- Email: [email protected]
Cartilage regeneration
Oral Maxillofacial Surg Clin N Am 14 (2002) 105 – 116
Cartilage regeneration Barbara D. Boyan, PhDa,b,c,*, David D. Dean, PhDa, Christoph H. Lohmann, MDa,d, Gabriele G. Niederauer, PhDe, Jacquelyn McMillan, MBChB, FRCSEd (Orth)a, Victor L. Sylvia, PhDa, Zvi Schwartz, DMD, PhDa,c,f a
Department of Orthopaedics, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229 – 3900, USA b Department of Biochemistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229 – 3900, USA c Department of Periodontics, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229 – 3900, USA d Department of Orthopaedics, University of Hamburg Eppendorf, D-20246 Hamburg, Germany e OsteoBiologics, Incorporated, 12500 Network, Suite 112, San Antonio, TX 78249, USA f Department of Periodontics, Hebrew University Hadassah, Jerusalem, Israel
Articular cartilage has been a primary focus of the emerging tissue engineering industry. The market for cartilage repair technologies is considerable for trauma and sports injuries and even greater for regeneration of cartilage in patients with arthritis. Articular cartilage degeneration is accompanied by morbidity, which leads to absenteeism and development of conditions associated with chronic pain. In severe cases, the loss of function is of sufficient magnitude that it is necessary to replace damaged joint tissue with a bioprosthesis.
Problems associated with tissue engineering of cartilage Despite these economic drivers, successful tissue engineering of articular cartilage has been elusive. Unfortunately, cartilage does not heal in the same manner as seen in other tissues, in part because it has only a rudimentary blood supply. When the cartilage is severed, the chondrocytes seal the exposed edges
* Corresponding author. E-mail address: [email protected] (B.D. Boyan).
of the wound and in effect create a new cartilage surface [2,66,72]. The two sides of the defect do not fuse, which creates a focal change in the way that the tissue experiences compressive loads. Similarly, when injuries occur that cause loss of a piece of cartilage, the chondrocytes in the surrounding tissue again seal off the edges of the defect site. There is a limited attempt at repair, but the tissue that forms within the defect tends to be fibrocartilage. There are several reasons why fibrocartilage forms within chondral defects. The source of cells is believed to be synoviocytes [39], which are fibroblastic and as such synthesize type I collagen. Even if chondroprogenitor cells migrate into the defect site, they must produce large amounts of matrix quickly to facilitate migration across large regions of space relative to the cell, and type I collagen is favored under such circumstances. Should the defect penetrate the subchondral plate, a clot is able to form within the defect site and serves as a scaffold for cell attachment and migration. Many of the cells that colonize such defects are derived from the vasculature and marrow stroma [62], however, and possess the capacity to differentiate into various mesenchymal cell types. In the absence of a sufficient supply of chondrogenic factors, these cells tend to select the default pathway and differentiate along a fibroblastic lineage [38,43,49,53,61].
1042-3699/02/$ – see front matter D 2002, Elsevier Science (USA). All rights reserved. PII: S 1 0 4 2 - 3 6 9 9 ( 0 2 ) 0 0 0 1 7 - 1
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Articular cartilage architecture In comparison with organs such as liver and kidney, cartilage is a deceptively simple tissue in that only one cell type is present. This is both strength and weakness with respect to tissue engineering. Although there is no need to design complex three-dimensional constructs that contain several different types of cells, the heterogeneity within the cartilage in terms of chondrocyte phenotype does require consideration. Cells at the top of the articular cartilage exhibit a flattened morphology. These cells form the barrier with the synovial fluid and provide a surface that can withstand shear. Most of the cartilage is hyaline in nature, with clonal populations of chondrocytes, each in its own lacunae, surrounded by a pericellular matrix that differs from the interterritorial matrix. The cells adjacent to the subchondral bone have yet another phenotype. These cells are hypertrophic and appear in many ways like a miniature growth plate. This region is called the tidemark because it is characterized by a distinctly different staining property, partly because of the difference in the extracellular matrix and the deposition of hydroxyapatite. This phenotypic heterogeneity of the chondrocytes is accompanied by a complex three-dimensional architecture provided by the extracellular matrix. Type II collagen fibrils delineate the form of the matrix, which results in overlapping gothic cathedral-like arches that separate columns of cells [19,40]. There is also secondary structure in the form of crossstruts. In addition to the collagen framework, there is a fine net formed of cartilage oligomeric matrix protein (COMP) throughout the interterritorial matrix [26,75]. Interspersed among the fibrillar proteins are proteoglycan aggregates, composed of a hyaluronic acid backbone to which the aggrecan monomer is connected via association with link protein [9]. The number of aggrecan monomers and the length of the hyaluronic acid chain vary with the articular cartilage zone. Aggrecan is decorated with sulfated glycosaminoglycan side chains that also vary in number and length. During embryonic bone formation, the articular cartilages form as remnants of cartilage anlagen as they are replaced with bone and marrow. The embryonic tissue is highly cellular, but postfetal cartilage is relatively acellular. The articular cartilage continues to grow as the long bones continue to lengthen; however, most cartilage repair is completed in adults, and by then, any mitosis in primary cartilages is infrequent. It is likely that chondrocytes retain some proliferative capacity, which is stimulated by injury. Even so, the neocartilage that forms within a chondral
defect has an extracellular matrix that is different from that of the cartilage surrounding the defect site. The architectural features of the matrix are honed over time and occur in response to directional loads that are placed on the tissue [67,73]. Even the types of proteoglycan in the extracellular matrix may differ in terms of core protein [18] and glycosaminoglycan sulfation [17,34]. The amount of hydration of the tissue and, consequently, the hydrostatic forces experienced by the cells differ [35,46]. The resistance of the tissue to compressive loads also differs [5]. The integration of the neocartilage with the surrounding cartilage is often ineffective, not only because of the differences in matrix composition but also because of the cartilage matrix sealing effect. This results in mechanical instability that further reduces the success of the repair tissue. In healthy joints, the base of the articular cartilage is ‘‘glued’’ to the subchondral bone in a manner that is similar to the way that the base of a growth plate is attached to the bony metaphysis. By calcifying their matrix, the chondrocytes in the tidemark region form an interlock with the mineralized bone. Disruption of this interface also contributes to mechanical instability. It is currently known that it is critical to have good repair of the subchondral plate to have good repair of the articular cartilage [44]. Unfortunately, the subchondral plate can be damaged through trauma, during surgery, or as a consequence of degenerative diseases. Tissue-engineering strategies must consider how to ensure that mineralization of the cartilage occurs in an appropriate manner and provide a method for regenerating the bone itself.
