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Health - Osteoarthritis AC Tissue Engineering and Insertion Into Joint

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TISSUE ENGINEERING: Part B Volume 16, Number 6, 2010 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.teb.2010.0 10.1089/ten.teb.2010.0191 191

Articular Cartilage: Structure and Regeneration Jose´ Becerra, Ph.D.,1,2,* Jose´ A. A Andrades, ndrades, Ph.D.,1,2,* Enrique Guerado, M.D., Ph.D.,2,3 Pla´ cido Zamora-Navas, Zamor a-Navas, M.D., Ph.D., Ph. D.,2,4 Jose´ M. Lo L o´ pez-Puer pez- Puertas, tas, M.D., 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 development of osteoarthritis. One method that has been proven to result in long-term repair or isolated lesions is autologous 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 compliant articulating surface to the bones in diarthrodial joints. It protects the joint by distributing loads applied to it, so preventing 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 fibercomposite material. Fibrils of predominantly type II collagen provide tensile reinforcing to a highly hydrated proteoglycan gel. The tissue typically comprises 70% water and it is the structuring and retention of this water by the proteoglycans and collagen that is largely responsible for the remarkable ability of the tissue to support compressive loads.

prevalent in women than in men and increases with age. 1–4 What can be done about the damaged joints in OA? Can they rthritis rthritis is a major challenge for the musculoskeletal  be repaired in the least or can one obtain complete regenerheal health th.. Cons Consid ider erab able le prog progre ress ss in the the trea treatm tmen entt of  ation of the cartilage with fidelity? rheumatoid rheumatoid arthritis, arthritis, an autoimmune autoimmune disorder, disorder, has been Repair is the rapid process to resolve an injury. The reachieved by the use of antagonists of tumor necrosis factor parative tissue is not identical to the original tissue and there and cognate receptors by judicious use of monoclonal anti- is no integr integrati ation on of repair repair tissue tissue with with the origin original al tissue tissue..  body therapeutics. On the other hand, osteoarthritis (OA) is a Regeneration is a relatively slow process that recapitulates complex multifactorial disease defying systematic diagnosis, development development and morphogene morphogenesis sis and restores restores completely completely and treatment. treatment.1 The integr integrity ity of articu articular lar cartil cartilage age (AC) (AC) structure and function, including integration of the new tisstructure structure is at the crux of the problem of OA. The symptoms symptoms sue seamlessly to the original. include, but not limited to joint pain, impaired and limited Regene Regenerat rative ive medici medicine ne is the emergi emerging ng discip disciplin linee of  movement and inflammation and tender joints. OA can be medicine based on advances in basic science of development local in certain joints or more generalized. One of the chal- and morphogenesis and science of biomaterials and stem cell lenges in OA is the distinction between the original symp-  biology. The three key ingredients for regenerative medicine toms and attendant sequelae of reparative response as, for and surgery surgery are the induct inductive ive signal signalss such such as bone bone mormorexample, in the synovium is increasingly difficult. The his- phogen phogeneti etics cs protei proteins ns (BMPs) (BMPs),, respon respondin ding g cells, cells, and the tologi tological cal signs signs are damage damage to AC, clefts in cartil cartilage age,, and scaffolding scaffolding of extracellu extracellular lar matrix matrix (ECM).5,6 Regenerative incursion of capillaries into the calcified tide mark. Although medicine is governed by biology, bioengineering, and bioall joints can be affected with OA, some joints are more mechanics. susceptibl susceptiblee such as hand, spine, hip, knee, and foot. What is The embryonic development and morphogenesis of carticertain in the epidemiology of OA is that the disease is more lage is initiated and regulated by BMPs. 5 The cartilage-derived Introduction



Laboratory of Bioengineering and Tissue Regeneration (LABRET-UMA), (LABRET-UMA), Department of Cell Biology, Genetics and Physiology, Faculty of  Sciences, University of Ma´ laga, Ma´ laga, Spain. 2 Networking Biomedical Research Center in Bioengineering, Bioengineering, Biomaterials and Nanomedicine Nanomedicine (CIBER-BBN), University of Ma´ Ma´ laga, Ma´ laga, Spain. 3 Department of Orthopaedic Surgery and Traumatology, Hospital Costa del Sol, Marbella, Spain. 4 Department of Orthopaedic Surgery and Traumatology, Universitary Hospital Virgen de la Victoria, Ma´ Ma´ laga, Spain. 5 Department of Orthopaedic Surgery and Traumatology, Hospital Universitario Virgen del Rocı´ Rocı ´o, Sevilla, Spain. 6 Department 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.




