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103(4):534–542

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measurements. J Periodontol 77(7):1261–1266

19. Mischkowski RA, Pulsfort R, Ritter L, Neugebauer J, BrochhagenHG, Keeve E, Zoller JE (2007) Geometric

accuracy of a newly developed cone-beam device for maxillofacial imaging. Oral Surg Oral Med Oral Pathol

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26. Reitz I, Hesse BM, Nill S, Tucking T, Oelfke U (2009) Enhancement of image quality with a fast iterative

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52(4):707–730

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93

Chapter 6

General Discussion

Chapter 6

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General Discussion

Currently, the world is experiencing an exceedingly high demand for functional bone grafts

to repair bone defects. It is perceived as a more promising approach to develop bone grafts

using the tissue engineering approach than harvesting autografts from patients. Through

tissue engineering bone grafts can be functionalized with specific properties so as to repair

bone defects with various complications, such as bone defects in patients with diabetes or

metabolic bone disorders, large bone defects beyond their self-healing capacity or locally

infected bone defects. Therefore, functionalized bone grafts, with osteoinductivity and/or

antibacterial activity, have been intensively studied and developed.

Many biomaterials with potential osteoinductivity have been reported and their

osteoinductivity was mainly evaluated by heterotopic bone formation in animal models.

Glass cylinders [1] and poly-hydroxyethylmethacrylate [2] were the first synthetic materials

associated with heterotopic bone formation. Composites, consisting of polylactide and HA

particles have however recently proven to be osteoinductive too [3, 4]. In the family of

metals, porous titanium has shown osteoinductivity alone [5], coated with a thin layer of

calcium phosphate [6] or in a construct with a calcium phosphate ceramic [7]. In contrast to

the limited number of reports on osteoinduction by polymers and metals, ceramics –

particularly CaP based biomaterials– have shown osteoinductive potential in a variety of

studies: HA [8, 9], β-TCP [10, 11] and BCP – the mixture of HA and TCP [12, 13]. This is

possibly because that the presence of a calcium phosphate source is a prerequisite for

heterotopic bone formation to occur. The liberation of Ca2+

, PO43-

, HPO42-

from the

material into the surroundings may cause precipitation of carbonated apatite that

incorporates calcium, phosphate and other ions (Mg2+

, Na+ , CO3

2-), as well as proteins,

and other organic compounds [14, 15], which are necessary for bone formation.

Apart from the chemical composition of the material, the geometry and macrostructural

properties have shown to play an important role in bone formation. In the case of

macrostructure, porosity is of great importance. Bone formation has never been observed

on a dense sintered ceramic, which does not degrade in vivo, whereas a ceramic with the

same chemical composition, but containing pores, induced bone formation [16, 17].

Generally, the importance of pores inside bone graft substitutes is related to the invasion of

the material by blood vessels, that bring along growth factors and oxygen as well as

osteogenic cells with the capacity to differentiate into osteoblasts [18]. Moreover, with the

presence of “protective areas” in the form of pores, heterotopic bone formation can occur

without being disturbed by high body fluid refreshments or mechanical forces due to

implant movement.

In addition to chemical composition and macrostructural properties, material surface

properties at micro- and nanoscale have shown to be of great importance for the

General discussion

95

osteoinductive potential of the material. Since the micropores (defined as pores with a

diameter smaller than 10 μm) [14, 16] enlarge the surface area, mineral deposition from the

body fluids are expected to be more pronounced – which may be beneficial for

osteoinduction to occur.

No matter what chemico-physical properties these potential osteoinductive materials

possess, all of them were designed to form new bone by recruiting more osteogenic cells

and/or growth factors. It is therefore logical to develop bone substitutes by directly adding

osteogenic cells and/or growth factors to them via tissue engineering. Bone grafts loaded

with osteogenic cells are regarded as materials with osteogenicity－the ability to produce

bone independently. In this thesis, we focus on osteoinductive bone grafts － the bone

substitutes loaded with growth factors.

Among all growth factors, BMPs are the most intensively studied. Both BMP2 and BMP7

are approved by the FDA to be used in the treatment of a variety of bone-related conditions

including spinal fusion and nonunion [19]. However, multiple questions regarding a

suitable carrier for BMPs, dosage, repeat exposure, carcinogenesis and long-term results

have hampered the potential benefits these molecules could offer for bone formation [20].

The present way of delivering BMP2 clinically, the superficial adsorption of BMP2 onto

bone filling materials [21], causes burst release and consequently the transient high local

concentration of BMP2. This kind of delivery of BMP2 is often associated with various

potential side effects such as an excessive stimulation of bone resorption and the induction

of bone formation at unintentional sites [22]. To maximize its osteoinductivity, BMP2

needs to be sustainedly delivered to target sites at a low concentration[23]. Guided by this

theory and given the necessity of a source of calcium and phosphate in the process of bone

formation, we used CaP as the carrier of BMP2 to developed CaP-based slow-delivery

system of BMP2, BMP2-cop.BioCaP (chapter 2) and BMP2-BioCaP (chapter 3). The

release kinetics of BMP2 in these materials showed a steady controlled release mode.

BMP2-cop.BioCaP proved to function as an effective osteoinducer to improve bone

regeneration in CSBDs, which are 8mm rat cranial defects in this thesis. Furthermore, as an

independent osteoinductive bone substitute, BMP2-BioCaP granules showed significant

ectopic bone formation in subcutaneous sites in rats.

Since BMPs are not bony tissue-specific [24], their localized (vs. systemic) and

release-controlled (vs. uncontrolled) delivery is necessary to prevent any undesired and

uncontrolled ectopic bone formation in non-bony tissues in the body [25]. Besides

BMP2-cop.BioCaP (chapter 2) and BMP2-BioCaP (chapter 3), there are therefore many

other systems developed to achieve a localized and release-controlled BMP2 delivery

system

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96

Alginate, is a non-immunogenic polysaccharide found abundantly in the surface of

seaweeds, have been used in a wide range of tissue engineering applications due to its

gel-forming properties [26]. BMPs can be encapsulated into alginate matrices and its

release mode can be modulated by different parameters such as particle size, viscosity and

chemical composition. Liew et al. [27] in a recent investigation found that particle size

affected the extent of burst release and the higher the viscosity the slower the encapsulant

release.

Unlike natural polymers and collagen, synthetic biodegradable polymers pose no danger of

immunogenicity or possibility of disease transmission. A number of synthetic

biodegradable polymeric delivery systems for BMPs were developed [28, 29]. Particularly,

Nano- and micro-particles from synthetic polymers have attracted much attention for the

localized and release-controlled delivery of growth factors due to their attractive tendency

to amass in sites of inflammation [30]. In a recent example of a combined localized and

release-controlled delivery system, poly D, L-lactide-co-gycolic acid (PLGA) nanospheres

immobilized onto prefabricated nanofibrous poly L-lactic acid (PLLA) scaffolds were used

to load and deliver rhBMP-7 [31, 32]. BMP7 delivered from nanospheres-scaffolds induced

significant ectopic bone formation while passive adsorption of the protein into the scaffold

resulted in failure of bone induction either due to the loss of protein bioactivity or its rapid

release from the scaffolds upon implantation in vivo.