Cartilage calcification and the tidemark Physiologic mineralization of cartilage involves chondrocyte hypertrophy and deposition of apatite crystals within the extracellular matrix. Both processes require turnover of the extracellular matrix. As the cells produce matrix, they also synthesize matrix processing enzymes and store them in inactive form as zymogens or together with inhibitors such as tissue inhibitor of metalloproteinase (TIMP) [14]. Other matrix processing enzymes are packaged in extracellular matrix vesicles and are released in active form upon signals from the cells. Because of the importance of maintaining a functional architecture, matrix turnover is slow in healthy tissue. At the tidemark, however, the rate of matrix degradation is believed to be increased, both to accommodate hypertrophy and to redesign the matrix for mineral deposition [21 – 23]. These cells produce increased numbers of matrix
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vesicles, which also provide sites for the initial formation of calcium phosphate crystals [55]. Successful tissue engineering must result in a cartilage in which these events occur in a regulated manner in time and space. Should the hypertrophy of the cells extend too far up into the hyaline cartilage, the possibility of osteophyte formation is increased, as is the likelihood of tissue destruction. If hypertrophy and matrix mineralization do not occur, then the repair cartilage is likely to delaminate.
Tissue engineering of cartilage This section of the article focuses on the engineering of articular cartilages at the ends of long bones. The mandibular condyle is a secondary cartilage and as such retains the ability to remodel throughout life. Relatively little is known about the cellular, biochemical, and structural features of the articular cartilage of the condyle that impinge on the success of tissue engineering in this tissue. For this reason, engineering strategies for the mandibular cartilage are not discussed. The need for cells The repair capacity for cartilage is low, because of the relatively low proliferative capacity of cells resident in the tissue and their low migratory ability. Because of this, the strategies for cartilage tissue engineering have focused on the delivery of cells to the defect site through various approaches. There are three different types of cartilage defects, each of which requires its own set of repair strategies. Partial-thickness defects Defects that do not extend to the subchondral plate are called ‘‘chondral’’ or ‘‘partial-thickness’’ defects. Surgeons are reluctant to use strategies that penetrate the subchondral plate for repair of these types of defects because to do so requires loss of additional cartilage. The usual approach is to remove undermined chondral flaps from the edges of the defect and leave stable cartilage undisturbed and hope for the best. In some instances, this is not a practical choice, yet the reluctance remains. The problems of how to maintain cells at the site or ‘‘glue’’ allograft to the existing cartilage have not yet been resolved. Hunziker and Rosenberg [39] have characterized the healing process of partial-thickness defects with respect to the cells that colonize the defect site and methods for enhancing their ability to differentiate into chondroblasts and ultimately become functional
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chondrocytes. Preparation of the chondral defect is critical. Not only is it necessary to remove the damaged cartilage but also healing is enhanced if the walls of the defect are etched with enzymes that remove the outer layer of cartilage matrix proteins. Host cells adhere more effectively to the tissue surface. They also have noted that delivery of transforming growth factor-beta (TGF-b) to the site in various time-release carriers also improves the differentiation of the colonizing cells into chondrocytes. Full-thickness defects When the cartilage defect extends to the subchondral plate, several strategies can be considered. The choice of strategies is related to the actual size of the defect. In humans, trauma and sports injuries result in defects that vary in size but tend to be focal. In contrast, arthritis leads to large regions of degenerate tissue and has the added complication of being chronic, involving the subchondral bone, and compromising the surrounding ‘‘healthy’’ cartilage tissue. The biggest problems to be resolved in treating full-thickness defects are the interface with the surrounding cartilage and the tendency of the neocartilage to delaminate. Microfracture. By far the most common tissue engineering strategy relies on microfracture of the subchondral plate, providing access of cells from the underlying vasculature and marrow stroma to migrate into the defect site. This technique has been popularized by Blevins et al. [8] and Steadman et al. [65] with reasonable success. After removing all damaged cartilage, the subchondral plate is penetrated at several sites. The blood that moves into the synovial space forms a clot in the defect and brings with it growth factors that can enhance tissue repair. Mesenchymal cells migrate onto and through the hematoma, and in the presence of the surrounding cartilage at least some of these cells differentiate into chondroblasts. The subchondral plate is repaired through endochondral ossification, the same mechanism that repairs bone fractures. The chondroblasts produce a cartilage matrix, but the quality of the matrix is variable at best. Eventually, fibrocartilage predominates. This approach can be successful clinically because the patient can return to function relatively pain free. Over time, the repair tissue may fail, but for some patients, it is adequate. Periosteum and perichondrium. The periosteum, the skin-like tissue that forms on the outer surface of bone, is a rich source of multipotent mesenchymal cells, including cells with the potential to become chondrocytes. O’Driscoll et al. [50 – 52] have pio-
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neered the use of periosteal autografts for treatment of chondral defects. By placing the periosteum in an inverted fashion in the defect site, the mesenchymal cells can migrate easily into the defect space. Their ability to migrate out of the site is reduced by the graft itself. Ideally, the periosteal cells differentiate into chondroblasts in the presence of chondrogenic signals from the surrounding cartilage. This technique has proved successful in some cases, but exactly what makes one case a success and another case a failure is not well understood. Pretreatment of the tissue with TGF-b improves chondrogenesis [45] for the same reasons as noted for partial-thickness defects. To improve on the concept, Amiel et al. [1] and Chu et al. [20] have suggested that perichondrial tissue may be a better source of cells. The perichondrium is an extension of the periosteum, but it overlies cartilage tissue. These investigators reasoned that it was likely to contain a greater percentage of chondroprogenitor cells. They have used perichondrium successfully as a source of cells for treatment of full-thickness defects. Again, treatment of the cells with TGF-b improved success. Cartilage cell therapy Another approach is to use committed chondrocytes rather than tissue that contains chondroprogenitor cells. Allografts and ex vivo tissue-engineered cartilage Cartilage autografts are in short supply; otherwise the patient would not need a tissue-engineering approach for repair of the defect. Allografts are used to cover large defects, with varying success [6,16]. One method that holds promise is the use of allografts produced by tissue engineering ex vivo. Several methods have been developed to achieve the goal of making a tissue in culture that has the properties of articular cartilage and integrates with the surrounding host cartilage and with the subchondral bone [68,71]. To accomplish this goal, several problems have emerged that have required innovative solutions, including the need for a reliable source of cells, the need for a structural scaffold on which to grow the cells in three dimensions, the contribution of mechanical force to the development of the tissue, and the development of novel bioreactors to achieve sufficient tissue in a reasonable period of time [36,56]. Despite considerable success in solving each of these problems, the resulting tissue still does not replicate the qualities of the native host tissue. The only Food and Drug Administration-approved cartilage cell therapy currently in use clinically in the
