health - osteoarthritis ac tissue engineering and insertion into joint

8/2/2019 Health - Osteoarthritis AC Tissue Engineering and Insertion Into Joint

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Articular Cartilage: Structure and Regeneration

Jose Becerra, Ph.D.,1,2,* Jose A. Andrades, Ph.D.,1,2,* Enrique Guerado, M.D., Ph.D.,2,3

Placido Zamora-Navas, M.D., Ph.D.,2,4 Jose M. Lopez-Puertas, M.D.,2,5 and A. Hari Reddi, Ph.D.1,6

Articular cartilage (AC) has no or very low ability of self-repair, and untreated lesions may lead to the devel-opment of osteoarthritis. One method that has been proven to result in long-term repair or isolated lesions isautologous chondrocyte transplantation. However, first generation of these cells implantation has limitations,and introducing new effective cell sources can improve cartilage repair. AC provides a resilient and compliantarticulating surface to the bones in diarthrodial joints. It protects the joint by distributing loads applied to it, sopreventing potentially damaging stress concentrations on the bone. At the same time it provides a low-friction-

bearing surface to enable free movement of the joint. AC may be considered as a visco- or poro-elastic fiber-

composite material. Fibrils of predominantly type II collagen provide tensile reinforcing to a highly hydratedproteoglycan gel. The tissue typically comprises 70% water and it is the structuring and retention of this water bythe proteoglycans and collagen that is largely responsible for the remarkable ability of the tissue to supportcompressive loads.

Introduction

Arthritis is a major challenge for the musculoskeletalhealth. Considerable progress in the treatment ofrheumatoid arthritis, an autoimmune disorder, has beenachieved by the use of antagonists of tumor necrosis factorand cognate receptors by judicious use of monoclonal anti-body therapeutics. On the other hand, osteoarthritis (OA) is acomplex multifactorial disease defying systematic diagnosis,and treatment.1 The integrity of articular cartilage (AC)structure is at the crux of the problem of OA. The symptomsinclude, but not limited to joint pain, impaired and limitedmovement and inflammation and tender joints. OA can belocal in certain joints or more generalized. One of the chal-lenges in OA is the distinction between the original symp-toms and attendant sequelae of reparative response as, forexample, in the synovium is increasingly difficult. The his-tological signs are damage to AC, clefts in cartilage, andincursion of capillaries into the calcified tide mark. Althoughall joints can be affected with OA, some joints are moresusceptible such as hand, spine, hip, knee, and foot. What iscertain in the epidemiology of OA is that the disease is more

prevalent in women than in men and increases with age.14

What can be done about the damaged joints in OA? Can theybe repaired in the least or can one obtain complete regener-ation of the cartilage with fidelity?

Repair is the rapid process to resolve an injury. The re-parative tissue is not identical to the original tissue and thereis no integration of repair tissue with the original tissue.Regeneration is a relatively slow process that recapitulatesdevelopment and morphogenesis and restores completelystructure and function, including integration of the new tis-sue seamlessly to the original.

Regenerative medicine is the emerging discipline ofmedicine based on advances in basic science of developmentand morphogenesis and science of biomaterials and stem cellbiology. The three key ingredients for regenerative medicineand surgery are the inductive signals such as bone mor-phogenetics proteins (BMPs), responding cells, and thescaffolding of extracellular matrix (ECM).5,6 Regenerativemedicine is governed by biology, bioengineering, and bio-mechanics.

The embryonic development and morphogenesis of carti-lage is initiated and regulated by BMPs.5 The cartilage-derived

1Laboratory of Bioengineering and Tissue Regeneration (LABRET-UMA), Department of Cell Biology, Genetics and Physiology, Faculty ofSciences, University of Malaga, Malaga, Spain.

2Networking Biomedical Research Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), University of Malaga, Malaga,Spain.

3Department of Orthopaedic Surgery and Traumatology, Hospital Costa del Sol, Marbella, Spain.4Department of Orthopaedic Surgery and Traumatology, Universitary Hospital Virgen de la Victoria, Malaga, Spain.5Department of Orthopaedic Surgery and Traumatology, Hospital Universitario Virgen del Roc o, Sevilla, Spain.6Department of Orthopaedic Surgery, The Ellison Center for Tissue Regeneration and Repair, University of California, Davis Medical

Center, Sacramento, California.*These two authors contributed equally to this work.