morphogenetic proteins are critical for AC differentiaThe organization of several cartilages, including AC, were 7,8 tion and the complete joint morphogenesis. BMPs and investigated using tissue sections, Picrosirius red staining, cartilage-derived morphogenetic proteins were isolated enzymatic digestions, and polarized microscopy. 20 Picrosiriusfrom bone and cartilage, respectively. The rules of archi- polarization method has an advantage over other methods tecture for regenerative medicine and surgery are an ad-  because it increases the resolution of light microscopy due to aptation and imitation of the rules of development biology the increase in natural birefringency of collagen fibrils when and morphogenesis and may be generally universal for they bind the Picrosirius Red dye. Colored birefringency is many tissues. 5 enhanced against a dark background, allowing the distincThe AC is adjacent to the subchondral bone. Yet, there is a tion of usually undetected thin fibrils. 21 Enzymatic digestion fundamental difference between the regenerative response of  using hyaluronidase removes proteoglycans and unmasks  bone and AC. Bone has supreme regenerative capacity. On collagen fibrils rendering them more accessible and therefore the other hand, AC is recalcitrant and feeble in its capacity   better stained.22 Using such methods several authors found for regeneration and even repair. What is the cellular and for AC in several species similar fibril distribution to the molecular basis for these distinct differences of regenerative original Benninghoff proposal: single gothic arches made by potential in these two adjacent and related tissues? First, vertically oriented fibrils in the deeper zone reaching osunlike bone, cartilage is avascular and is devoid of nerve teochondral boundary and horizontal orientation in the susupply. Second, as they lack vascular supply there are no perficial zone. Similar collagen fibril distribution has been immediate early repair responses with monocytes and mac- reported by Nieminen et al. using quantitative magnetic rerophages to injury. Recent work on mesenchymal stem cells sonsnce imaging and polarized light microscopic study in (MSCs) demonstrated that blood vessels and associated   bovine AC.23 pericytes support tissue regeneration and homeostasis.9 The three-dimensional structure of collagen in bovine AC Perhaps the absence of vasculature and attendant lack of  was discovered with scanning electron microscopy using a pericytes may explain in part lack of regeneration. modification of a technique.24,25 Enzymatic digestion of the The AC has very distinct anisotropy and distinct polarity. proteoglycans defined the underlying collagen structure but The superficial zone has flattened chondrocytes. The surface was incomplete to maintain tissue integrity. In the middle zone secretes the superficial zone protein (SZP) also known and superficial zones, collagen was organized in a layered or as lubricin.10–15 The middle zone chondrocytes secrete col- leaf-like manner. The orientation was vertical in the interlagen II and the proteoglycan aggrecan. The deep zone of AC mediate zone, curving to become horizontal and parallel to has a mineralized ECM with the distinct tidemark, on the the articular surface in the superficial zone. Each leaf consubchondral bone. The distinct functional zones of surface, sisted of a fine network of collagen fibrils. Adjacent leaves middle, and deep has to be faithfully regenerated with merged or were closely linked by bridging fibrils and were complete fidelity. This is the main challenge in AC regenerarranged according to the split-line pattern. The surface layer ation. The next section will discuss the structure of the AC (lamina splendens) was morphologically distinct. As collawith special emphasis on collagen fibrils orientation in each gen is an integral component of cartilage matrix its organiof the three zones of AC. zation is critical for cartilage formation, growth, repair and regeneration. 24 Following a similar pattern of collagen distribution in rabbit and in human were described novel Structure of AC Collagen Fibrils: Arrangement, aspects from cryo and modified chemical preparation techPolarization, and Distribution niques for scanning electron microscopy. 26,27 They focused The ECM of the AC is a specialized connective tissue the studies on the radial zone where a special distribution of  consisting of a hydrated proteoglycan gel that resists com- collagen fibrils forming columns in the regions surrounding pression reinforced by a network of collagen fibrils. Collagen the rows of chondrocytes, the so-called chondrons. These is an important component of the AC, representing around columns of 1–3 mm diameter each, have densely packed 50% of its dry weight and being the most important con- collagen fibrils. These fibrils were arranged radially; some stituent to provide tensile strength. Nevertheless, its distri- were straight of 30 nm and others in an opposed spiral ar  bution and organization remains a matter of controversy. rangement of 10 nm, with regularly repeating patterns. The Perhaps the strong interaction with proteoglycans and the aggrecan component of the ECM could be contained in such high level of hydration presents challenge to observe the columns. The load bearing property of the tissue was exmatrix by classical optical microscopy. Recent technological plained by the directed flow and containment of the interimprovements have contributed to our increasing under- stitial fluid, modulated by the protein–carbohydrate complexes, standing of cartilage organization. along collagen bound tubular structures. The possible reason The pioneering work of Benninghoff established the con- why such structure was not described earlier may be that it cept that collagen fibrils are oriented vertically in the deeper is not preserved by aldehyde fixation followed by dehydralayers of AC, 13 twisting into arches at the intermediate lay- tion, the method commonly used for tissue preparation for ers, and assuming a horizontal disposition in the superficial electron microscopy. layer.15 Benninghoff’s concept has ever since received wide There is no unanimity about the diameter of the collagen support especially by investigators using polarization mi- fibrils; they ranged in diameter between 30 and 110 nm.20 croscopy. In the last century, several authors, using different Type IX collagen interacts with collagen II and other IX fimicroscopic methods, postulated specific orientation for  bers.25 In addition, there is potential crosslinking between collagen fibrils in AC. However, the methodological limita- collagens II and IX.27 The collagen IX binds to collagen II. 28–30 tions and the intricate organization of collagen fibrils con- A similar D-periodic banding has been observed in chick tinue to be challenging. 16–19 embryo sternal and bovine AC. 29–31