United States is the direct delivery of autologous chondrocytes to the defect site. This methodology was pioneered by Brittberg et al. [15] and has been the subject of several studies over the past 5 years. The concept is simple. Cartilage is removed surgically from a non – weight-bearing region of the joint to be treated, and the cells isolated from that cartilage are cultured ex vivo through four passages. This practice is critical because of the low cellularity of cartilage. It is necessary to expand the number of cells to increase the likelihood of repopulating the defect with chondrocytes. At the same time of this surgery, the defect site is prepared. Periosteal tissue is removed from the bone and used to create a seal over the surface of the defect. Currently, this periosteal flap is sutured, but the process of suturing itself leads to defects in the cartilage, so attempts to develop a glue to attach the periosteum to the cartilage surface are also underway. While the cells are in culture, the surgical site is allowed to heal. When the cells are ready, they are injected into the defect site arthroscopically. Technically, the periosteal seal is intended to retain the cells within the defect. Practically, this does not occur. Second-look arthroscopies show that the periosteum can be dislodged and that the cells tend to migrate out of the defect. When the treatment does work, however, the results are promising. Whether they hold up in the long term is not yet known. Studies using dogs and goats suggest that this neocartilage brings with it the same problems as seen with other repair strategies [13,24]. It also is not clear whether the repair cartilage is the consequence of the cells in the periosteum, the cells that are injected, or some combination of the two. More recently, investigators have begun to grow cartilage in multilayers on membranes [41] and to transfer the construct to the defect site rather than to inject cell suspensions. This technique may not require the use of a periosteal seal, because the cells are in effect anchored to the underlying membrane. The limitation is the attachment to the subchondral bone. All of the cell strategies are limited by the tendency of chondrocytes to lose their chondrogenic phenotype in monolayer culture. More than 20 years ago, Benya et al. [7] noted that articular chondrocytes lose their ability to synthesize type II collagen and produce sulfated proteoglycan when they were grown in monolayer culture. The chondrocyte phenotype was restored when the cells were grown in suspension culture or in various kinds of gels. Alginate was particularly effective at preserving these cell characteristics [31]. The reasons for this are not entirely understood, but it is clear that retention of the rounded articular chondrocyte morphology is impor-
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tant. Unfortunately, cells grow slowly using these methods, which makes it difficult to achieve sufficient expansion. Recently, alginate has been used to microencapsulate articular chondrocytes after culture in monolayer and found that the microencapsulation process restored the ability of the cells to produce type II collagen and sulfated proteoglycan (A. Sambanis, unpublished observation). This opens up the possibility of expanding the cells in monolayer culture and then preparing them for injection via microencapsulation. Alternatively, articular chondrocytes can be expanded in bioreactors. Various methods are being developed for this purpose. Currently, the cells tend to form spherical nodules in these sorts of culture systems and develop three-dimensional architecture within the nodules, including hypertrophic chondrocytes at the center [28,69,74]. Whether these nodules are suitable for use in treating full-thickness defects is not yet clear. Another approach is to use chondrocytes that do not undergo such marked loss of phenotype in vitro. The authors’ laboratory has used hyaline chondrocytes from the resting zone of the costochondral cartilage of the rib for this purpose. Although these cells are not identical to articular chondrocytes, they are not yet in the endochondral lineage per se, and they produce a proteoglycan-rich extracellular matrix through four passages in culture [10]. Although they do produce
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type I collagen as an adaptation to monolayer culture, they also continue to produce type II collagen [10], and when they are implanted in nude mouse muscle in vivo, they form cartilage nodules that are indistinguishable from normal hyaline cartilage [11]. One problem with using chondrocytes is their tendency to undergo hypertrophy (Fig. 1). To increase the number of chondrocytes while at the same time control their differentiation along the endochondral pathway is an elusive goal of cartilage cell therapy. One approach is to pretreat autologous chondrocytes with growth factors that modulate one or more of the phenotypic characteristics of the cells. Unfortunately, growth factors are pleiotropic, affecting different cells in different ways. They may affect the same cell differentially depending on the cell’s state of maturation within its lineage cascade. This is the case with chondrocytes. As shown in Table 1, cells from the resting zone of costochondral cartilage respond to various regulatory factors in a manner that is distinct from the response of growth zone cells, which are derived from the prehypertrophic and upper hypertrophic zones of the rat costochondral growth plate. Table 1 is a summary of several experiments performed in the authors’ laboratory using factors and hormones that are known to regulate cartilage and bone or that are being tested for their effectiveness in various musculoskeletal tissue-engineering applications [42,43,58 – 60]. Many of the factors increase
Fig. 1. Rat costochondral cartilage resting zone chondrocytes were loaded on a polylactic acid/polyglycolic acid porous foam scaffold and implanted for 8 weeks in the calf muscle of a nude mouse. Chondrogenesis has occurred throughout the scaffold space and where the scaffold has resorbed, the cartilage is in direct contact with the surrounding muscle. Chondrocytes at the center of the neocartilage are producing a cartilage matrix and some of the cells are undergoing hypertrophy. Residual scaffold is evident as a clear area, due to its dissolution during processing. (Haematoxylin and eosin, original magnification 10).
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Table 1 Cell maturation and chondrocyte response to bioactive factors EMD
TP508
PDGF-BB
IGF-1
TGF-b1
FGF-2
BMP-2
1,25
24,25
Resting zone cells Proliferation Alkaline phosphatase [35S]-sulfate incorporation Response to 1,25-D3
" # — ND
— # " ND
" — " —
" " " —
" " " "
" " " ND
" — " "
# — — ND
"# " " "
Growth zone cells Proliferation Alkaline phosphatase [35S]-sulfate incorporation
" — #
" — —
ND ND ND
ND ND ND
" " "
ND ND ND
" " —
# " "
" — —
Abbreviations: FGF-2, basic fibroblast growth factor; IGF-1, insulin-like growth factor; ND, not done; 1,25, 1,25 dihydroxyvitamin D3; 24,25, 24,25 dihydroxyvitamin D3.