TISSUE ENGINEERING: Part BVolume 16, Number 6, 2010 Mary Ann Liebert, Inc.DOI: 10.1089/ten.teb.2010.0191

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morphogenetic proteins are critical for AC differentia-tion and the complete joint morphogenesis.7,8 BMPs andcartilage-derived morphogenetic proteins were isolatedfrom bone and cartilage, respectively. The rules of archi-tecture for regenerative medicine and surgery are an ad-aptation and imitation of the rules of development biologyand morphogenesis and may be generally universal formany tissues.5

The AC is adjacent to the subchondral bone. Yet, there is afundamental difference between the regenerative response ofbone and AC. Bone has supreme regenerative capacity. Onthe other hand, AC is recalcitrant and feeble in its capacityfor regeneration and even repair. What is the cellular andmolecular basis for these distinct differences of regenerativepotential in these two adjacent and related tissues? First,unlike bone, cartilage is avascular and is devoid of nervesupply. Second, as they lack vascular supply there are noimmediate early repair responses with monocytes and mac-rophages to injury. Recent work on mesenchymal stem cells(MSCs) demonstrated that blood vessels and associatedpericytes support tissue regeneration and homeostasis.9

Perhaps the absence of vasculature and attendant lack of

pericytes may explain in part lack of regeneration.The AC has very distinct anisotropy and distinct polarity.

The superficial zone has flattened chondrocytes. The surfacezone secretes the superficial zone protein (SZP) also knownas lubricin.1015 The middle zone chondrocytes secrete col-lagen II and the proteoglycan aggrecan. The deep zone of AChas a mineralized ECM with the distinct tidemark, on thesubchondral bone. The distinct functional zones of surface,middle, and deep has to be faithfully regenerated withcomplete fidelity. This is the main challenge in AC regener-ation. The next section will discuss the structure of the ACwith special emphasis on collagen fibrils orientation in eachof the three zones of AC.

Structure of AC Collagen Fibrils: Arrangement,

Polarization, and Distribution

The ECM of the AC is a specialized connective tissueconsisting of a hydrated proteoglycan gel that resists com-pression reinforced by a network of collagen fibrils. Collagenis an important component of the AC, representing around50% of its dry weight and being the most important con-stituent to provide tensile strength. Nevertheless, its distribution and organization remains a matter of controversy.Perhaps the strong interaction with proteoglycans and thehigh level of hydration presents challenge to observe thematrix by classical optical microscopy. Recent technologicalimprovements have contributed to our increasing under-

standing of cartilage organization.The pioneering work of Benninghoff established the con-

cept that collagen fibrils are oriented vertically in the deeperlayers of AC,13 twisting into arches at the intermediate lay-ers, and assuming a horizontal disposition in the superficiallayer.15 Benninghoffs concept has ever since received widesupport especially by investigators using polarization mi-croscopy. In the last century, several authors, using differentmicroscopic methods, postulated specific orientation forcollagen fibrils in AC. However, the methodological limita-tions and the intricate organization of collagen fibrils con-tinue to be challenging.1619

The organization of several cartilages, including AC, wereinvestigated using tissue sections, Picrosirius red staining,enzymatic digestions, and polarized microscopy.20 Picrosirius-polarization method has an advantage over other methodsbecause it increases the resolution of light microscopy due tothe increase in natural birefringency of collagen fibrils whenthey bind the Picrosirius Red dye. Colored birefringency isenhanced against a dark background, allowing the distinc-

tion of usually undetected thin fibrils.21

Enzymatic digestionusing hyaluronidase removes proteoglycans and unmaskscollagen fibrils rendering them more accessible and therefore better stained.22 Using such methods several authors foundfor AC in several species similar fibril distribution to theoriginal Benninghoff proposal: single gothic arches made byvertically oriented fibrils in the deeper zone reaching os-teochondral boundary and horizontal orientation in the su-perficial zone. Similar collagen fibril distribution has beenreported by Nieminen et al. using quantitative magnetic re-sonsnce imaging and polarized light microscopic study inbovine AC.23