We have to bear in mind that not only material properties themselves, but also other factors

have an great impact when studying osteoinduction: for instance the animal model and

implantation site. Yang and co-workers tested the performance of sintered BCP ceramics in

five different animal models at heterotopic locations in a single study. Until day 120, in rats,

rabbits and goats, only dense fibrous connective tissue encapsulating the ceramics and loose

connective tissue inside the pores were observed – without signs of bone formation.

However, in dogs and pigs, bone formation was found in implants retrieved as early as 45

days after implantation. Extensive amounts of bone were found at day 120 mainly in the

pores of the materials implanted in pigs [12]. This study showed that larger animals yielded

more bone than smaller ones, with exception of the goat where no bone formation was

observed.

Several studies have also investigated the osteoinductive capacity of a material, depending

on the implantation site. No obvious bone formation was found after four months of

subcutaneous implantation of a BCP ceramic in goats, whereas intramuscularly, bone was

induced in seven out of ten implants in the same animals [33]. These studies suggest that at

intramuscular locations, bone formation occurs more frequently – or at least at a higher rate.

In this thesis, although we used the more challenging animal model – the rat and the more

challenging implantation sites – subcutaneous implantation, we still gained satisfactory

General discussion

97

bone regeneration using our materials – BMP2-cop.BioCaP (chapter 2) and BMP2-BioCaP

(chapter 3). This supports our hypothesis on the osteoinductivity of our materials.

Although these models were chosen in such a way that they resembled the clinical situation

as closely as possible, only clinical trials will be able to provide the proof for the relevance

of osteoinductivity in human patients. Although ectopic bone formation in animals is used

to judge if a material is osteoinductive material, we need to further consider the influence of

the animal model and implantation sites when studying osteoinductive materials

To repair bone defects with local infection, it is necessary to produce bone-filling-materials

with both osteoinductivity and antibacterial properties. The most common way is to

encapsulate both osteoinductive agents and antibiotics into carrying materials. Mostly, both

the encapsulated osteoinductive agent and antibiotic are released simultaneously and are

largely dependent on the permeability and the degradability of the carrying materials.

Calcium sulfate, a widely used bone-defect-filling material, is also frequently adopted as an

antibiotic carrier for the treatment of infected bone defects [34]. It has many advantages

such as low price, a high level of biodegradability, good biocompatibility [35] and high

osteoconductivity. Wang et al. used calcium sulfate to carry BMP2 and vancomycin using

internal co-encapsulation [36]. In a bone defect in the proximal tibia, this material

significantly augmented new bone formation compared to the control [36]. Besides the

advantages of calcium sulfate, we should also bear in mind is that this kind of material

usually forms a solid block and lacks of porous structure, which may hinder the ingrowth of

bone tissues.

Compared to simultaneous release of osteoinductive agents and antibiotics, it is more

preferable to deliver the osteoinductive and antibacterial drugs from the bone-filling

materials according to the aim of the clinical application and optimal delivery mode of each

drug. For example, an antibacterial drug is encapsulated into a carrying material while in

the same system an osteoinductive drug superficially adsorbed onto its surface, or vice

versa. The two carrying modes can realize different aims: the former mode is mainly aimed

for promoting bone regeneration with a prevention of potential infection, while the latter

mode is mainly aimed for suppressing an existing bacterial activity and thereafter

promoting bone regeneration. Most of the current studies with a mixed carrying mode for

BMP2 and antibacterial drugs focused on bone regeneration with the prevention of

potential infection. Song et al. developed a pHEMA [(poly(2-hydroxyethyl

methacrylate)]/nHA (nanocrystalline hydroxyapatite) composite [37]. In this composite,

nHA was added to enhance the osteoconductivity of the composite. The encapsulated

vancomycin was released in a sustained manner over 2 weeks, which could significantly

inhibit the growth of Escherichia coli. The BMP2 preabsorbed onto the

pHEMA-nHA-vancomycin composite was continuously released over 8 days, which

induced osteogenic differentiation of C2C12 cells [38]. In critical rat femoral segmental

Chapter 6

98

defects, the authors showed that the pHEMA-nHA-vancomycin-BMP2 composites could

achieve full bridging with substantially mineralized callus and partial restoration of

torsional strength [39]. On the other hand, as abovementioned, the superficially adsorption

is less favorable for the osteoinductive efficiency of BMP2. In another study, the authors

tried to modify the carrying material to slow down the release of the superficially adsorbed

BMP2. Zhou et al. used zein, a major starch storage protein found in corn, as a carrying

material for antibacterial HACC and BMP2 [40]. 10% HACC was encapsulated into zein,

which showed a strong antibacterial effect without significantly compromising cell

proliferation. Different amounts of mesoporous silica SBA-15 nanoparticles were added

into zein in order to provide large and highly ordered pores and uniform tunable channels

[41]. The release of the superficially adsorbed BMP2 was significantly slowed down with

the higher ratio of mesoporous silica SBA-15 nanoparticles. In a radial bone defect model

(20 mm in length and 5 mm in diameter) in rabbits, zein-HACC-S20-BMP2 composite

almost fully repaired the bone marrow cavity after 12 weeks. The authors concluded that

Silica/HACC/zein scaffolds with both antibacterial and osteoinductive activities had an

immense potential in orthopedics and other biomedical applications [42].

Unlike the mode mentioned above, in chapter 3 we designed the BMP2-BioCaP/HACC

complex with an osteoinductive drug encapsulated into a carrying material and an

antibacterial drug superficially adsorbed onto its surface, aiming to suppress existing

bacterial activity and thereafter promoting bone regeneration. HACC, a strong antibacterial

drug was adsorbed on the BMP2-BioCaP granules so as to achieve a burst release, which

was designed to rapidly kill residual bacteria in infected bone defects without resulting in

bacterial resistance. On the other hand, to function as an effective osteoinductive agent,

BMP2 needs to be released slowly and continuously at a low concentration [23]. Based on

this theory, we developed BMP2-BioCaP granules [43] where BMP2 was internally

incorporated into the BioCaP granules. BMP2 was therefore released slowly and

continuously with the undergoing degradation of BioCaP granules in vivo. The bone

formation observed in the BMP2-BioCaP/HACC complex also supports the optimal

delivery mode of BMP2.We therefore conclude that BMP2-BioCaP/HACC complex is a

sequential release system with a burst release of a powerful antibacterial

agent-Hydroxypropyltrimethylammonium chloride chitosan (HACC) followed by a

controlled release of BMP2. This release system is in line with the optimal delivery mode

of both HACC and BMP2, which makes BMP2-BioCaP/HACC complex be able to rapidly

kill residual bacteria and thereafter induce new bone formation so as to repair infected bone

defects.