proliferation of the hyaline-like resting zone cells. Of those factors that stimulate proliferation, some also stimulate proteoglycan production based on the incorporation of radiolabeled sulfate into glycosaminoglycans, which indicates that cartilage matrix synthesis is enhanced. Most of the factors also promote endochondral differentiation of the cells, however. Alkaline phosphatase specific activity is increased, and this enzyme, which is associated with extracellular organelles called matrix vesicles, is involved in calcification. Resting zone cells normally respond primarily to the vitamin D metabolite 24R,25(OH)2D3, whereas the more mature growth zone cells respond primarily to 1a,25(OH)2D3. When resting zone cells are treated with TGF-b1, bone morphogenetic protein2, or 24R,25(OH)2D3 for extended periods of time, however, responsiveness to1a,25(OH)2D3 is upregulated and responsiveness to 24R,25(OH)2D3 is lost, which indicates that these cells not only have become more differentiated but also have acquired a growth zone chondrocyte phenotype [58 – 60]. The factors that show the most promise for pretreatment of chondrocytes are factors that stimulate proliferation and cartilage matrix production but retard or inhibit endochondral differentiation. Of all of the factors that the authors have tested to date, the best candidates seem to be Emdogain (EMD) (Biora, Inc., Malmo, Sweden), which increases the pool of chondroprogenitor cells [12,57], TP508 (Chrysalin, Chrysalis, BioTechnology, Inc, Galveston, TX), which enhances matrix synthesis but not differentiation, and platelet-derived growth factor-BB (PDGF-BB), which stimulates proliferation and matrix synthesis, but not endochondral maturation [42]. To test whether these in vitro assays are relevant to in vivo behavior, the authors pretreated resting zone chondrocytes for 4 hours or 24 hours with PDGF-BB
before implantation in nude mouse muscle. For these experiments, they used scaffolds provided by OsteoBiologics, Inc. (San Antonio, TX). After treatment, the cells were loaded onto the scaffolds and implanted bilaterally in the calf muscles. Implanted tissue was examined histomorphometrically for the presence of new cartilage and the degree of chondrocyte hypertrophy at 4 and 8 weeks. The results showed that PDGF-BB pretreatment increased the amount of neocartilage at 8 weeks and prevented chondrocyte hypertrophy; however, the shorter exposure to the growth factor was more effective than the longer exposure [43]. This result shows that the type of factor used is important, and it indicates that the pretreatment regimen may be critical. Finally, several other cell-based strategies are in various states of development. Mesenchymal stem cells (MSCs) were first used by Wakitani et al. [70] to repair full-thickness defects in rabbits. These multipotent cells differentiated into chondrocytes and formed neocartilage within the experimental defects that was comparable to neocartilage formed by committed chondrocytes. The effectiveness of the strategy was improved through the use of gels to create MSCbased constructs [54,64]. The use of MSCs is a powerful technology because of their high proliferative capacity and their ability to differentiate into tissues. There are some caveats, however. For example, recent studies suggest that the phenotype of MSCs can vary, depending on the physiology of the donor [47]. Even taking this into consideration, MSCs hold great promise. One of the most innovative uses is the use of fat as a source of MSCs [25,76]. MSCs in fat possess the ability to differentiate into chondrocytes when treated with growth factors that induced chondrogenesis, such as TGF-b. Similarly, MSCs isolated from perichondrium can be used if pretreated
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Fig. 2. Mosaic arthroplasty showing the placement of osteochondral cores within a defect. (Courtesy of Smith and Nephew, Endoscopy Division, Andover, MA.)
with TGF-b or when transfected with plasmids that contain TGF-b1 DNA [32]. Osteochondral defects In instances in which the subchondral bone is damaged, a core of cartilage and underlying bone is removed. If the defect is small enough, healing occurs by a process similar to that described for microfracture. In larger defects, the repair of the subchondral bone is inadequate to support the repair cartilage, and the result is poor. For these types of defects, mosaic arthroplasty is preferred. Mosaic arthroplasty. Mosaic arthroplasty relies on the use of an osteochondral plug from a region of the articular cartilage that is normally not required for weight bearing [36,37]. The surgeon may remove several cores of cartilage and bone from this site and then place them in a mosaic-like fashion in the defect site on the weight-bearing region of the joint (Fig. 2). This method works well because the cartilage in each plug remains viable, but problems arise because the transplanted cartilage does not fuse with the surrounding host tissue. Just as the edges of the cartilage defect site seal, so do the edges of the cartilage plugs. The defect site is osteochondral and permits clot formation and osteochondral repair around and between each plug. This repair tissue is fibrocartilage as would be the case for microfracture. The final result is an improvement over simply allowing the larger osteochondral defect to heal via clot formation and mesenchymal cell defferentiation, but the me-
chanical instabilities created by the variation in tissue type may predispose this strategy to failure. Cell-based therapies. Cell-based therapies also are used for osteochondral repair, but they depend on a suitable delivery device because the subchondral plate is not present to serve as a substrate. All of the same cell strategies as described for full-thickness defects with respect to cell source are applicable. The physical properties of the scaffold are critical to ensure appropriate healing of the defect with bone and cartilage, however. Simple biodegradable felts work well for growing cartilage allografts ex vivo for use in full-thickness defects [27,29,36], because all that is required is a structural support for the cells. In contrast, the scaffolds used in osteochondral defects also must possess mechanical properties. In small defects, a singlephase implant is possible, because the marrow stromal cells that populate the scaffold quickly form new bone and provide a substrate for the overlying neocartilage. In larger defects, however, two-phase implants are preferred, with each phase mimicking the structural and mechanical properties of the tissue with which it interfaces. Various versions of a two-phase implant are in development. Frenkel et al. [30] used type I collagen as the structural base of the implant, varying the density of the collagen fiber mesh, with looser fibers in the cartilage phase and more compacted fibers in the bone phase. At the interface the network of fibers is so dense that it serves as a barrier for migration of
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Fig. 3. Placement of a two-phase polylactic acid/polyglycolic acid osteochondral implant in the condyle. The stiffer bone phase interfaces with the subchondral bone, whereas the cartilage interfaces with a scaffold that mimics its mechanical properties. In this particular implant, the cartilage phase is covered with a surface that is designed to reduce shear stress. (Courtesy of OsteoBiologics, Inc., San Antonio, TX.)