The three-dimensional structure of collagen in bovine ACwas discovered with scanning electron microscopy using a

modification of a technique.24,25

Enzymatic digestion of theproteoglycans defined the underlying collagen structure butwas incomplete to maintain tissue integrity. In the middleand superficial zones, collagen was organized in a layered orleaf-like manner. The orientation was vertical in the inter-mediate zone, curving to become horizontal and parallel tothe articular surface in the superficial zone. Each leaf con-sisted of a fine network of collagen fibrils. Adjacent leavesmerged or were closely linked by bridging fibrils and werearranged according to the split-line pattern. The surface layer(lamina splendens) was morphologically distinct. As colla-gen is an integral component of cartilage matrix its organi-zation is critical for cartilage formation, growth, repairand regeneration.24 Following a similar pattern of collagen

distribution in rabbit and in human were described novelaspects from cryo and modified chemical preparation tech-niques for scanning electron microscopy.26,27 They focusedthe studies on the radial zone where a special distribution ofcollagen fibrils forming columns in the regions surroundingthe rows of chondrocytes, the so-called chondrons. Thesecolumns of 13mm diameter each, have densely packedcollagen fibrils. These fibrils were arranged radially; somewere straight of 30 nm and others in an opposed spiral ar-rangement of 10 nm, with regularly repeating patterns. Theaggrecan component of the ECM could be contained in suchcolumns. The load bearing property of the tissue was ex-plained by the directed flow and containment of the inter-stitial fluid, modulated by the proteincarbohydrate complexes,

along collagen bound tubular structures. The possible reasonwhy such structure was not described earlier may be that itis not preserved by aldehyde fixation followed by dehydra-tion, the method commonly used for tissue preparation forelectron microscopy.

There is no unanimity about the diameter of the collagenfibrils; they ranged in diameter between 30 and 110 nm.20

Type IX collagen interacts with collagen II and other IX fi-bers.25 In addition, there is potential crosslinking betweencollagens II and IX.27 The collagen IX binds to collagen II.2830

A similar D-periodic banding has been observed in chickembryo sternal and bovine AC.2931

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Rieppo et al. observed birefringence, orientation, andparallel collagen fibril network after autologous chondrocytetransplantation.31 The repair tissue lacks the typical collagennetwork organization of AC. Collagen fibril orientation isparallel to the surface throughout the entire thickness ofcartilage and the normal phenotype of AC is not achieved.Changes in the collagen architecture and spatial collagencontent during AC growth and maturation in pig showed

classic Benninghoff architecture. Perhaps one possible ex-planation for the appearance of cartilage is adaptation tojoint loading. The finding in pig may have significance to thehuman. Shirazi and Shirazi-Adl32 have investigated in hu-man knee joint the role of deeper vertical fibrils of collagen inAC mechanics through finite element methods. They hy-pothesize that those fibrils play a crucial role in cartilagemechanics by supporting and protecting the tissue underphysiological loading conditions.

The surface of the superficial zone is stained by PicrosiriusRed more strongly than the interior (Fig. 1). The surface re-gion fibrils are oriented parallel to the surface and the par-allel fibers appear to intersect. The polarization opticspermits the observation of birefringence in gradation in both

positions of polarization going from less in one position tomore when the specimen is turned 458 left or right. The in-ner areas of superficial zone are initially dark and increasesbirefringency with turn of the slide. After papain treatmentto remove proteoglycans there is a generalized increase inbirefringency as a consequence of unmasking collagen fibrils.In the middle zone increased birefringency is observed withpolarized light. The territorial matrix in the pericellular areasdemonstrated weak birefringence under polarized light. Theappearance of the cells after papain treatment decreasesmetachromasia and the territorial matrix shows the appear-ance of a Maltese cross, which move around the cells whenthe slide turned, which demonstrating a heterogenous col-lagenous organization. The interterritorial zone exhibits

thicker collagenous fibrils with an oblique pattern criss-crossing at right angles to each other. The schematic depic-tion (Fig. 2a) demonstrates that the fibers are parallel in thesurface and in the layer close to middle layer the obliquecollagen fibers interspersed within vertical regions. Similarfibril organization also is observed in the deep zone. In thecourse of repair one observes a disorganized fibril organi-zation (Fig. 2b). Ideally, one would like to observe completeregeneration with optimal fibril orientation (Fig. 2c).

As collagen is an integral component of cartilage matrix,its organization is critical for cartilage formation, growth,repair, and regeneration.

Transforming Growth Factor-bs Family

and Cartilage Zonal Organization

and Differentiation and Metabolism

The AC has a distinct zonal organization that is intimatelylinked to function. The superficial zone cartilage (SZC), themiddle zone cartilage (MZC), and the deep zone cartilage(DZC) are generally identified in AC.33 The AC in additionhas a calcified cartilage zone that has mineralized ECM andis distinguished at the interface with DZC by the so-calledtide mark, which in most histological stains such as toluidine blue has different staining properties from the metachro-matic middle and deep zones (MZC and DZC). The entire

AC is intimately associated with subchondral bone by themost characteristic interdigitation of the calcified cartilagezone into the bone.