Limitations and future perspective

General discussion

99

At this point the question remains on how to progress growth factor-based bone tissue

engineering strategies into widespread adoption in clinical practice. For this to happen, a

number of limitations and further investigations need to be considered. Firstly, it is

important to bear in mind that our osteoinductive biomaterials (BMP2-cop.BioCaP and

BMP2-BioCaP) are available in the shape of granules. They can induce satisfactory bone

formation in defects with intact bony walls. However it is very difficult for them to induce

bone formation in an environment without supporting bony walls, such as alveolar ridge in

need of augmentation or bone nonunion. Their clinical application in bone tissue

engineering is therefore limited and further effort is needed to

produce defect-matching scaffolds, for example by applying 3-dimensional printing

technique.

Secondly, although bone formation in an ectopic model, such as subcutaneous pockets in

rats, is considered as golden standard to confirm the osteoinductivity of a biomaterial, it

fails to exam if the biomaterial can functionally repair bone defects. In other words, we also

need bone-defect models to evaluate if the repaired bone tissue can perform the original

function.

Thirdly, because the pathogenesis of infections in infected bone defects is a complex

process and involves interactions between the pathogen, biomaterial, and host, the in-vitro

assays do not account for host defense and some other in-vivo factors. In our ongoing

studies, in-vivo infected-bone-defect models will be utilized to evaluate the antibacterial

efficacy of BMP2-BioCaP/HACC complex.

Conclusion

In summary, it is clear that current strategies discussed in this thesis are less than ideal, but

they are useful explorations in the field of bone tissue engineering. Based on the discoveries

in this thesis, we have not only favorable preclinical outcomes for our osteoinducer, the

osteoinductive and antibacterial biomaterials, but also a reliable radiological method ─

CBCT to clinically evaluate bone regeneration. It builds a solid foundation for the

translation of our osteoinductive and antibacterial biomaterials into clinical practice.

Chapter 6

100

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engineering. Biomaterials, 2003. 24(13): p. 2287-93.

29. Saito, N., et al., Synthetic biodegradable polymers as drug delivery systems for bone morphogenetic

proteins. Adv Drug Deliv Rev, 2005. 57(7): p. 1037-48.

30. Lee, S.H. and H. Shin, Matrices and scaffolds for delivery of bioactive molecules in bone and cartilage

tissue engineering. Adv Drug Deliv Rev, 2007. 59(4-5): p. 339-59.

31. Wei, G., et al., The enhancement of osteogenesis by nano-fibrous scaffolds incorporating rhBMP-7

nanospheres. Biomaterials, 2007. 28(12): p. 2087-96.

32. Ma, P.X., Biomimetic materials for tissue engineering. Adv Drug Deliv Rev, 2008. 60(2): p. 184-98.

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33. Habibovic, P., et al., Osteoinduction by biomaterials--physicochemical and structural influences. J Biomed

Mater Res A, 2006. 77(4): p. 747-62.

34. Kanellakopoulou, K., et al., Treatment of experimental osteomyelitis caused by methicillin-resistant

Staphylococcus aureus with a synthetic carrier of calcium sulphate (Stimulan) releasing moxifloxacin. Int J

Antimicrob Agents, 2009. 33(4): p. 354-9.

35. Sanicola, S.M. and S.F. Albert, The in vitro elution characteristics of vancomycin and tobramycin from

calcium sulfate beads. J Foot Ankle Surg, 2005. 44(2): p. 121-4.

36. Wang, Y., et al., Assessing the character of the rhBMP-2- and vancomycin-loaded calcium sulphate

composites in vitro and in vivo. Arch Orthop Trauma Surg, 2011. 131(7): p. 991-1001.

37. Song, J., et al., Elastomeric high-mineral content hydrogel-hydroxyapatite composites for orthopedic

applications. J Biomed Mater Res A, 2009. 89(4): p. 1098-107.

38. Li, X., et al., pHEMA-nHA encapsulation and delivery of vancomycin and rhBMP-2 enhances its role as a

bone graft substitute. Clin Orthop Relat Res, 2013. 471(8): p. 2540-7.

39. Skelly, J.D., et al., Vancomycin-bearing synthetic bone graft delivers rhBMP-2 and promotes healing of

critical rat femoral segmental defects. Clin Orthop Relat Res, 2014. 472(12): p. 4015-23.

40. Strobel, C., et al., Sequential release kinetics of two (gentamicin and BMP-2) or three (gentamicin, IGF-I

and BMP-2) substances from a one-component polymeric coating on implants. J Control Release, 2011.

156(1): p. 37-45.

41. Dai, C., et al., Osteogenic evaluation of calcium/magnesium-doped mesoporous silica scaffold with

incorporation of rhBMP-2 by synchrotron radiation-based muCT. Biomaterials, 2011. 32(33): p. 8506-17.

42. Zhou, P., et al., Enhanced bone tissue regeneration by antibacterial and osteoinductive silica-HACC-zein

composite scaffolds loaded with rhBMP-2. Biomaterials, 2014. 35(38): p. 10033-45.

43. Liu, T., et al., Cell-mediated BMP-2 release from a novel dual-drug delivery system promotes bone

formation. Clin Oral Implants Res, 2014. 25(12): p. 1412-21.

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Chapter 7

General Summary

Chapter 7

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General Summary

Nowadays, many patients with missing teeth or bone defects have gained satisfactory

outcomes from the development of dental implants and bone substitutes. However, for

patients with well-recognized risk factors, such as diabetes, metabolic bone disorders, or

with bone defects that are too large to self-heal, or with locally infected bone defects, we

are in significant need to improve the properties of dental implants and bone substitutes to

achieve more satisfactory osseointegration and bone regeneration.

To some extent, osseointegration of dental implants and bone regeneration caused by bone

substitutes share the same essence, which is forming new bone along the material─either

dental implants or bone substitutes. Based on what we understand about the three key

factors (Osteogenic cells, scaffolds, growth factors) in bone tissue engineering, dental

implants and bone substitutes can function as scaffolds in the process of bone formation, we

believe that it can be an effective strategy to introduce growth factors to these scaffolds in

order to improve bone formation─either osseointegration or bone formation.

Based on this theory, our group has developed various osteoinductive materials by

introducing a well-known and effective growth factor─BMP2 to many kinds of calcium

phosphate materials. Firstly, we developed a BMP2-incorporated biomimetic calcium

phosphate coating which can be applied to functionalize biomaterials, such as dental

implants. The biomaterial is first immersed in a 5-fold-concentrated simulated body fluid

for 24 hours at 37°C to gain a fine, dense layer of amorphous calcium phosphate on it.