chondrocytes down into the bone phase or for migration of marrow stromal cells up into the carti-
lage phase. This design is intended to mimic the barrier provided by the subchondral bony plate. When articular chondrocytes are precultured on the implant before its placement in an osteochondral defect, healing is excellent in animal models. This design also has been produced as a biodegradable implant constructed from polylactic acid fibers [33]. The implant is anchored in the site with small polylactic acid pins. The authors have designed a two-phase implant that not only varies pore size, as would be achieved by modulating the packing density of collagen fibers, but also varies mechanical properties of each phase [4]. This implant is constructed using polylactic acid and polyglycolic acid to create a foam that is stiffer in the bone phase than it is in the cartilage phase. Studies that use rabbits as the animal model show that the material properties of the neocartilage are comparable to those of the surrounding host cartilage when implants of this design are used, particularly when TGF-b1 is incorporated into the cartilage phase. The importance of retaining the mechanical properties of such an implant over the initial healing period was demonstrated in a study using goats as the animal model [3]. Compression of the scaffold also led to premature compression of the neocartilage and failure of the repair strategy, however. In vitro studies were conducted to show that fiber reinforcement can be used to tailor the mech-
Fig. 4. Typical histological appearance near the center of an osteochondral defect that was treated using a two-phase implant to which chondrocytes had been added prior to implantation. The type of experimental defect used in this study was ‘‘critical sized’’ so complete healing across the entire defect space was not anticipated. Subchondral bone is restored and there is excellent integration of neocartilage with the surrounding host tissue. Defects treated with implants that were not preloaded with cells also had a similar appearance. (Courtesy of OsteoBiologics, Inc., San Antonio, TX.)
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anical properties of porous, resorbable scaffolds for optimal performance in an articular cartilage environment [63]. A study using a goat femur model to test these newly developed synthetic, resorbable multiphase implants designed specifically for osteochondral cartilage repair showed the value of this approach [48]. In this screening study, four different multiphase implants with varying mechanical and physical properties were randomly implanted in weight-bearing (high load environment) (Fig. 3) and non – weight-bearing (low load environment), 3-mm diameter defects (n = 64) of goat distal femurs. The implants were assessed for their effectiveness as cartilage repair scaffolds after 16 weeks of healing using gross, biomechanical, and histologic evaluations. All implants were tested as scaffold alone (cell-free) and loaded with autologous costochondral chondrocytes. Qualitative histologic evaluations showed that all groups had a high percentage of hyaline cartilage and good bony restoration, with new tissue integrating well with the native cartilage (Fig. 4). Defect healing in the condyle was significantly higher than in the patellar groove, but there were no differences in healing because of implant type. In weight-bearing sites, the quality of the neocartilage was equally good regardless of whether cells were added to the scaffolds before implantation. This observation suggests that cells resident in the host cartilage may contribute to the formation of neocartilage at the interface of the scaffold and the native tissue when the mechanical environment is favorable, thereby achieving the goal of integration. Marrow stromal cells also can differentiate into hyaline and hypertrophic chondrocytes in an appropriate manner, again when attaching to a physical substrate that is like that of the natural tissue.
Summary This article has shown the problems and challenges of tissue engineering cartilage and has presented the current strategies that are under investigation. The specific characteristics of the tissue are advantages and disadvantages. Surgeries can be performed arthroscopically, but the lack of a robust intrinsic healing response hampers the effectiveness of the therapy. We have not yet solved the problem of the choice of cells, nor have we identified all of the requisites for optimal scaffold design. Efforts thus far have focused on small defects in relatively healthy patients. How aging, disease, and pharmacologic intervention will modify the effectiveness of tissue-engineered cartilage,
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whether it is produced in vivo or ex vivo, is still unknown. Despite the problems, the advances made over the past 5 years suggest that the challenges will be met.
Acknowledgements The authors thank Sandra Messier for her assistance in the preparation of the manuscript, and they acknowledge the support of the National Institutes of Health (US PHS grants DE-08603 and DE-05937). The authors also thank Dr. Frank Barry, Osiris Therapeutics, Inc. (Baltimore, MD) for his assistance in the preparation of the manuscript; Smith and Nephew (Andover, MA) for their contribution of Fig. 2; and OsteoBiologics, Inc. (San Antonio, TX) for their contribution of Figs. 3 and 4.
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[51] O’Driscoll SW, Keeley FW, Salter RB. Durability of regenerated articular cartilage produced by free autogenous periosteal grafts in major full-thickness defects in joint surfaces under the influence of continuous passive motion: a follow-up report at one year. J Bone Joint Surg Am 1988;70:595 – 606. [52] O’Driscoll SW, Salter RB. The induction of neochondrogenesis in free intra-articular periosteal autografts under the influence of continuous passive motion: An experimental investigation in the rabbit. J Bone Joint Surg Am 1984;66:1248 – 57. [53] Perka C, Schultz O, Spitzer RS, Lindenhayn K. The influence of transforming growth factor beta-1 on mesenchymal cell repair of full-thickness cartilage defects. J Biomed Mater Res 2000;52:543 – 52. [54] Ponticiello MS, Schinagl RM, Kadiyala S, Barry FP. Gelatin-based resorbable sponge as a carrier matrix for human mesenchymal stem cells in cartilage regeneration therapy. J Biomed Mater Res 2000;52:246 – 55. [55] Rees JA, Ali SY. Ultrastructural localisation of alkaline phosphatase activity in osteoarthritic human articular cartilage. Am Rheum Dis 1988;47:747 – 53. [56] Schreiber RE, Ilten-Kirby BM, Dunkelman NS, Symons KT, Rekettye LM, Willoughby J, et al. Repair of osteochondral defects with allogeneic tissue engineered cartilage implants. Clin Orthop 1999;367(suppl):382 – 95. [57] Schwartz Z, Carnes DL Jr, Pulliam R, Lohmann CH, Sylvia VL, Liu Y, et al. Porcine fetal enamel matrix derivative stimulates proliferation but not differentation of pre-osteoblastic 2T9 cells, inhibits proliferation and stimulates differentation of osteoblast-like MG63 cells, and increases proliferation and differentation of normal human osteoblast NHOst cells. J Periodontol 2000;71:1287 – 96. [58] Schwartz Z, Dean DD, Walton JK, Brooks BP, Boyan BD. Treatment of resting zone chondrocytes with 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] induces differentiation into a 1,25-(OH)2D3-responsive phenotype characteristic of growth zone chondrocytes. Endocrinology 1995;136:402 – 11. [59] Schwartz Z, Sylvia VL, Liu Y, Dean DD, Boyan BD. Treatment of resting zone chondrocytes with bone morphogenetic protein-2 induces maturation into a phenotype characteristic of growth zone chondrocytes by downregulating responsiveness to 24,25-(OH)2D3 and upregulating responsiveness to 1,25-(OH)2D3. Endocrine 1998;9:273 – 80. [60] Schwartz Z, Sylvia VL, Liu Y, Dean DD, Boyan BD. Treatment of resting zone chondrocytes with transforming growth factor-b1 induces differentiation into a phenotype characteristic of growth zone chondrocytes by downregulating responsiveness to 24,25-(OH)2D3 and upregulating responsiveness to 1,25-(OH)2D3. Bone 1998;23:465 – 70. [61] Sellers RS, Zhang R, Glasson SS, Kim HD, Peluso D, D’Augusta DA, et al. Repair of articular cartilage defects one year after treatment with recombinant human bone morphogenetic protein-2 (rhBMP-2). J Bone Joint Surg Am 2000;82:151 – 60.