Rieppo et al. observed birefringence, orientation, and AC is intimately associated with subchondral bone by the parallel collagen fibril network after autologous chondrocyte most characteristic interdigitation of the calcified cartilage transplantation.31 The repair tissue lacks the typical collagen zone into the bone. network organization of AC. Collagen fibril orientation is The middle and DZC has the collagen II and the proteoparallel to the surface throughout the entire thickness of  glycan aggrecan and several noncollagenous proteins, incartilage and the normal phenotype of AC is not achieved. cluding but not limited to cartilage oligomeric matrix protein Changes in the collagen architecture and spatial collagen and cartilage intermediate larger protein. 34 It is important to content during AC growth and maturation in pig showed keep in mind that the superficial, middle, and deep zones are classic Benninghoff architecture. Perhaps one possible ex- a continuum, and therefore there are an expected transitional planation for the appearance of cartilage is adaptation to areas between the SZC, MZC, and DZC. The functional as joint loading. The finding in pig may have significance to the pects of the various zones in the AC are an important conhuman. Shirazi and Shirazi-Adl 32 have investigated in hu- sideration in exploring the AC regeneration. man knee joint the role of deeper vertical fibrils of collagen in SZP is secreted by the SZC and is a mucinous glycoprotein AC mechanics through finite element methods. They hy- with a covalently attached proteoglycan chain. The SZP is pothesize that those fibrils play a crucial role in cartilage homologous to lubricin, a glycoprotein secreted by synomechanics by supporting and protecting the tissue under vium and first purified from synovial fluid. SZP/lubricin is physiological loading conditions. secreted by both the superficial zone chondrocytes and syThe surface of the superficial zone is stained by Picrosirius novium, and therefore the relative regulation of these cellular Red more strongly than the interior (Fig. 1). The surface re- sources by morphogens and growth factors is critical. 35 Lugion fibrils are oriented parallel to the surface and the par-  bricin/SZP is known to function as a boundary lubricant in allel fibers appear to intersect. The polarization optics diathroidal joints and plays a role in reducing the coefficient permits the observation of birefringence in gradation in both of friction in the opposing gliding surfaces or AC in all joints. positions of polarization going from less in one position to SZP and lubricin are encoded by the same gene, proteogly8 more when the specimen is turned 45 left or right. The in- can 4 ( prg4). Mutation in the prg4 gene has been attributed to ner areas of superficial zone are initially dark and increases the Camtodactyly-arthropathy-coxa-vara-pericarditis syn birefringency with turn of the slide. After papain treatment drome.34,35 A key feature of this syndrome is alterations in to remove proteoglycans there is a generalized increase in articular surface and attendant degradation of AC and  birefringency as a consequence of unmasking collagen fibrils. noninflammatory early onset joint failure. Thus, the funcIn the middle zone increased birefringency is observed with tional importance of SZP/lubricin is demonstrated by the polarized light. The territorial matrix in the pericellular areas pathophysiology of the joints in the Camtodactyly-arthropathydemonstrated weak birefringence under polarized light. The coxa-vara-pericarditis syndrome. appearance of the cells after papain treatment decreases Why focus on the surface of AC in arthritis? The first metachromasia and the territorial matrix shows the appear- changes at the onset of OA occur at the superficial zone of  ance of a Maltese cross, which move around the cells when AC.36–41 There is loss of cellular SZP immunostaining in the slide turned, which demonstrating a heterogenous col- degenerative AC in menisectomized sheep model of early lagenous organization. The interterritorial zone exhibits OA.42 In addition, there is a strong association between the thicker collagenous fibrils with an oblique pattern criss- loss of boundary lubrication and damage of AC in an excrossing at right angles to each other. The schematic depic- perimental model of OA in rabbits induced by transection of  tion (Fig. 2a) demonstrates that the fibers are parallel in the anterior and posterior cruciate ligaments. 43 Therefore, SZP surface and in the layer close to middle layer the oblique plays a critical role in joint physiology and is an attractive collagen fibers interspersed within vertical regions. Similar target for systematic investigations especially of the regulafibril organization also is observed in the deep zone. In the tory biology of the superficial zone of AC. course of repair one observes a disorganized fibril organiRegulation of SZP accumulation is critical not only for zation (Fig. 2b). Ideally, one would like to observe complete homeostasis and maintenance of AC, but also for the tissue regeneration with optimal fibril orientation (Fig. 2c). engineering and regenerative medicine of functional AC. As collagen is an integral component of cartilage matrix, SZP accumulation by superficial zone chondrocytes was its organization is critical for cartilage formation, growth, enhanced by transforming growth factor- b (TGF-b) in bovine repair, and regeneration. cartilage. BMP-7 stimulated the accumulation of SZP in both explant cultures of SZC from bovine AC and in monolayer cultures of chondrocytes derived from SZC.44 In view of this, Transforming Growth Factor-bs’ Family a systematic study of TGF-b/BMP superfamily members and Cartilage Zonal Organization was conducted on the responses of cells from SZC and syand Differentiation and Metabolism noviocytes in calf cartilage. 45 The BMP/TGF-b superfamily The AC has a distinct zonal organization that is intimately includes BMPs, TGF- b, growth/differentiation factors, and linked to function. The superficial zone cartilage (SZC), the activins. TGF-b isoforms 1, 2, and 3 were potent in stimumiddle zone cartilage (MZC), and the deep zone cartilage lating SZP secretion by both superficial zone chondrocytes (DZC) are generally identified in AC. 33 The AC in addition and synoviocytes. 46 SZP is mainly secreted by SZC but not has a calcified cartilage zone that has mineralized ECM and middle and deep zones even in the presence of TGF- b. The is distinguished at the interface with DZC by the so-called response of superficial zone chondrocytes and synovium to tide mark, which in most histological stains such as toluidine TGF-b isoforms is dose dependent and is biphasic. High dose   blue has different staining properties from the metachro- (30ng/mL) was inhibitory compared to the optimal dose matic middle and deep zones (MZC and DZC). The entire (1–3 ng/mL). The biological actions of TGF-b isoforms are