The middle and DZC has the collagen II and the proteo-glycan aggrecan and several noncollagenous proteins, in-cluding but not limited to cartilage oligomeric matrix proteinand cartilage intermediate larger protein.34 It is important tokeep in mind that the superficial, middle, and deep zones are

a continuum, and therefore there are an expected transitionalareas between the SZC, MZC, and DZC. The functional as-pects of the various zones in the AC are an important con-sideration in exploring the AC regeneration.

SZP is secreted by the SZC and is a mucinous glycoproteinwith a covalently attached proteoglycan chain. The SZP ishomologous to lubricin, a glycoprotein secreted by syno-vium and first purified from synovial fluid. SZP/lubricin issecreted by both the superficial zone chondrocytes and sy-novium, and therefore the relative regulation of these cellularsources by morphogens and growth factors is critical.35 Lu-bricin/SZP is known to function as a boundary lubricant indiathroidal joints and plays a role in reducing the coefficientof friction in the opposing gliding surfaces or AC in all joints.

SZP and lubricin are encoded by the same gene, proteogly-can 4 (prg4). Mutation in the prg4 gene has been attributed tothe Camtodactyly-arthropathy-coxa-vara-pericarditis syn-drome.34,35 A key feature of this syndrome is alterations inarticular surface and attendant degradation of AC andnoninflammatory early onset joint failure. Thus, the func-tional importance of SZP/lubricin is demonstrated by thepathophysiology of the joints in the Camtodactyly-arthropathy-coxa-vara-pericarditis syndrome.

Why focus on the surface of AC in arthritis? The firstchanges at the onset of OA occur at the superficial zone ofAC.3641 There is loss of cellular SZP immunostaining indegenerative AC in menisectomized sheep model of earlyOA.42 In addition, there is a strong association between the

loss of boundary lubrication and damage of AC in an ex-perimental model of OA in rabbits induced by transection ofanterior and posterior cruciate ligaments.43 Therefore, SZPplays a critical role in joint physiology and is an attractivetarget for systematic investigations especially of the regula-tory biology of the superficial zone of AC.

Regulation of SZP accumulation is critical not only forhomeostasis and maintenance of AC, but also for the tissueengineering and regenerative medicine of functional AC.SZP accumulation by superficial zone chondrocytes wasenhanced by transforming growth factor-b (TGF-b) in bovinecartilage. BMP-7 stimulated the accumulation of SZP in bothexplant cultures of SZC from bovine AC and in monolayercultures of chondrocytes derived from SZC.44 In view of this,

a systematic study of TGF-b/BMP superfamily memberswas conducted on the responses of cells from SZC and sy-noviocytes in calf cartilage.45 The BMP/TGF-b superfamilyincludes BMPs, TGF-b, growth/differentiation factors, andactivins. TGF-b isoforms 1, 2, and 3 were potent in stimu-lating SZP secretion by both superficial zone chondrocytesand synoviocytes.46 SZP is mainly secreted by SZC but notmiddle and deep zones even in the presence of TGF-b. Theresponse of superficial zone chondrocytes and synovium toTGF-b isoforms is dose dependent and is biphasic. High dose(30ng/mL) was inhibitory compared to the optimal dose(13 ng/mL). The biological actions of TGF-b isoforms are

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FIG. 1. Structure of human artic-ular cartilage. Human articular car-tilage sections were stained withPicrosirius and hematoxylin. (ac)depict the normal structure of hu-man articular cartilage. The threezones are observed: superficial,middle, and deep. In addition, thetransition zones can be identified

between each of the main zones.Light microscopy demonstrates (a)a metachromasia throughout theextracellular matrix (ECM) (violetcolor), except in the superficial zonewhere cells appear flattened andfusiform forming a thin layer. Thesurface of the superficial zone ismore intensely stained by Picrosir-ius Red than the deep layer. Polar-ization microscopy (b, c) depicts inthis surface region collagen fibrilsoriented parallel to the surface intwo types of bundles intersectingbetween them can be observed.