Serving as a seeding substratum for the following deposition, the biomaterial with the first

amorphous layer is then immersed in a supersaturated calcium phosphate solution with

BMP2 for 48 hours at 37°C to gain a more substantial crystalline layer with incorporated

BMP2. This biomimetic coating was proven by the success in introducing osteoinductivity

to a broad range of biomaterials, such as metallic, inorganic, polymeric materials that have

completely different geometries, topographies and surface chemistries. Albeit so, this type

of biomimetic coating on bone-defect-filling materials has the limitation that its growth

relies highly on the proper surface roughness and/or active surface chemistry of the bone-

defect-filling materials. We therefore modified the biomimetic coating technique and

developed a novel granule to break through these limitations. The process of preparing the

two-layer coating is repeated three times to obtain a novel 3-dimensional layer-by-layer

assembled BMP2-coprecipitated biomimetic calcium phosphate granules (BMP2-

cop.BioCaP). As an osteoinducer, it functions perfectly to introduce osteoinductivity to

osteoconductive bone substitutes, simply by being mixed with them, thus enhancing bone

regeneration. However, it cannot be used as an independent bone-defect-filling material

because it degrades so fast that it cannot function as scaffold for osteogenic cells to attach

and form new bone. For this reason, we modified the protocol for preparation of BMP2-

General Summary

105

cop.BioCaP and successfully developed a novel BMP2-incorporated biomimetic calcium

phosphate (BMP2-BioCaP). Compared to BMP2-cop.BioCaP, it showed a slower

degradation rate which matched better with the ingrowth of bone tissue in the process of

bone regeneration.

This thesis is built on the various applications of these three generations of biomimetic

materials. Firstly, we’ve already conducted intensive research on the BMP2-incorporated

biomimetic calcium phosphate coating in previous studies. In this thesis, besides this

coating, we also reviewed many other coatings to enhance and accelerate the

osseointegration of metallic implants. Secondly, we examined the function of BMP2-

cop.BioCaP as an osteoindcuer in a preclinical study. We combined BMP2-cop.BioCaP

with clinically often used osteoconductive bone substitute─biphasic calcium phosphate

(BCP) in order to introduce osteoinducivity to the bone substitutes and evaluate if it can

improve bone regeneration in critical-sized bone defects (CSBD). Thirdly, we modified the

BMP2-BioCaP as not only an osteoinductive bone substitute, but also an antibiotic carrier.

The hypothesis was that it can function as an osteoinductive and antibacterial biomaterial

system for treatment of infected critical-sized bone defects. Both in-vitro and in-vivo

experiments are performed to evaluate its osteoinductivity and antibacterial activity.

The aims described in the first chapter are addressed as follows:

1. To introduce osteoinductivity to clinically used BCP by a novel osteoinducer and

evaluate if it can improve bone regeneration in CSBD. Furthermore, to explore the

mechanism of the osteoinducer enhancing bone regeneration.

BCP is a clinically often used bone-defect-filling material with excellent osteoconductivity.

However, it may be not enough for some cases in clinical practice, such as patients

associated with diabetes, local osteoporosis and metabolic bone disorder which can

compromise bone healing. It is often mixed with autologous bone to gain osteoinductivity

for advanced bone regeneration. Due to the limitations of applying autologous bone, we

previously developed the BMP2-cop.BioCaP in aim to replace it, which can slowly release

BMP2 at a steady rate from the 3rd

day until 35th

day. In chapter 2, when repairing a CSBD

model─8mm rat cranial defect, BCP mixed with BMP2-cop.BioCaP or autologous bone

showed a comparable amount of newly formed bone at both 4 and 12 weeks, which was

significantly more than BCP alone. This confirmed that BMP2-cop.BioCaP, a potential

osteoinducer, can function as effectively as autologous bone to improve bone regeneration

in CSBD.

To better understand the mechanism of BMP2-cop.BioCaP improving bone regeneration,

we need to get deeper insights into the results. In group of BCP alone, the new bone was

only found on the periphery of the bone defect in contact with the host bone, which was

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named as osteoconductive bone. Whereas, in groups of BCP combined with BMP2-

cop.BioCaP or autologous bone, newly formed bone was found not only osteoconductive

bone, but also osteoinductive bone─the new bone in the center of the bone defect without

any contact with the host bone. What’s more, osteoinductive bone grew in time till 12

weeks as observed in this study, but osteoconductive bone stopped growing after 4 weeks.

Therefore, we believe that the mechanism of BMP2-cop.BioCaP improving bone

regeneration in CSBD was that (1) besides the bone-growing center along host bone lining

the defect, it brought a new bone-growing center in the middle of defect; (2) it prolonged

grow profile of newly formed bone thanks to the spontaneous growth of osteoinductive

bone.

2. To develop an antibacterial and osteoinductive biomaterial for treatment of infected

CSBD and to evaluate its antibacterial activity and osteoinductivity in vitro and in vivo.

Treatment of infected critical-sized bone defects remains a great challenge in orthopedic

and oral and maxillofacial surgery because of the residual bacteria that is impossible to

completely eliminate by debridement and beyond-self-healing bone defects. To overcome

these two difficulties and consequently repair the infected critical-sized bone defects, we

believe the ideal local biomaterial system should meet the following requirements: 1) The

biomaterial system can function as both antibiotic carrier and bone substitute, to not only

clear the infection but to also contribute to the subsequent bone regeneration process; 2)

The antibiotics used for local delivery should have a broad spectrum of activity and a low

rate of bacteria resistance; 3) The antibiotic should also be delivered to its optimal

concentration, at which it reaches the balance between cellular toxicity and antibacterial

activity; 4) Osteoinductive bone grafts are more favorable for improving bone regeneration;

5) The release kinetics of antibiotic and osteoinductive agents should meet their optimal

delivery mode respectively.

In chapter 3, we used the osteoinductive BMP2-BioCaP as the carrier of a powerful

antibacterial agent─hydroxypropyltrimethylammonium chloride chitosan (HACC) to

fabricate a BMP2-BioCaP/HACC complex. HACC showed strong antibacterial acitivity to

MRSA. Although many antibiotics are naturally associated with the issue of their

cytotoxicity, we could still determine the most balanced concentration of HACC─40μg/mL,

at which HACC can kill bacterial without harming pre-osteoblasts. At this concentration,

HACC also did not show negative influence on proliferation and BMP2-induced

differentiation of pre-osteoeblasts. The corresponding amount of HACC was therefore

loaded on the BMP2-BioCaP to fabricate an antibacterial and non-cytotoxic BMP2-

BioCaP/HACC complex. The release kinetic of this complex confirmed it as a sequential

release system: burst release of HACC and followed by controlled release of BMP2. Given

that long-term usage of low-dosage antibiotics is highly associated with bacterial resistance,

we logically speculate that burst release of a powerful antibiotic to kill possible site-

General Summary

107

infection related bacteria. What’s more, it is consensus that slow release BMP2 at a

relatively low concentration is best for its effectiveness. Therefore, it delivered both HACC

and BMP2 at their optimal delivery mode respectively, which guaranteed its antibacterial

activity and osteoinducvitity in vivo. We observed that new bone formation was induced by

the BMP2-BioCaP/HACC complex in a model of subcutaneous sites in rats.