FIG. 1. Structure of human articular cartilage. Human articular cartilage sections were stained with Picrosirius and hematoxylin. (a–c) depict the normal structure of human articular cartilage. The three zones are observed: superficial, middle, and deep. In addition, the transition zones can be identified  between each of the main zones. Light microscopy demonstrates (a) a metachromasia throughout the extracellular matrix (ECM) (violet color), except in the superficial zone where cells appear flattened and fusiform forming a thin layer. The surface of the superficial zone is more intensely stained by Picrosirius Red than the deep layer. Polarization microscopy (b, c) depicts in this surface region collagen fibrils oriented parallel to the surface in two types of bundles intersecting  between them can be observed. Thus, one can observe with two different angles of polarization (turning the specimen 45 8 from right to left) birefringency in both positions, although less in one (b) than the other (c). Moreover, the inner part of the superficial zone appears dark in the first location of  the lens (b) but with strong birefringency in the other (c). Thus, the outer part is reinforced with a minor extra bundles of collagen fi brils running parallel to the surface  but forming a certain angle with the main fibrils of that area. Next, the histological sections were treated with the proteolytic enzyme papain. Papain-digested slides (d–f) show, in general, an increase in birefringency, demonstrating an interaction of collagens with proteoglycans. The surface of the superficial zone does not show enhancement of birefringency after loss of proteoglycans by papain digestion (e, f). After papain digestion metachromasia disappears as a consequence of the removal of proteoglycans. The ECM shows a generalized red color (d). A transition area between the superficial zone and the middle one can be detected as coarse and intense red stained fibrils, which present intense birefringency when the sections are seen under polarized light (e). These fibrils are intertwined and oriented in perpendicular directions. Below the transition area, the middle zone occupies most of the articular cartilage. Here the cells are round and are embedded in ECM distinguishable with territorial matrix, around the cells, and the interterritorial space, between the cells. In control conditions without polarization, the interterritorial ECM appears to have few collagen fibrils (b), except in the zone close to surface where the polarized light depicts fibrils running parallel 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 Maltese cross image. The papain digestion unmasks a dense area of collagen fibrils. The Maltese cross appears more clearly around the cells and the interterritorial spaces are crossed by thick fibrils of collagen, which run parallel but in two predominant orientations: oblique to the surface forming a right angle between them (e). When the specimen turned 45 8 from right to left more 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 at the bottom area (d), corresponding with thick bundles of collagen fibrils running parallel between them but oriented in two main directions, which form a narrow angle ( <308), being one of them (the stronger one) perpendicular to the surface. This arrangement of the fibrils can be distinguished when the specimen is turned 45 8 from right to left (e, f). The intensity of the   birefringency increases close to the subchondral bone (f).