Thus, one can observe with twodifferent angles of polarization(turning the specimen 458 fromright to left) birefringency in bothpositions, although less in one (b)than the other (c). Moreover, theinner part of the superficial zoneappears dark in the first location ofthe lens (b) but with strong bi-refringency in the other (c). Thus,the outer part is reinforced with aminor extra bundles of collagen fi-brils running parallel to the surfacebut forming a certain angle with themain fibrils of that area. Next, thehistological sections were treatedwith the proteolytic enzyme papain.Papain-digested slides (df) show,in general, an increase in bi-refringency, demonstrating an in-teraction of collagens withproteoglycans. The surface of thesuperficial zone does not show enhancement of birefringency after loss of proteoglycans by papain digestion (e, f). Afterpapain digestion metachromasia disappears as a consequence of the removal of proteoglycans. The ECM shows a generalizedred color (d). A transition area between the superficial zone and the middle one can be detected as coarse and intense redstained fibrils, which present intense birefringency when the sections are seen under polarized light (e). These fibrils areintertwined and oriented in perpendicular directions. Below the transition area, the middle zone occupies most of thearticular cartilage. Here the cells are round and are embedded in ECM distinguishable with territorial matrix, around thecells, and the interterritorial space, between the cells. In control conditions without polarization, the interterritorial ECMappears to have few collagen fibrils (b), except in the zone close to surface where the polarized light depicts fibrils runningparallel and oriented obliquely to the surface (b). When the polarization filter turned 458 from right to left more birefringency

can be observed in upper part of the middle zone (c). The territorial area presents weak birefringence in a typical Maltesecross image. The papain digestion unmasks a dense area of collagen fibrils. The Maltese cross appears more clearly aroundthe cells and the interterritorial spaces are crossed by thick fibrils of collagen, which run parallel but in two predominantorientations: oblique to the surface forming a right angle between them (e). When the specimen turned 458 from right to leftmore birefringence can be observed, indicating a predominant orientation of the bundles of the fibrils in this middle zone (f ).The articular cartilage adjacent to subchondral bone is the deep zone. Chondrocytes are slightly flattened, arranged in rows,oriented almost perpendicular to the surface (a, d). Papain digestion shows a more intense red staining with Picrosirius Red atthe bottom area (d), corresponding with thick bundles of collagen fibrils running parallel between them but oriented in twomain directions, which form a narrow angle (


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dependent on TGF-b receptor I kinase as specific inhibitors ofthe kinase blocked the response.45 Members of the BMPfamily (BMP-2, 4, and 7, and growth/differentiation factor-5)demonstrated a distinct differential response in SZC chon-drocytes and synoviocytes in the bovine joint. The synovio-cytes were much more responsive to BMPs. Similarly,synoviocytes were more sensitive to activins A, B, and ABcompared to chondrocytes from the superficial zone. These

observations demonstrate the differential regulation betweensuperficial zone and synoviocytes in the joint, indicating thedifferences among the various compartments of the jointsuch as AC and synovium. In addition, there may be a di-vision of labor among the BMP/TGF-b superfamily; the su-perficial zone is more responsive to TGF-b isoforms, whereasthe middle zone is mainly regulated by BMPs.

The function of AC is intimately linked to biomechanics.Mechanical loading is critical for homeostasis of musculo-skeletal tissues, including AC. AC performs the biomechan-ical function of load support and lubrication with minimalwear and little or no damage in animals and humans. Dy-namic shear stimulates SZP. The molecular basis of me-chanotransduction in AC is beginning to be understood. The

SZP expression pattern is dependent on the geometry of thefemoral condyle surface and anatomical location. The ante-rior sites secreted more SZP in both lateral and medialfemoral condyles. The biomechanical assays of SZP accu-mulation were confirmed and corroborated by immuno-localization of SZP in the various anatomical locations of thelateral and medial femoral condyles.47 Again, the highestSZP were in anterior region compared to the posterior sites.The distribution of maximum contact pressures was associ-ated with high SZP content. Anatomical regions of highcontact pressure were consistently located in the anteriorregion. In addition, shear loading of the anterior medial

condyle increased significantly SZP secretion compared toposterior sites of the same femoral condyle. The SZP responseto shear-loading was abolished in the presence of TGF-b typeI receptor kinase inhibitor, demonstrating the critical role ofTGF-b signaling in mediating mechanotransduction.48 Thus,these experiments demonstrate a new role for TGF-b sig-naling pathway in joint lubrication and is mediated by cel-lular mechanotransduction in superficial zone of AC. The