3. To review the biological process of osseointegration and offer an overview of the

coatings designed for improving osseointegration of metallic biomaterials

Like introducing BMP2 into bone substitute materials is an efficient and effective way for

mateirals to gain osteoinductivity, osseointegration of metallic biomateirals can also be

improved by incorporating BMP2 into metallic biomaterials coating. In chapter 4, we

firstly reviewed the biological process of osseointegration at molecular level. The first step

of the body’s reaction to a biomaterial is the adsorption of the molecules onto the surface of

biomaterials from the surrounding fluid, which is followed by attachment and proliferation

of mesenchymal stem cells. Thanks to various growth factors, the multipotent mesenchymal

stem cells can differentiate into osteoblasts. Thirdly, osteoblasts produce cross-linked

collagen to form the organic matrix of bone, which is then calcified to form a mineralized

bone matrix. The fourth stage is the remodeling phase, in which the woven bone is

transformed into lamellar bone and osseointegration of metallic implants is finally

established. Various kinds of coatings on implant surfaces have been designed to regulate

these four phases to promote osseointegration, such as extracellular matrix for improving

recognition and adhesion of cells, BMP2 for enhancing proliferation and differentiation of

pre-osteoblasts fluoride for promoting mineralization and matrix, bisphosphonates for

regulating bone remodeling and so on. However, most of them are on the stage of

preclinical studies, only hydroxyapatite and bisphosphonate coatings have been evaluated

in clinical trials.

4. To evaluate the accuracy of measuring bone thickness surrounding dental implants

and the reliability of assessing existence and completion of osseous integration of

augmentation material using a Cone beam computered tomography (CBCT).

In clinical practice, CBCT is widely used for pre-operative assessment of bone quantity and

quality in the region of interest, and for post-operative evaluation of bone regeneration after

sinus lift augmentation and osseointegration of dental implants. In chapter 5, the accuracy

of CBCT was evaluated by comparing the same measurements with golden

standard─histological images. The results indicated that this CBCT system allows reliable

measurements of peri-implant bone thickness at an accuracy of half a millimeter and

assessing the existence and integration of bone augmentation materials. However, it is not

possible to evaluate whether the implant is covered completely by hard tissue.

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109

Hoofdstuk 7

Samenvatting

110

Samenvatting

Hoewel veel patiënten baat hebben bij de huidig beschikbare tand en bot vervangende

implantaten is er toch nog een vrij grote groep die vanwege medische problemen, zoals

diabetes, te grote (kaak)botdefecten, of ontstoken kaakbot, daarmee niet goed geholpen

kunnen worden. Er is dus behoefte aan implantaten, botvervangers en botregeneratie

materialen die zowel een betere integratie als herstel van botherstel (regeneratie) laten zien:

beide aspecten berusten overigens op eenzelfde eigenschap, namelijk het vormen van nieuw

bot op het grensvlak van bot – implantaat en bot - regeneratie materiaal.

Een van de methodes om nieuwvorming van bot te bevorderen is gelegen in de toepassing

van biologische groeifactoren zoals het reeds geruime tijd bekende en klinisch toegepaste

BMP-2. We hebben in onze groep een nieuwe methode ontwikkeld om een efficiënt

gebruik van dit BMP-2 in de tandheelkunde (orale implantologie) in bot regeneratie

mogelijk te maken door dit BMP-2 niet als stand-alone materiaal te gebruiken maar als

deklaag op voornoemde tandwortel en bot implantaten. In principe volgen we de natuur –

vandaar ook de naam “biomimetic coating” – zoals die ook in humaan bot (en zelfs door

schelpdieren, maar dan met carbonaat in plaats van fosfaat!) met veel succes wordt

toegepast. De deklaag bestaat in principe uit een dun (ca 1/20 ste millimeter) laagje

gemineraliseerd calcium fosfaat met ongeveer dezelfde minerale samenstelling als

menselijk tand en bot weefsel: in dit laagje bevindt zich de benodigde hoeveelheid BMP-2.

De methode is in principe eenvoudig: we brengen het te bedekken implantaat in een

waterige oplossing, bij kamertemperatuur, die zowel de benodigde calcium als fosfaat ionen

bevat als de vereiste concentratie aan BMP-2 moleculen. Na het te coaten implantaat in de

vloeistof te hebben ondergedompeld, manipuleren we deze oplossing zodanig, dat de

opgeloste componenten als een coating neerslaan op het implantaat oppervlak. We kunnen

de exacte eigenschappen van deze coating, zoals de dikte, de samenstelling, oplosbaarheid,

de hoeveelheid geïncorporeerd BMP2, zodanig regelen dat de coating na implantatie

gedurende een bij het implantaat en de patiënt behorende tijdsduur de gewenste

hoeveelheid BMP2 per tijdseenheid afgeven aan de weefsels rondom het implantaat.

In dit proefschrift noemen we deze methode de BMP2-BioCaP coating. In de praktijk bleek

daarnaast de behoefte te bestaan om een coating te hebben die niet alleen preciezer met de

tijd wisselende hoeveelheden BMP2 afgeeft maar daarnaast ook antibiotica kan vrijmaken

ter bestrijding van infecties rondom het implantaat. Dit bereiken we door meerdere (‘layer-

by-layer”) coatings aan te brengen, ieder met verschillende oplossnelheden, met

verschillende hoeveelheden BMP2, al of niet met antibiotica. De op deze wijze verkregen

“multipurpose” coating hebben we in dit proefschrift verder uitontwikkeld zodat we

uiteindelijk een geheel nieuwe deklaag tot onze beschikking hebben. Een coating die niet

alleen stevig genoeg was en goed hechtte aan het implantaat oppervlak, maar daarnaast ook

Samenvatting

111

nog in staat was na implantatie van het implantaat of botvervanger in een geprogrammeerd

tempo op te lossen zodat precies de goede hoeveelheid BMP-2 op de juiste tijd in de ruimte

tussen implantaat en omliggend (kaak)bot kan worden vrijgemaakt. Dit zet dan aan tot het

nieuw vormen van bot ter vervanging van de aangebrachte coating. Deze nieuwe

“multipurpose coating” hebben we de naam BMP2-BioCaP gegeven.