FIG. 2. A schematic description of  the collagen fiber orientation in the human articular cartilage. The normal structure (a) is represented as Figure 1. In (b), the appeared repair tissue in the defect is shown, whereas in (c) it represents the ideal desired outcome of the defect. In the normal cartilage the fibrils are parallel to the surface of the cartilage. Just beneath the parallel fibrils there are obliquely oriented fibrils interspersed with more vertical fibrils. In the middle zone the oblique fibrils are at *458. In the deep zone there are rows of  columnar cells with vertical fibrils. The oblique fibrils can also be observed in the deep zone as in the middle zone. Also depicted in the ‘‘repair’’ state disorganized fibril organization. On the other hand, in the ‘‘regeneration’’ the structure of collagen fibrils is identical to the ‘‘normal’’ articular cartilage not only  because of thenormalcelland matrix organization but also because of the complete integration of new cartilage with the old cartilage.

dependent on TGF-b receptor I kinase as specific inhibitors of  the kinase blocked the response.45 Members of the BMP family (BMP-2, 4, and 7, and growth/differentiation factor-5) demonstrated a distinct differential response in SZC chondrocytes and synoviocytes in the bovine joint. The synoviocytes were much more responsive to BMPs. Similarly, synoviocytes were more sensitive to activins A, B, and AB compared to chondrocytes from the superficial zone. These observations demonstrate the differential regulation between superficial zone and synoviocytes in the joint, indicating the differences among the various compartments of the joint such as AC and synovium. In addition, there may be a division of labor among the BMP/TGF- b superfamily; the superficial zone is more responsive to TGF- b isoforms, whereas the middle zone is mainly regulated by BMPs. The function of AC is intimately linked to biomechanics. Mechanical loading is critical for homeostasis of musculoskeletal tissues, including AC. AC performs the biomechanical function of load support and lubrication with minimal wear and little or no damage in animals and humans. Dynamic shear stimulates SZP. The molecular basis of mechanotransduction in AC is beginning to be understood. The SZP expression pattern is dependent on the geometry of the femoral condyle surface and anatomical location. The anterior sites secreted more SZP in both lateral and medial femoral condyles. The biomechanical assays of SZP accumulation were confirmed and corroborated by immunolocalization of SZP in the various anatomical locations of the lateral and medial femoral condyles. 47 Again, the highest SZP were in anterior region compared to the posterior sites. The distribution of maximum contact pressures was associated with high SZP content. Anatomical regions of high contact pressure were consistently located in the anterior region. In addition, shear loading of the anterior medial

condyle increased significantly SZP secretion compared to posterior sites of the same femoral condyle. The SZP response to shear-loading was abolished in the presence of TGF- b type I receptor kinase inhibitor, demonstrating the critical role of  TGF-b signaling in mediating mechanotransduction. 48 Thus, these experiments demonstrate a new role for TGF-b signaling pathway in joint lubrication and is mediated by cellular mechanotransduction in superficial zone of AC. The middle and deep zones were refractory to shear loading, further illustrating that there apparently a gradient of response originating from the surface to deep zones. The SZC is therefore critical in mechanotransduction and in joint lu brication. The science of tribology deals with lubrication and wear in various surface boundary lubrication regimens. A new field of biotribology has emerged dealing with biological surfaces in sliding contact combining concepts of friction, wear, and lubrication of opposing interacting surfaces such as the gliding AC surfaces in the joint.49 Thus, the emerging new findings in AC are also critical for regeneration and restoration of damaged AC in OA by tissueengineered cartilage. It is critical that attention be focused on the characteristics of low coefficient of friction and high resistance to wear in regenerative medicine and tissue engineering of AC. Cell Therapy for Cartilage Regeneration

During the last decade there has been an exponential increase in research activity in the field of cartilage tissue engineering. AC is seen as an ideal candidate for a tissue engineering approach to tissue regeneration. Trauma to the AC surface of the joint represents a challenging clinical problem because of the very limited ability of  this tissue to self-repair. A number of surgical protocols are