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Cartilage regeneration Barbara D. Boyan, PhDa,b,c,*, David D. Dean, PhDa, Christoph H. Lohmann, MDa,d, Gabriele G. Niederauer, PhDe, Jacquelyn McMillan, MBChB, FRCSEd (Orth)a, Victor L. Sylvia, PhDa, Zvi Schwartz, DMD, PhDa,c,f a
Department of Orthopaedics, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229 – 3900, USA b Department of Biochemistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229 – 3900, USA c Department of Periodontics, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229 – 3900, USA d Department of Orthopaedics, University of Hamburg Eppendorf, D-20246 Hamburg, Germany e OsteoBiologics, Incorporated, 12500 Network, Suite 112, San Antonio, TX 78249, USA f Department of Periodontics, Hebrew University Hadassah, Jerusalem, Israel
Articular cartilage has been a primary focus of the emerging tissue engineering industry. The market for cartilage repair technologies is considerable for trauma and sports injuries and even greater for regeneration of cartilage in patients with arthritis. Articular cartilage degeneration is accompanied by morbidity, which leads to absenteeism and development of conditions associated with chronic pain. In severe cases, the loss of function is of sufficient magnitude that it is necessary to replace damaged joint tissue with a bioprosthesis.
Problems associated with tissue engineering of cartilage Despite these economic drivers, successful tissue engineering of articular cartilage has been elusive. Unfortunately, cartilage does not heal in the same manner as seen in other tissues, in part because it has only a rudimentary blood supply. When the cartilage is severed, the chondrocytes seal the exposed edges
* Corresponding author. E-mail address: [email protected] (B.D. Boyan).
of the wound and in effect create a new cartilage surface [2,66,72]. The two sides of the defect do not fuse, which creates a focal change in the way that the tissue experiences compressive loads. Similarly, when injuries occur that cause loss of a piece of cartilage, the chondrocytes in the surrounding tissue again seal off the edges of the defect site. There is a limited attempt at repair, but the tissue that forms within the defect tends to be fibrocartilage. There are several reasons why fibrocartilage forms within chondral defects. The source of cells is believed to be synoviocytes [39], which are fibroblastic and as such synthesize type I collagen. Even if chondroprogenitor cells migrate into the defect site, they must produce large amounts of matrix quickly to facilitate migration across large regions of space relative to the cell, and type I collagen is favored under such circumstances. Should the defect penetrate the subchondral plate, a clot is able to form within the defect site and serves as a scaffold for cell attachment and migration. Many of the cells that colonize such defects are derived from the vasculature and marrow stroma [62], however, and possess the capacity to differentiate into various mesenchymal cell types. In the absence of a sufficient supply of chondrogenic factors, these cells tend to select the default pathway and differentiate along a fibroblastic lineage [38,43,49,53,61].
1042-3699/02/$ – see front matter D 2002, Elsevier Science (USA). All rights reserved. PII: S 1 0 4 2 - 3 6 9 9 ( 0 2 ) 0 0 0 1 7 - 1
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Articular cartilage architecture In comparison with organs such as liver and kidney, cartilage is a deceptively simple tissue in that only one cell type is present. This is both strength and weakness with respect to tissue engineering. Although there is no need to design complex three-dimensional constructs that contain several different types of cells, the heterogeneity within the cartilage in terms of chondrocyte phenotype does require consideration. Cells at the top of the articular cartilage exhibit a flattened morphology. These cells form the barrier with the synovial fluid and provide a surface that can withstand shear. Most of the cartilage is hyaline in nature, with clonal populations of chondrocytes, each in its own lacunae, surrounded by a pericellular matrix that differs from the interterritorial matrix. The cells adjacent to the subchondral bone have yet another phenotype. These cells are hypertrophic and appear in many ways like a miniature growth plate. This region is called the tidemark because it is characterized by a distinctly different staining property, partly because of the difference in the extracellular matrix and the deposition of hydroxyapatite. This phenotypic heterogeneity of the chondrocytes is accompanied by a complex three-dimensional architecture provided by the extracellular matrix. Type II collagen fibrils delineate the form of the matrix, which results in overlapping gothic cathedral-like arches that separate columns of cells [19,40]. There is also secondary structure in the form of crossstruts. In addition to the collagen framework, there is a fine net formed of cartilage oligomeric matrix protein (COMP) throughout the interterritorial matrix [26,75]. Interspersed among the fibrillar proteins are proteoglycan aggregates, composed of a hyaluronic acid backbone to which the aggrecan monomer is connected via association with link protein [9]. The number of aggrecan monomers and the length of the hyaluronic acid chain vary with the articular cartilage zone. Aggrecan is decorated with sulfated glycosaminoglycan side chains that also vary in number and length. During embryonic bone formation, the articular cartilages form as remnants of cartilage anlagen as they are replaced with bone and marrow. The embryonic tissue is highly cellular, but postfetal cartilage is relatively acellular. The articular cartilage continues to grow as the long bones continue to lengthen; however, most cartilage repair is completed in adults, and by then, any mitosis in primary cartilages is infrequent. It is likely that chondrocytes retain some proliferative capacity, which is stimulated by injury. Even so, the neocartilage that forms within a chondral
defect has an extracellular matrix that is different from that of the cartilage surrounding the defect site. The architectural features of the matrix are honed over time and occur in response to directional loads that are placed on the tissue [67,73]. Even the types of proteoglycan in the extracellular matrix may differ in terms of core protein [18] and glycosaminoglycan sulfation [17,34]. The amount of hydration of the tissue and, consequently, the hydrostatic forces experienced by the cells differ [35,46]. The resistance of the tissue to compressive loads also differs [5]. The integration of the neocartilage with the surrounding cartilage is often ineffective, not only because of the differences in matrix composition but also because of the cartilage matrix sealing effect. This results in mechanical instability that further reduces the success of the repair tissue. In healthy joints, the base of the articular cartilage is ‘‘glued’’ to the subchondral bone in a manner that is similar to the way that the base of a growth plate is attached to the bony metaphysis. By calcifying their matrix, the chondrocytes in the tidemark region form an interlock with the mineralized bone. Disruption of this interface also contributes to mechanical instability. It is currently known that it is critical to have good repair of the subchondral plate to have good repair of the articular cartilage [44]. Unfortunately, the subchondral plate can be damaged through trauma, during surgery, or as a consequence of degenerative diseases. Tissue-engineering strategies must consider how to ensure that mineralization of the cartilage occurs in an appropriate manner and provide a method for regenerating the bone itself.