We vergelijken in dit proefschrift in een literatuurstudie onze beide coatings met andere,

soms klinisch al toegepaste implantaatoppervlaktes, waarvan geclaimd wordt dat ze

osseointegratie vertonen. Daarna bestuderen en vergelijken we onze beide (BMP2-BioCaP

en BMP2-cop.BioCaP) coatings met elkaar. Aangezien we ook geïnfecteerde botdefecten

willen herstellen door gebruik van deze botregeneratie materialen is ons in-vitro en in-

vivo onderzoek tevens gericht op de inbouw van antibiotica; en dit is alleen mogelijk met

onze nieuwe “layer-by-layer” BMP2-BioCap coating.

Hoofdstuk 1 en 2 bespreekt een specifiek botimplantaat, namelijk het BCP (het in het

lichaam oplosbare, ofwel resorbeerbare, biphasic calcium fosfaat), dat reeds geruime tijd in

de handel is en met redelijk succes klinische toepassing vindt. Ondanks deze goede

eigenschappen is BCP niet in staat zonder hulp van buitenaf – autoloog bot, ofwel bot van

de patiënt zelf – voldoende nieuw bot te regenereren. BCP wordt derhalve veelvuldig

gemengd met autologe botsnippers om vermogen heeft om (1) grotere defecten en (2)

defecten bij niet geheel gezonde patiënten (diabetes, osteoporose) te behandelen. Gebleken

is dat BCP korrels gemengd met BCP korrels met BMP2-cop.BioCaP hetzelfde resultaat

geeft als BCP gemengd met autologe botsnippers, en dat beide toevoegingen beter zijn dan

alleen BCP. Het essentiële verschil met alleen BCP is dat beide mengsels niet alleen

nieuwvorming van bot geven in contact met het eigen bot, maar ook osteoinductie vertonen,

dat wil zeggen dat er ook bot gevormd wordt in het centrum van het te vullen botdefect, een

plaats waar geen eigen bot aanwezig is – kortom een betere en snellere vulling van het

defect. En het essentiële voordeel van onze compositie is natuurlijk dat we geen autoloog

bot meer nodig hebben!

Echter, met een enkelvoudige met BMP2 geïncorporeerde CaP deklaag, het BMP2 BMP2-

cop.BioCaP, kunnen we het infectieprobleem niet aanpakken. Immers daarvoor moet er

behalve de botgroeifactor BMP2 een geschikt antibioticum bij. De vraag is nu hoe we

concentratie en afgifte kinetiek daarvan zorgvuldig kunnen definiëren.

Deze vraag behandelen we in Hoofdstuk 3: Het osteoinductieve BMP2-BioCaP voorzien

we opeenvolgend van deklagen met respectievelijk BMP2 en antibioticum als actieve

component. Daarvoor hebben we gekozen voor HACC (hydroxypropyltrimethylammonium

chloride chitosan) als model: HACC is een breed spectrum antibioticum tegen MRSA,

S.Aureus, en P.gingivalis. We slaagden er in om BMP2-BioCaP te laden met HACC

zodanig dat het resulterende BMP2-BioCaP/HACC complex enerzijds de activiteit van het

112

BMP2 niet beïnvloedde en dat anderzijds het afgifte patroon snel was voor het antibioticum

en langzaam voor BMP2, zodanig dat beide actieve componenten optimaal tot hun recht

kwamen. Een en ander bevestigden we in een dierproefmodel.

Waar we ons in het vorige Hoofdstuk 3 gericht hebben op BCP als een te verbeteren

botimplantaat is onze aandacht in Hoofdstuk 4 op de vraag hoe een metalen implantaat

kunnen voorzien van zo’n coating. We bespreken daartoe enkele essentiële aspecten van het

osseointegratie proces, waaruit we de conclusie trekken dat na een aantal tussenstappen -

adsorptie van biomoleculen uit de omringende vloeistof, gevolgd door aanhechting en

proliferatie van mesenchymale stamcellen, die differentiëren tot osteoblasten, waarna

uiteindelijk het nieuwe bot gevormd wordt. Coatings op het metaal die dit proces

ondersteunen zijn tot dusver op CaP gebaseerd, en in onze groep betreft dat het eerder

besproken BMP2-BioCaP. Echter, er zijn tot dusverre geen klinische studies verricht met

deze gecoate metaal implantaten. Voor zo’n klinische studie is vereist dat de bot ingroei

rondom een (tand)implantaat nauwkeurig kan worden gemeten. Omdat we zo’n

meetmethode ook nodig hebben voor het meten van het effect van onze botimplantaten (de

met BMP2-cop.BioCaP of BMP2-BioCaP gecoate korrels BCP) op de ingroei in

botdefecten, is het van groot belang zo’n meetmethode ter beschikking te hebben.

Dat doel is het onderwerp van Hoofdstuk 5. We toetsten of de zogeheten CBCT methode –

een afkorting van Cone Beam Computerized Tomography – daarvoor geschikt is. De reden

dat we voor deze methode kozen is dat CBCT in de tandheelkundige implantologie reeds

veelvuldig toegepast in de evaluatie van sinus lift chirurgie en osseointegratie van metalen

implantaten. We hebben deze methode onderworpen aan een zorgvuldige histologische

evaluatie, en onze conclusie is dat we met een nauwkeurigheid van een halve mm een goed

inzicht moeten kunnen krijgen in de effectiviteit van onze gecoate bot en tandimplantaten,

dat wil zeggen met BMP2 en al dan niet met antibioticum. De conclusie van dit hoofdstuk

is dan ook dat we alles in huis hebben om een goede klinische proef te beginnen.

Als we de algehele conclusie in enkele woorden willen samenvatten, zou dat kunnen

worden omschreven als volgt: ons onderzoek heeft een tweetal nieuwe strategieën

geformuleerd voor effectiever (kaak)bot vullers en sneller ingroeiende tandimplantaten,

namelijk door specifieke CaP coatings toe te passen met botgroei versnellende factoren

(BMP2) en ontstekingsremmende antibiotica (HACC). En van praktisch nut is dat we

beschikken over een getoetste CBCT methode om deze effecten te meten.

113

Acknowledgement

Dankwoord

114

The outcome of my four-year PhD project and this thesis could not have been accomplished

without the support of many individuals. Besides the honor of collaborating with them, I

would like to express my appreciation for their great support and help.

First and foremost, I would like to express my deepest gratitude to my promoter Prof.

Daniel Wismeijer. Dear Daniel, thank you for offering me the opportunity to do research

in ACTA. I am proud of being a member in your department, one of the top groups in the

field of implantology. Thank you for providing me opportunities to learn from various

conferences, training courses and lectures. I have learnt so much from you, not only from

your broad knowledge and solid expertise in implanology, but also from your critical

thinking and hard working attitude. Your extraordinary achievements as a worldwide

famous implantologist encouraged me and set a benchmark for my career.