currently in use for the treatment of AC defects. In abrasion arthroplasty,50 for instance, the subchondral bone is perforated by drilling to promote bleeding into the defect, with the result that there is formation of bone and fibrous repair tissue. In the microfracture technique51 the exposed subchondral bone is microfractured (‘‘picked’’) to promote localized bleeding. Moreover, repair techniques such as microfracture, which introduce cells into the joint, have unpredictable clinical outcomes as they produce a fibrocartilage tissue that degenerates with time. Another approach involves the use of allografts 52 where cartilage lesions are filled with grafts of donor-derived osteochondral fragments. However, these procedures are restricted by the availability of suitable donor tissue. In mosaicplasty52 cylindrical osteochondral plugs are harvested from nonload-bearing sites in the affected joint and pressed into place within the osteochondral defect, creating an autograft ‘‘mosaic’’ to fill the lesion. Autologous chondrocyte implantation (ACI) therapy52 represents another approach, where a chondral biopsy is taken from a donor site at the time of clinical examination. One of the major relevant issue in this field as been the recent acceptance of ChondroCelectÒ (by TiGenix) as a cell-based medicinal product consisting of chondrocytes that are taken from a healthy region of the patient’s cartilage, grown outside the body, and then re-implanted during an ACI surgical procedure. The ACI procedure with a membrane of collagen (collagens I and III) is called MACI. Chondrocytes, enzymatically released from the retrieved tissue, are expanded in monolayer culture, and subsequently implanted in a second procedure beneath the periosteal membrane or the MACI membrane, which is sutured to the cartilage adjacent to the defect and sealed with fibrin glue. All of these approaches offer exciting opportunities for the regeneration of cartilage defects. However, the long-term outcome may be uncertain and there are may be other disadvantages associated with the harvest site, even when it is some distance from the lesion. In this regard, nowadays it is well known that lesion size, activity level, and age were the influencing parameters of the outcome of AC repair surgery.53 Lesions >2.5cm2 should be treated with ACI or osteochondral autologous transplantation, whereas microfracture is a good first-line treatment option for smaller ( <2.5cm2) lesions. Patients who are active show better results after ACI or osteochondral autologous transplantation when compared with microfracture. In this regard, studies discussed by Knutsen et al. conclude that both ACI and microfracture provide satisfactory results in 77% of the patients at 5 years with no significant difference in the clinical and radiographic results. 54 However, because there are limitations, these have been the driving force behind the emerging field of AC tissue engineering, whose approaches may provide an ideal alternative to the current surgical treatments for cartilage repair. Implantation of cells with both chondrogenic and osteogenic potential derived from the bone marrow for the treatment of  osteochondral lesions represents another approach that might result in persistent, functional restoration of the AC. Chondrogenic cells that are in more abundant supply can be used for cartilage tissue engineering. Identification of a suitable cell population for cartilage tissue regeneration is the critical first step in the process. Articular chondrocytes are the most obvious choice. To obtain large numbers of allogeneic chondrocytes for culture of autologous cartilage con-


structs, it is necessary to be able to expand the cells in culture. Different groups have carried out experiments to assess whether ovine chondrocytes could be expanded in monolayer and retain their ability to produce cartilage in the culture system.55 Aging of the cells is an issue when these autogenous cell based techniques are applied to aged people. Chondrocytes obtained from the aged have much lower ability to repair cartilage than young ones.56 For cartilage regeneration in aged people, using cell type with greater potential, such as  bone marrow stromal progenitor cells, may be perhaps more promising,54 as it has been demonstrated recently where age did not made a difference in outcomes in the use of bone marrow-derived MSCs in patients older than 45 years.57 There are also a prevalence in the use of MSCs from synovial origin rather than bone marrow in terms of chondrogenesis as it has been demonstrated by several authors with porcine58 and human multipotent MSCs isolated from the synovial membrane of knee joints.59,60 These cells may be superior as a potential source of MSCs and play a role in the regenerative response during OA. 61 More recently, attention has been focused on human embryonic stem cells as they have the ability to self-renew and differentiate into any cell lineage of the three germ layers, therefore holding great promise for regenerative medicine applications. In this term, Toh et al. describe a micromass culture system as a model system to study chondrogenic differentiation and modulate cartilage-specific matrix gene expression in a distinctive manner.62 The development of effective therapies for OA has been slow, and today most recommended medications alleviate symptoms without altering the course of the disease. In many cases, joint replacement surgery is the best option to restore joint function. The observations made by several authors63 on the characteristics of stem cells from OA patients indicated that stem cell therapy might be an effective approach in impeding the degenerative changes in the OA  joint. A number of studies have been carried out involving the delivery of stem cells to the goat knee joint following medial menisectomy, resection of the anterior cruciate ligament, or a combination of the two.64 This procedure, in combination with a defined exercise regime, leads to the development of lesions in the joint that are characteristic of  OA. Transduced cells with green fluorescent protein have   been injected and the general approach was to deliver the cells as a suspension by intra-articular injection, thus avoiding the need for arthrotomy and the placement of a scaffold.65 Injected cells were retained within the joint and also recovered from synovial fluid in a viable form. The cells colonized soft tissue surfaces, primarily those of synovial origin rather than the AC. Injection of MSCs into destabilized, osteoarthritic joints resulted in marked remodeling of  the medial meniscus, which had been totally removed during surgery. 66 The new tissue that formed has a hyaline-like appearance and focal areas of type II collagen similar to developing rabbit meniscus. Related to this tissue regeneration is a marked chondroprotective effect of the MSCs injection. These observations highlight the potential therapeutic benefit of injected MSCs in an osteoarthritic joint by regenerating neomeniscal tissue to stabilize the joint and protect the articular surfaces against progressive degeneration.67 Wakitani and others show the first clinical trial using