middle and deep zones were refractory to shear loading,further illustrating that there apparently a gradient of re-sponse originating from the surface to deep zones. The SZCis therefore critical in mechanotransduction and in joint lu-brication. The science of tribology deals with lubrication andwear in various surface boundary lubrication regimens. Anew field of biotribology has emerged dealing with biolog-ical surfaces in sliding contact combining concepts of fric-tion, wear, and lubrication of opposing interacting surfacessuch as the gliding AC surfaces in the joint.49 Thus, theemerging new findings in AC are also critical for regenera-tion and restoration of damaged AC in OA by tissue-engineered cartilage. It is critical that attention be focused onthe characteristics of low coefficient of friction and high re-

sistance to wear in regenerative medicine and tissue engi-neering of AC.

Cell Therapy for Cartilage Regeneration

During the last decade there has been an exponential in-crease in research activity in the field of cartilage tissue en-gineering. AC is seen as an ideal candidate for a tissueengineering approach to tissue regeneration.

Trauma to the AC surface of the joint represents a chal-lenging clinical problem because of the very limited ability ofthis tissue to self-repair. A number of surgical protocols are

FIG. 2. A schematic description ofthe collagen fiber orientation in thehuman articular cartilage. The nor-mal structure (a) is represented asFigure 1. In (b), the appeared repairtissue in the defect is shown, whereasin (c) it represents the ideal desiredoutcome of the defect. In the normalcartilage the fibrils are parallel to the

surface of the cartilage. Just beneaththe parallel fibrils there are obliquelyoriented fibrils interspersed withmore vertical fibrils. In the middlezone the oblique fibrils are at *458.In the deep zone there are rows ofcolumnar cells with vertical fibrils.The oblique fibrils can also be ob-served in the deep zone as in themiddle zone. Also depicted in therepair state disorganized fibril or-ganization. On the other hand, in theregeneration the structure of col-lagen fibrils is identical to the nor-mal articular cartilage not only

because of thenormalcelland matrixorganization but also because of thecomplete integration of new cartilagewith the old cartilage.

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currently in use for the treatment of AC defects. In abrasionarthroplasty,50 for instance, the subchondral bone is perfo-rated by drilling to promote bleeding into the defect, withthe result that there is formation of bone and fibrous repairtissue. In the microfracture technique51 the exposed sub-chondral bone is microfractured (picked) to promotelocalized bleeding. Moreover, repair techniques such as mi-crofracture, which introduce cells into the joint, have un-

predictable clinical outcomes as they produce a fibrocartilagetissue that degenerates with time. Another approach in-volves the use of allografts52 where cartilage lesions are filledwith grafts of donor-derived osteochondral fragments.However, these procedures are restricted by the availabilityof suitable donor tissue. In mosaicplasty52 cylindrical os-teochondral plugs are harvested from nonload-bearing sitesin the affected joint and pressed into place within the os-teochondral defect, creating an autograft mosaic to fill thelesion. Autologous chondrocyte implantation (ACI) therapy52

represents another approach, where a chondral biopsy istaken from a donor site at the time of clinical examination.One of the major relevant issue in this field as been the recentacceptance of ChondroCelect (by TiGenix) as a cell-based

medicinal product consisting of chondrocytes that are takenfrom a healthy region of the patients cartilage, grown out-side the body, and then re-implanted during an ACI surgicalprocedure. The ACI procedure with a membrane of collagen(collagens I and III) is called MACI. Chondrocytes, enzy-matically released from the retrieved tissue, are expanded inmonolayer culture, and subsequently implanted in a secondprocedure beneath the periosteal membrane or the MACImembrane, which is sutured to the cartilage adjacent to thedefect and sealed with fibrin glue. All of these approachesoffer exciting opportunities for the regeneration of cartilagedefects. However, the long-term outcome may be uncertainand there are may be other disadvantages associated withthe harvest site, even when it is some distance from the le-

sion. In this regard, nowadays it is well known that lesionsize, activity level, and age were the influencing parametersof the outcome of AC repair surgery.53 Lesions >2.5cm2

should be treated with ACI or osteochondral autologoustransplantation, whereas microfracture is a good first-linetreatment option for smaller (