I also deeply appreciate Dr. Yuelian Liu. Dear Maria, you have been supportive from the

first day since I began working in ACTA. Ever since, you have supported me not only

academically but also emotionally by guiding me through the rough road to finish this

thesis. You are a great mentor for work. Thank you for encouraging me to apply the IADR

award when I thought it was almost not possible, for inspiring me to be confident of writing

grant proposal, for offering me opportunity to attend various conferences, for preparing me

for the public presentations. During the most difficult times of writing this thesis, you gave

me the support and freedom I needed to move on. Apart from work, you are much more

than a supervisor to me. Instead, you are more like a caring parent. I am so happy that we

can share those important moments of my life: the first article accepted, my wedding, etc.

Whenever and wherever I have problems, you have always been supportive and trying your

best to help me. I really can not thank you enough for everything.

To Prof. Klass de Groot: Dear Klass, thank you for your contribution on the Dutch

Summary of this thesis. Moreover, I enjoyed the discussion we had in those group research

meetings, not only for the insight and knowledge, but also for the way you think. Although

we did not meet often, I have learnt so much from you.

To Prof. Ernst Hunziker: Dear Ernst, it was so inspiring to talk with you. Although your

questions were always sharp and critical, it helped to make me aware of the weakness in my

knowledge system. To answer your questions, I always had to struggle to think out of box,

which inspired me with many new ideas and gave me insights on how to understand, think

and solve the problems I had in research. I believe that the “torture” I had from your

questions is a catalyst to make my research more mature. Moreover, I really enjoyed our

conversation about philosophy, again it was inspiring, joyful and comforting.

To Dr. Gang Wu: Dear Gang, you led me to the field of science and research from the very

beginning of experiment design and even reading chemical labels. Your critical thinking in

Acknowledgement

115

research and writing helped me make enormous progress in the past years. You always

taught me all technical details very patiently, encouraged me to try more new experiments.

It was amazing when you help me analyze data and make it into an insightful and

interesting story. I sincerely appreciate it. Jing, thank you and Gang for giving me those

very useful tips of living in the Netherlands when I first arrived. You made my starting in a

new country much easier.

To Dr. Dongmei Deng. Dear Dongmei, thank you for offering me your support on my

antibacterial experiments without hesitation. By working together with you, I personally

experienced your rigorous attitude towards science. It is worthwhile to learn for the rest of

my life.

To Prof. Zhuofan Chen: Dear Prof. Chen, thank you for enlightening me the first glance

of research, especially for leading me into the thriving world of dental implantology. I owe

my most sincere gratitude to you. You helped me to correct my first English email, taught

to convert my thinking and behavior from a pre-mature student to an independent

researcher and always corrected my little misbehavior patiently. Thank you a lot for those

philosophy you taught me, such as the spirit of give and take to cooperate with people, to

put myself in other’s shoes. I still benefit from them every day.

To my nice supervisors in Germany: Dear Prof. Jürgen Becker, thank you for accepting as

a guest researcher and guiding me into the international research world. I would like to

express my deep appreciation to you and Mrs Becker for organizing everything for me

when I was in Düsseldorf, Germany. You created the chance for me to learn cell biopsy,

showed me complicated cases of implantology, supported me to join the course of animal

experiments and built up a project specially for me, which became my first scientific article.

Although that was my first time abroad, when you picked up from the airport and later I

saw Mrs Becker have already prepared bed linings and kitchenware for me in my dormitory,

I indeed felt like home. I really can not thank you enough for everything. Without you

opening an international research world for me, I would not have been encouraged to

pursue a PhD in Europe. Dear Prof. Frank Schwarz and Dr. Andreas Künzel, thank you

for helping me finish my first article published in an international journal. Your valuable

supervision, constant encouragement and constructive comments made me confident to

explore on the path of research.

I would like to express my deep gratitude to Prof. Vincent Everts, Prof. Jenneke Klein-

Nulend, Prof. Sue Gibbs, Dr. Cees Kleverlaan, Dr. Astrid Bakker, for support,

comments, suggestions and encouragements throughout my study

My warm gratitude to my professional support in the lab: Dear Cor, Jolanda, Dirk-Jan,

Teun, Ton (Schoenmaker), Ton (Bronckers) Ineke, Behrouz, Marion, Kamran, Leo,

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Arie, Thank you for all your professional support in the past years. Thanks for all the

shared coffee times, lunch and cake times. It is a pleasure to work around you. Special

thanks for Liquan to organize animal experiment in China. Without your help, my animal

study would not have gone so smoothly.

I would like to express my sincere gratitude to my colleague from clinical part: Dear

Kostas, you are always very supportive and willing to share your knowledge whenever I

have questions. I really appreciate it and enjoy talking with you. Dear Afsheen, thank you

for your insightful comments on my draft article and for improving my academic writing

skills. I still keep my first draft and your comments, which constantly reminded me how I

should be careful with the scientific descriptions, and your encouragement and comfort to

me.

To my lovely officemates: Ceylin and Nowal, we sat next to each other in one office for

almost four years, we had all kinds of chat about work, life, culture difference, religion and

of course babies. I enjoyed it and missed it. Especially Ceylin, your stories of raising two

babies during a PhD really encouraged me a lot. You are a role model for me to perfectly

balance work and life.

To my dearest senior fellows: Tie and Xingnan, you took care of me since the first day I

arrived, especially Tie even started to help me apply this PhD position before my arrival.

You shared your experience of work with me and listened to my complaints about

everything, just like two elder brothers to me.

Besides this PhD title, the most precious gain is the friendship from a group of fun girls.

Thank you, Francis, Ana, Alejandra, Yixuan and Yu for organizing the surprising

bachelor party. The day we spent in Den Bosch is one of my most unforgettable days. I will

keep the cup with our photo, and of course our precious friendship forever. Dear Francis,

thank you for even flying to China for my wedding and being my maid of honor. We had so

many to share, preparing the newsletters together for ACTApro, all those girls talks,

gossips and romantic stories….I am and will always be cherishing our friendship for my

lifetime.

I would like to thank all my PhD colleagues. Thank you for creating friendly and cozy

atmosphere in the past years. Thank you for help me with all kinds of problems in the lab

and sharing funny jokes in the coffee room. Thank you, Patrick，Hessam, Bas, Yixuan,

Sara, Angela, Yi, Fei, Ana (Milheiro), Mahshid, Beatric, Jenny, Janak, Marjolein,

Rozita, Ben, Thijs, Caroline, Xin(Zhang), Xiao (Zhang) for your company along this

PhD journey. Dear Patrick, thank you for being a close and reliable friend, listening to me

and always trying to help me. It is a great pleasure to know you. Dear Hui (Chen), you are

Acknowledgement

117

not only a colleague, but also a sweet roomie. I enjoyed the one year when we shared an

apartment. You are such a lovely girl.