autologous culture-expanded bone marrow MSCs to repair chondrocytes.74 In that study, no differentiation into muscle AC.68 Subsequently, they performed this procedure in about was obtained when cells from a low passage were used, 40 patients demonstrating the safety of the procedure. Later whereas with later passages, cartilage-derived muscle fibers on, they demonstrated improvement in clinical symptoms were obtained, confirming the amplification of adult articuwith a hyaline-like type of cartilage tissue in young, active lar chondrocytes in vitro results in a population of cells with patients,69 although the repair cartilage was not hyaline progenitor properties. cartilage in normal age individuals. To regenerate AC by cell Biomaterial scaffolds provide the chondrogenic cells transplantation, it is essential that cells proliferate without with a microenvironment, where they survive, multiply, and losing their capacity for differentiation. To find appropriate produce ECM to constitute regenerated cartilage. Although conditions, different culture conditions, mechanical stresses, the cellular products are expected to replace the degradable growth factors, and gene transfection have all been explored,  biomaterial, the process is usually time-consuming and the  but these have not yet been applied clinically. 70 scaffold should be implanted before completion of the proOne might consider the phenotypic plasticity as an in vitro cess. The biomaterials thus play the role of a vehicle to artefact as the chondrocytes are being exposed to and arti- transfer cells and therefore should be compatible with the ficial environment and being extensively modified during native tissue around the recipient site. 76 Many natural subtheir expansion in monolayer. However, plasticity between stances are suitable as the cell-carrying scaffold for cartilage different phenotypes is a common phenomenon during em- engineering, including fibrin, agarose, alginate, collagen,  bryonic development of tissues and organs. Using this fea- chitosan, and hyaluronan. Many of these are hydrogels and ture, the same cells can be reprogrammed to serve several can be designed as injectable in their liquid form, which functions. Also, the plasticity is preserved and has critical  blends well with chondrogenic cells.77 After being injected functions in adult amphibians such as salamanders, as ob- into the recipient site, they set by gelation to fill in any shape served in the regeneration of an amputated limb. 71 and size of cartilage defect. In vivo, plasticity is mainly seen during the embryonic Either chondrocytes or stem cells are used to constitute development of organs and tissues, for example, the shift engineered cartilage useful to regenerate damaged tissue, from endothelial-like cells to mesenchymal cells during the and in vitro manipulation of the cells is necessary in most of  embryonic formation of synovial joints. This change is made the currently available systems. When the constructed carfrom the cells that will form the AC, the interzonal cells. tilage tissue is considered for clinical used, the safety of the Initially, these cells have characteristics similar to the endo- whole process needs to be debated and the cost is high. The thelial cells lining blood vessels and, after condensation, will entire process has to be conducted with expensive laboratory round up and start to secrete cartilage matrix proteins. The facilities that meet the high standard of good tissue practice. shift from endothelial to mesenchymal cells has further been In addition, all reagents involved in the process should be shown to persist in the formation of adult heart valves. 72 proven as safe for human use. More complicated manipuIt is a well-established fact that during monolayer ex- lation of the cells will arouse more concern that the cells pansion of chondrocytes in vitro, the cell population losses its may be affected in unknown ways. When developing a phenotype and starts to express primitive embryonic mark- system to regenerate cartilage for clinical application, one ers.73,74 This dedifferentiation process is not unique for should always consider the safety and efficacy of the rechondrocytes but a rather common phenomenon observed in generated tissue. many types of cells and tissues. 75–77 Another possible exAs a result of the variable and unpredictable clinical explanation to the change in expression during the expansion is periences in cartilage regeneration in the past, biotechnology that the culture conditions are favoring certain cell subpop- has been introduced to this field for evidence-based develulations, for example, transit-amplifying cells, and that these opment of a solution. The knowledge to date supports that cells are able to respond to the culture conditions more AC is best repaired with autologous engineered cartilage, rapidly. and a considerable research has been carried out to improve During redifferentiation of dedifferentiated adult chon- cartilage regeneration. Although the efficacy of regeneration drocytes expanded in human serum, chondrocytes do ex- has much improved in the laboratory and animal studies, press genes that have been described in the initiation of  most findings have not been investigated for their clinical chondrogenesis during limb formation.77 This shows, in safety and performance. Further studies should highlight addition to the phenotypic plasticity, 77,78 that the cells revert their clinical relevance to facilitate the development of  to a primitive stage during the expansion in vitro. Culture- products applicable to humans. expanded chondrocytes are able to redifferentiate when One needs to organize currently available knowledge to replaced into three-dimensional environment under appro- develop clinically applicable models of cartilage regenerapriate culture conditions. 79–81 This ability of redifferentiation tion, on the basis of autogenous chondrogenic cell implanis particularly important when the cells need to be trans- tation. A clinically applicable model of cartilage regeneration planted into patients. To better define the chondrogenic should be safe, efficient, and simple. It can be completed in a phenotype and to try to optimize the cellular recovery for single seed-and-implant surgery procedure, which decreases clinical cell transplantation, several attempts have been made the surgical risks and complications from repetitive operato characterize culture-expanded chondrocytes at the mo- tions of conventional autologous chondrocytes implantation. If the site of repair allows an arthroscopic approach, the lecular level.80–84 During the expansion of human chondrocytes, the degree surgery can be done in a minimally invasive manner within a of dedifferentiation has been correlated to the number of cell short time, estimated at 1 h. By avoiding the complex treatdivision or passages.73–75 This was a typical characteristic ment of the autogenous cells in vitro, the safety of the prodemonstrated in the study of myogenic differentiation of  cedure can be improved and the cost reduced.