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autologous culture-expanded bone marrow MSCs to repairAC.68 Subsequently, they performed this procedure in about40 patients demonstrating the safety of the procedure. Lateron, they demonstrated improvement in clinical symptomswith a hyaline-like type of cartilage tissue in young, activepatients,69 although the repair cartilage was not hyalinecartilage in normal age individuals. To regenerate AC by celltransplantation, it is essential that cells proliferate without

losing their capacity for differentiation. To find appropriateconditions, different culture conditions, mechanical stresses,growth factors, and gene transfection have all been explored,but these have not yet been applied clinically.70

One might consider the phenotypic plasticity as an in vitroartefact as the chondrocytes are being exposed to and arti-ficial environment and being extensively modified duringtheir expansion in monolayer. However, plasticity betweendifferent phenotypes is a common phenomenon during em-bryonic development of tissues and organs. Using this fea-ture, the same cells can be reprogrammed to serve severalfunctions. Also, the plasticity is preserved and has criticalfunctions in adult amphibians such as salamanders, as ob-served in the regeneration of an amputated limb.71

In vivo, plasticity is mainly seen during the embryonicdevelopment of organs and tissues, for example, the shiftfrom endothelial-like cells to mesenchymal cells during theembryonic formation of synovial joints. This change is madefrom the cells that will form the AC, the interzonal cells.Initially, these cells have characteristics similar to the endo-thelial cells lining blood vessels and, after condensation, willround up and start to secrete cartilage matrix proteins. Theshift from endothelial to mesenchymal cells has further beenshown to persist in the formation of adult heart valves.72

It is a well-established fact that during monolayer ex-pansion of chondrocytes in vitro, the cell population losses itsphenotype and starts to express primitive embryonic mark-ers.73,74 This dedifferentiation process is not unique for

chondrocytes but a rather common phenomenon observed inmany types of cells and tissues.7577 Another possible ex-planation to the change in expression during the expansion isthat the culture conditions are favoring certain cell subpop-ulations, for example, transit-amplifying cells, and that thesecells are able to respond to the culture conditions morerapidly.

During redifferentiation of dedifferentiated adult chon-drocytes expanded in human serum, chondrocytes do ex-press genes that have been described in the initiation ofchondrogenesis during limb formation.77 This shows, inaddition to the phenotypic plasticity,77,78 that the cells revertto a primitive stage during the expansion in vitro. Culture-expanded chondrocytes are able to redifferentiate when

replaced into three-dimensional environment under appro-priate culture conditions.7981 This ability of redifferentiationis particularly important when the cells need to be trans-planted into patients. To better define the chondrogenicphenotype and to try to optimize the cellular recovery forclinical cell transplantation, several attempts have been madeto characterize culture-expanded chondrocytes at the mo-lecular level.8084

During the expansion of human chondrocytes, the degreeof dedifferentiation has been correlated to the number of celldivision or passages.7375 This was a typical characteristicdemonstrated in the study of myogenic differentiation of

chondrocytes.74 In that study, no differentiation into musclewas obtained when cells from a low passage were used,whereas with later passages, cartilage-derived muscle fiberswere obtained, confirming the amplification of adult articu-lar chondrocytes in vitro results in a population of cells withprogenitor properties.

Biomaterial scaffolds provide the chondrogenic cellswith a microenvironment, where they survive, multiply, and

produce ECM to constitute regenerated cartilage. Althoughthe cellular products are expected to replace the degradablebiomaterial, the process is usually time-consuming and thescaffold should be implanted before completion of the pro-cess. The biomaterials thus play the role of a vehicle totransfer cells and therefore should be compatible with thenative tissue around the recipient site.76 Many natural sub-stances are suitable as the cell-carrying scaffold for cartilageengineering, including fibrin, agarose, alginate, collagen,chitosan, and hyaluronan. Many of these are hydrogels andcan be designed as injectable in their liquid form, whichblends well with chondrogenic cells.77 After being injectedinto the recipient site, they set by gelation to fill in any shapeand size of cartilage defect.