Cartilage calcification and the tidemark Physiologic mineralization of cartilage involves chondrocyte hypertrophy and deposition of apatite crystals within the extracellular matrix. Both processes require turnover of the extracellular matrix. As the cells produce matrix, they also synthesize matrix processing enzymes and store them in inactive form as zymogens or together with inhibitors such as tissue inhibitor of metalloproteinase (TIMP) [14]. Other matrix processing enzymes are packaged in extracellular matrix vesicles and are released in active form upon signals from the cells. Because of the importance of maintaining a functional architecture, matrix turnover is slow in healthy tissue. At the tidemark, however, the rate of matrix degradation is believed to be increased, both to accommodate hypertrophy and to redesign the matrix for mineral deposition [21 – 23]. These cells produce increased numbers of matrix
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vesicles, which also provide sites for the initial formation of calcium phosphate crystals [55]. Successful tissue engineering must result in a cartilage in which these events occur in a regulated manner in time and space. Should the hypertrophy of the cells extend too far up into the hyaline cartilage, the possibility of osteophyte formation is increased, as is the likelihood of tissue destruction. If hypertrophy and matrix mineralization do not occur, then the repair cartilage is likely to delaminate.
Tissue engineering of cartilage This section of the article focuses on the engineering of articular cartilages at the ends of long bones. The mandibular condyle is a secondary cartilage and as such retains the ability to remodel throughout life. Relatively little is known about the cellular, biochemical, and structural features of the articular cartilage of the condyle that impinge on the success of tissue engineering in this tissue. For this reason, engineering strategies for the mandibular cartilage are not discussed. The need for cells The repair capacity for cartilage is low, because of the relatively low proliferative capacity of cells resident in the tissue and their low migratory ability. Because of this, the strategies for cartilage tissue engineering have focused on the delivery of cells to the defect site through various approaches. There are three different types of cartilage defects, each of which requires its own set of repair strategies. Partial-thickness defects Defects that do not extend to the subchondral plate are called ‘‘chondral’’ or ‘‘partial-thickness’’ defects. Surgeons are reluctant to use strategies that penetrate the subchondral plate for repair of these types of defects because to do so requires loss of additional cartilage. The usual approach is to remove undermined chondral flaps from the edges of the defect and leave stable cartilage undisturbed and hope for the best. In some instances, this is not a practical choice, yet the reluctance remains. The problems of how to maintain cells at the site or ‘‘glue’’ allograft to the existing cartilage have not yet been resolved. Hunziker and Rosenberg [39] have characterized the healing process of partial-thickness defects with respect to the cells that colonize the defect site and methods for enhancing their ability to differentiate into chondroblasts and ultimately become functional
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chondrocytes. Preparation of the chondral defect is critical. Not only is it necessary to remove the damaged cartilage but also healing is enhanced if the walls of the defect are etched with enzymes that remove the outer layer of cartilage matrix proteins. Host cells adhere more effectively to the tissue surface. They also have noted that delivery of transforming growth factor-beta (TGF-b) to the site in various time-release carriers also improves the differentiation of the colonizing cells into chondrocytes. Full-thickness defects When the cartilage defect extends to the subchondral plate, several strategies can be considered. The choice of strategies is related to the actual size of the defect. In humans, trauma and sports injuries result in defects that vary in size but tend to be focal. In contrast, arthritis leads to large regions of degenerate tissue and has the added complication of being chronic, involving the subchondral bone, and compromising the surrounding ‘‘healthy’’ cartilage tissue. The biggest problems to be resolved in treating full-thickness defects are the interface with the surrounding cartilage and the tendency of the neocartilage to delaminate. Microfracture. By far the most common tissue engineering strategy relies on microfracture of the subchondral plate, providing access of cells from the underlying vasculature and marrow stroma to migrate into the defect site. This technique has been popularized by Blevins et al. [8] and Steadman et al. [65] with reasonable success. After removing all damaged cartilage, the subchondral plate is penetrated at several sites. The blood that moves into the synovial space forms a clot in the defect and brings with it growth factors that can enhance tissue repair. Mesenchymal cells migrate onto and through the hematoma, and in the presence of the surrounding cartilage at least some of these cells differentiate into chondroblasts. The subchondral plate is repaired through endochondral ossification, the same mechanism that repairs bone fractures. The chondroblasts produce a cartilage matrix, but the quality of the matrix is variable at best. Eventually, fibrocartilage predominates. This approach can be successful clinically because the patient can return to function relatively pain free. Over time, the repair tissue may fail, but for some patients, it is adequate. Periosteum and perichondrium. The periosteum, the skin-like tissue that forms on the outer surface of bone, is a rich source of multipotent mesenchymal cells, including cells with the potential to become chondrocytes. O’Driscoll et al. [50 – 52] have pio-
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neered the use of periosteal autografts for treatment of chondral defects. By placing the periosteum in an inverted fashion in the defect site, the mesenchymal cells can migrate easily into the defect space. Their ability to migrate out of the site is reduced by the graft itself. Ideally, the periosteal cells differentiate into chondroblasts in the presence of chondrogenic signals from the surrounding cartilage. This technique has proved successful in some cases, but exactly what makes one case a success and another case a failure is not well understood. Pretreatment of the tissue with TGF-b improves chondrogenesis [45] for the same reasons as noted for partial-thickness defects. To improve on the concept, Amiel et al. [1] and Chu et al. [20] have suggested that perichondrial tissue may be a better source of cells. The perichondrium is an extension of the periosteum, but it overlies cartilage tissue. These investigators reasoned that it was likely to contain a greater percentage of chondroprogenitor cells. They have used perichondrium successfully as a source of cells for treatment of full-thickness defects. Again, treatment of the cells with TGF-b improved success. Cartilage cell therapy Another approach is to use committed chondrocytes rather than tissue that contains chondroprogenitor cells. Allografts and ex vivo tissue-engineered cartilage Cartilage autografts are in short supply; otherwise the patient would not need a tissue-engineering approach for repair of the defect. Allografts are used to cover large defects, with varying success [6,16]. One method that holds promise is the use of allografts produced by tissue engineering ex vivo. Several methods have been developed to achieve the goal of making a tissue in culture that has the properties of articular cartilage and integrates with the surrounding host cartilage and with the subchondral bone [68,71]. To accomplish this goal, several problems have emerged that have required innovative solutions, including the need for a reliable source of cells, the need for a structural scaffold on which to grow the cells in three dimensions, the contribution of mechanical force to the development of the tissue, and the development of novel bioreactors to achieve sufficient tissue in a reasonable period of time [36,56]. Despite considerable success in solving each of these problems, the resulting tissue still does not replicate the qualities of the native host tissue. The only Food and Drug Administration-approved cartilage cell therapy currently in use clinically in the