624 The Future: Challenges and Opportunities

The foregoing discussion of the structure of the AC, the zonal organization, the regulation and mechanotransduction, and joint lubrication sets the stage for contemplating the future challenges and opportunities for regeneration and tissue engineering of AC. This brief review has by design focused on structure, fibril-orientation and the cell shape, TGF- b signaling pathways, and mechanotransduction. It is well known to most students and practioners of tissue engineering that it requires triad of signals, stem cells, and scaffolds. 9 In future, there will   be continuous refinement of the scaffolds for the tissue assembly during tissue engineering. The native AC is a durable tissue that, unless ravaged by arthritis, lasts a life time in humans. The AC is an engineering marvel in the human body. It is truly outstanding that the ECM of cartilage has predominantly collagen II providing tensile strength and the electronegative proteoglycan aggrecan, which permits movement of water with the matrix to ensure electroneutrality. This represents an advantage for an efficient function with a minimal energy expense. AC has significant biomechanical properties, including compressive modulus of 0.79MPa, a shear modulus of  0.69 MPa, and a tensile modulus with a range from 0.3 to 10 MPa.78 Thus, a dynamic hydrogel of cartilage ECM is critical for the biochemical, cellular, and biomechanical function. The recent advances in nanomaterials bodes well for nanoscaffolds for AC regenerative medicine. Therefore, we enter the realms of nanomaterials and nanomedicine for the benefit of patients with OA desperately looking for solutions for the painful disease. 79 Thus, regenerative medicine of AC and tissue engineering presents critical challenges for the future and presents outstanding opportunities. Cells to be used for an efficient regenerative medicine should be chosen. Undifferentiated versus differentiated or predifferentiated chondrocytes will be the choice. A permanent solution will come when the new tissue built in the defect is of the same nature and is perfectly integrated in the whole structure in any pathology and in any age. Only in such s way structure and function will be fully recovered. In conclusion, the recent advances in biotechnology, nanotechnology, and nanomaterials bode well for an optimistic and bright future for regenerative medicine of AC. Acknowledgments

The authors thank P. Jime´nez-Palomo for his excellent technical assistance. This work was supported by grants from the Banco Bilbao-Vizcaya-Argentaria Foundation (FBBVA, Chair in Biomedicine 2007 to A.H. Reddi), the Ministry of Science and Technology (BIO2009-13903-C02-01), the Ministry of Science and Innovation (FIS PI06/1855, PLE2009-0163, FIS PI10/2529), Red TerCel (Institute of Health Carlos III), and the Andalusian AutonomousGovernment (P07-CVI-2781, PAIDI, and BIO-217). Disclosure Statement

No competing financial interests exist. References 1. Brandt, K.D., and Fife R.S. Ageing in relation to the pathogenesis of osteoarthritis. Clin Rheum Dis 12, 117, 1986.

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Address correspondence to:  Jose´  A. Andrades, Ph.D. Laboratory of Bioengineering and Tissue Regeneration (LABRET-UMA) Department of Cell Biology, Genetics and Physiology Faculty of Sciences, University of Ma´ laga  Ma´ laga 29071 Spain E-mail: [email protected] Received: April 3, 2010   Accepted: September 13, 2010 Online Publication Date: October 28, 2010

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