I also would like to express my gratitude to my teachers and friends in China. Although

from such a long distance, your constant support and encouragement made each step I took

more steady and confident.

亲爱的爸爸妈妈，我今年三十岁，博士毕业。二十多年来，一直在读书，从你们壮

年到退休。这些年来，无论我做什么决定，你们都相信我，支持我，祝福我。年纪

大一些，才能明白“世上只有父母的爱是无条件的”，无论我遇到任何难处要开口

请你们帮忙，你们总是毫不犹豫的应承下来，尽心尽力的为我做到最好，却从不对

我提任何要求。而今，我三十而立，有了小家庭，也将为人父母，更能明白你们的

付出和不易。从今后换我们来照顾你们，保护你们。

感谢公公婆婆对我们工作生活的关怀。你们不但是我们的精神导师，还给我们的生

活提供了切实的支持。感谢你们的帮助，让我们在异国他乡也生活的舒适惬意。不

管我们在哪，我们的家都是你们的家。

最后，感谢我的丈夫朱秋实。我常说在荷兰最大的收获除了读博士，就是与你相遇、

相识、相知，然后在异国他乡相濡以沫，相依为命。你总是在我沮丧的时候，安慰

鼓励我。在我遇到难处的时候帮我出谋划策。我常常任性，你总是宽容。难以想象，

如果没有你，我要如何才能熬过这四年，如何才能写到这论文的致谢部分。谢谢你

给了我一个家！

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Curriculum Vitae

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Personal information

Name： Dongyun Wang

Date of birth：1985. Dec. 22

Nationality: Chinese

Telephone： +31 616662749

E-mail： [email protected]

Education and professional experience

2012-2016: PhD candidate at Department of oral implantology and prosthetics, ACTA, UV

and UvA University, the Netherlands

2009-2012: Master student of clinical dentistry in implant dentistry at Guanghua school of

stomatology, Sun Yat-sen University, China

2010-2011: Guest researcher in Department of Oral Surgery, Westdeutsche Kieferklinik

Heinrich Heine University, Düsseldorf, Germany

2004-2009: Bachelor student of dentistry, Guanghua school of stomatology, Sun Yat-sen

University, China

Memberships of professional societies

Member of International Association for Dental Research

Member of global Osteology Community

Publication list

1. Dongyun Wang, Andreas Künzel, Vladimir Golubovic, Ilya Mihatovic, Gordon John,

Zhuofan Chen, Jüngen Becker, Frank Schwartz. Accuracy of peri-implant bone

thickness and validity of assessing bone augmentation material using cone beam

computed tomography. Clinical oral investigations. 2013, 17:1601-1609

2. Dongyun Wang, Andreas Künzel, Vladimir Golubovic, Ilya Mihatovic, Gordon John,

Zhuofan Chen, Jüngen Becker, Frank Schwartz. More about accuracy of peri-implant

bone thickness and validity of assessing bone augmentation material using cone beam

Gurriculum Vitae

121

computed tomography. Clinical oral investigations. 2013, 17:1787–1788 (Letter to the

editor)

3. Dongyun Wang, Gang Wu, Xingnan Lin, Yuelian Liu. Coatings for osseointegration of

metallic biomaterials. In: Cuie Wen. Surface coating and modification of metallic

biomaterials. Elsevier. 2015, 11, 345-358

4. Dongyun Wang, Afsheen Tabassum, Gang Wu, Liquan Deng, Daniel Wismeijer,

Yuelian Liu. Bone regeneration in critical-sized bone defect enhanced by introducing

osteoinductivity to biphasic calcium phosphate granules. Clinical oral implants

research. 2016, 00, 1-10

5. Dongyun Wang, Yuelian Liu, Yi Liu, Liquan Deng, Sebastian A.J. Zaat, Daniel

Wismeijer, Gang Wu. A novel bone-defect-filling material with sequential antibacterial

and osteoinductive properties for repairing infected bone defects. (In preparation)

6. Xingnan Lin, Klass de Groot, Dongyun Wang, Qingang Hu, Daniel Wismeijer,

Yuelian Liu. A Review Paper on Biomimetic Calcium Phosphate Coatings. The Open

Biomedical Engineering Journal. 2015, 9: (Suppl 1-M4) 40-48

7. Xin Zhang, Tie Liu, Xingnan Lin, Dongyun Wang, Yi Liu, Yuanliang Huang, Yuelian

Liu. Combination of icariin and BMP-2: a potential osteoinductive compound for bone

tissue engineering. (In preparation)

8. Xingnan Lin, Dongyun Wang, Arjen. J. van Wijk, William. G.M. Geraets, Yuelian Liu.

Comparison of the planimetry and point-counting method for the estimation the area of

interest: a gold standard study. (In preparation)

9. Dongyun Wang, Quan Liu, Zetao Chen, Zhuofan Chen. The effect of oorcine

hydroxyapatite on the behavior of MG63 osteoblast-like cell lines. Journal of Clinical

Stomatology. 2012, 5: 265-267 (Chinese)

Presentations and awards

1. Dongyun Wang, Afsheen Tabassum, Gang Wu, Liquan Deng, Daniel Wismeijer, and

Yuelian Liu. Osteoinducer improves osteogenic efficacy of calcium phosphate bone

substitute. 93rd General Session & Exhibition of the IADR, Boston, USA. March, 2015

(Poster presentation)

Heraeus Kulzer travel award (one of five awards worldwide)

122

2. Dongyun Wang, Afsheen Tabassum, Gang Wu, Liquan Deng, Daniel Wismeijer, and

Yuelian Liu. Osteoinducer Improves Osteogenic Efficacy of Calcium Phosphate Bone

Substitute. 6th Annual MOVE Research Meeting, Amsterdam the Netherlands.

February 2015 (Poster presentation)

3. Dongyun Wang, Gang Wu, Afsheen Tabassum, Daniel Wismeijer and Yue Liu.

Biomimetic osteoinducer promotes bone regeneration in a rat cranial critical-sized

defect model. IADR/PER Congress, Dubrovnik, Croatia. September 2014 (Oral

presentation)

4. Dongyun Wang, Yuelian Liu, Yi Liu, Liquan Deng, Sebastian A.J. Zaat, Daniel

Wismeijer, Gang Wu. In-vitro and in-vivo characterization of a novel bone-defect-

filling BMP2-BioCaP/HACC complex with sequentially antibacterial and

osteoinductive properties. International Osteology Symposium, Monaco. April 2016

(Poster presentation)

phd thesis

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