Either chondrocytes or stem cells are used to constituteengineered cartilage useful to regenerate damaged tissue,and in vitro manipulation of the cells is necessary in most ofthe currently available systems. When the constructed car-tilage tissue is considered for clinical used, the safety of thewhole process needs to be debated and the cost is high. Theentire process has to be conducted with expensive laboratoryfacilities that meet the high standard of good tissue practice.In addition, all reagents involved in the process should beproven as safe for human use. More complicated manipu-lation of the cells will arouse more concern that the cellsmay be affected in unknown ways. When developing asystem to regenerate cartilage for clinical application, oneshould always consider the safety and efficacy of the re-

generated tissue.As a result of the variable and unpredictable clinical ex-

periences in cartilage regeneration in the past, biotechnologyhas been introduced to this field for evidence-based devel-opment of a solution. The knowledge to date supports thatAC is best repaired with autologous engineered cartilage,and a considerable research has been carried out to improvecartilage regeneration. Although the efficacy of regenerationhas much improved in the laboratory and animal studies,most findings have not been investigated for their clinicalsafety and performance. Further studies should highlighttheir clinical relevance to facilitate the development ofproducts applicable to humans.

One needs to organize currently available knowledge to

develop clinically applicable models of cartilage regenera-tion, on the basis of autogenous chondrogenic cell implan-tation. A clinically applicable model of cartilage regenerationshould be safe, efficient, and simple. It can be completed in asingle seed-and-implant surgery procedure, which decreasesthe surgical risks and complications from repetitive opera-tions of conventional autologous chondrocytes implantation.If the site of repair allows an arthroscopic approach, thesurgery can be done in a minimally invasive manner within ashort time, estimated at 1 h. By avoiding the complex treat-ment of the autogenous cells in vitro, the safety of the pro-cedure can be improved and the cost reduced.



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The Future: Challenges and Opportunities

The foregoing discussion of the structure of the AC, thezonal organization, the regulation and mechanotransduc-tion, and joint lubrication sets the stage for contemplating thefuture challenges and opportunities for regeneration andtissue engineering of AC.

This brief review has by design focused on structure,fibril-orientation and the cell shape, TGF-b signaling path-ways, and mechanotransduction. It is well known to moststudents and practioners of tissue engineering that it requirestriad of signals, stem cells, and scaffolds.9 In future, there willbe continuous refinement of the scaffolds for the tissue as-sembly during tissue engineering.

The native AC is a durable tissue that, unless ravaged byarthritis, lasts a life time in humans. The AC is an engineeringmarvel in the human body. It is truly outstanding that theECM of cartilage has predominantly collagen II providingtensile strength and the electronegative proteoglycan ag-grecan, which permits movement of water with the matrix toensure electroneutrality. This represents an advantage for anefficient function with a minimal energy expense.

AC has significant biomechanical properties, includingcompressive modulus of 0.79MPa, a shear modulus of0.69 MPa, and a tensile modulus with a range from 0.3 to10 MPa.78 Thus, a dynamic hydrogel of cartilage ECM iscritical for the biochemical, cellular, and biomechanicalfunction. The recent advances in nanomaterials bodes wellfor nanoscaffolds for AC regenerative medicine. Therefore,we enter the realms of nanomaterials and nanomedicine forthe benefit of patients with OA desperately looking for so-lutions for the painful disease.79 Thus, regenerative medicineof AC and tissue engineering presents critical challenges forthe future and presents outstanding opportunities.

Cells to be used for an efficient regenerative medicineshould be chosen. Undifferentiated versus differentiated orpredifferentiated chondrocytes will be the choice. A perma-nent solution will come when the new tissue built in thedefect is of the same nature and is perfectly integrated in thewhole structure in any pathology and in any age. Only insuch s way structure and function will be fully recovered.

In conclusion, the recent advances in biotechnology,nanotechnology, and nanomaterials bode well for an opti-mistic and bright future for regenerative medicine of AC.

Acknowledgments

The authors thank P. Jimenez-Palomo for his excellent tech-nical assistance. This work was supported by grants from theBanco Bilbao-Vizcaya-Argentaria Foundation (FBBVA, Chair inBiomedicine 2007 to A.H. Reddi), the Ministry of Science and

Technology (BIO2009-13903-C02-01), the Ministry of Scienceand Innovation (FIS PI06/1855, PLE2009-0163, FIS PI10/2529),Red TerCel (Institute of Health Carlos III), and the AndalusianAutonomousGovernment (P07-CVI-2781, PAIDI, and BIO-217).

Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Jose A. Andrades, Ph.D.

Laboratory of Bioengineeringand Tissue Regeneration (LABRET-UMA)

Department of Cell Biology, Genetics and PhysiologyFaculty of Sciences, University of Malaga

Malaga 29071Spain

E-mail: [email protected]

Received: April 3, 2010Accepted: September 13, 2010

Online Publication Date: October 28, 2010



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health - osteoarthritis ac tissue engineering and insertion into joint

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