United States is the direct delivery of autologous chondrocytes to the defect site. This methodology was pioneered by Brittberg et al. [15] and has been the subject of several studies over the past 5 years. The concept is simple. Cartilage is removed surgically from a non – weight-bearing region of the joint to be treated, and the cells isolated from that cartilage are cultured ex vivo through four passages. This practice is critical because of the low cellularity of cartilage. It is necessary to expand the number of cells to increase the likelihood of repopulating the defect with chondrocytes. At the same time of this surgery, the defect site is prepared. Periosteal tissue is removed from the bone and used to create a seal over the surface of the defect. Currently, this periosteal flap is sutured, but the process of suturing itself leads to defects in the cartilage, so attempts to develop a glue to attach the periosteum to the cartilage surface are also underway. While the cells are in culture, the surgical site is allowed to heal. When the cells are ready, they are injected into the defect site arthroscopically. Technically, the periosteal seal is intended to retain the cells within the defect. Practically, this does not occur. Second-look arthroscopies show that the periosteum can be dislodged and that the cells tend to migrate out of the defect. When the treatment does work, however, the results are promising. Whether they hold up in the long term is not yet known. Studies using dogs and goats suggest that this neocartilage brings with it the same problems as seen with other repair strategies [13,24]. It also is not clear whether the repair cartilage is the consequence of the cells in the periosteum, the cells that are injected, or some combination of the two. More recently, investigators have begun to grow cartilage in multilayers on membranes [41] and to transfer the construct to the defect site rather than to inject cell suspensions. This technique may not require the use of a periosteal seal, because the cells are in effect anchored to the underlying membrane. The limitation is the attachment to the subchondral bone. All of the cell strategies are limited by the tendency of chondrocytes to lose their chondrogenic phenotype in monolayer culture. More than 20 years ago, Benya et al. [7] noted that articular chondrocytes lose their ability to synthesize type II collagen and produce sulfated proteoglycan when they were grown in monolayer culture. The chondrocyte phenotype was restored when the cells were grown in suspension culture or in various kinds of gels. Alginate was particularly effective at preserving these cell characteristics [31]. The reasons for this are not entirely understood, but it is clear that retention of the rounded articular chondrocyte morphology is impor-
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tant. Unfortunately, cells grow slowly using these methods, which makes it difficult to achieve sufficient expansion. Recently, alginate has been used to microencapsulate articular chondrocytes after culture in monolayer and found that the microencapsulation process restored the ability of the cells to produce type II collagen and sulfated proteoglycan (A. Sambanis, unpublished observation). This opens up the possibility of expanding the cells in monolayer culture and then preparing them for injection via microencapsulation. Alternatively, articular chondrocytes can be expanded in bioreactors. Various methods are being developed for this purpose. Currently, the cells tend to form spherical nodules in these sorts of culture systems and develop three-dimensional architecture within the nodules, including hypertrophic chondrocytes at the center [28,69,74]. Whether these nodules are suitable for use in treating full-thickness defects is not yet clear. Another approach is to use chondrocytes that do not undergo such marked loss of phenotype in vitro. The authors’ laboratory has used hyaline chondrocytes from the resting zone of the costochondral cartilage of the rib for this purpose. Although these cells are not identical to articular chondrocytes, they are not yet in the endochondral lineage per se, and they produce a proteoglycan-rich extracellular matrix through four passages in culture [10]. Although they do produce
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type I collagen as an adaptation to monolayer culture, they also continue to produce type II collagen [10], and when they are implanted in nude mouse muscle in vivo, they form cartilage nodules that are indistinguishable from normal hyaline cartilage [11]. One problem with using chondrocytes is their tendency to undergo hypertrophy (Fig. 1). To increase the number of chondrocytes while at the same time control their differentiation along the endochondral pathway is an elusive goal of cartilage cell therapy. One approach is to pretreat autologous chondrocytes with growth factors that modulate one or more of the phenotypic characteristics of the cells. Unfortunately, growth factors are pleiotropic, affecting different cells in different ways. They may affect the same cell differentially depending on the cell’s state of maturation within its lineage cascade. This is the case with chondrocytes. As shown in Table 1, cells from the resting zone of costochondral cartilage respond to various regulatory factors in a manner that is distinct from the response of growth zone cells, which are derived from the prehypertrophic and upper hypertrophic zones of the rat costochondral growth plate. Table 1 is a summary of several experiments performed in the authors’ laboratory using factors and hormones that are known to regulate cartilage and bone or that are being tested for their effectiveness in various musculoskeletal tissue-engineering applications [42,43,58 – 60]. Many of the factors increase
Fig. 1. Rat costochondral cartilage resting zone chondrocytes were loaded on a polylactic acid/polyglycolic acid porous foam scaffold and implanted for 8 weeks in the calf muscle of a nude mouse. Chondrogenesis has occurred throughout the scaffold space and where the scaffold has resorbed, the cartilage is in direct contact with the surrounding muscle. Chondrocytes at the center of the neocartilage are producing a cartilage matrix and some of the cells are undergoing hypertrophy. Residual scaffold is evident as a clear area, due to its dissolution during processing. (Haematoxylin and eosin, original magnification 10).
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Table 1 Cell maturation and chondrocyte response to bioactive factors EMD
TP508
PDGF-BB
IGF-1
TGF-b1
FGF-2
BMP-2
1,25
24,25
Resting zone cells Proliferation Alkaline phosphatase [35S]-sulfate incorporation Response to 1,25-D3
" # — ND
— # " ND
" — " —
" " " —
" " " "
" " " ND
" — " "
# — — ND
"# " " "
Growth zone cells Proliferation Alkaline phosphatase [35S]-sulfate incorporation
" — #
" — —
ND ND ND
ND ND ND
" " "
ND ND ND
" " —
# " "
" — —
Abbreviations: FGF-2, basic fibroblast growth factor; IGF-1, insulin-like growth factor; ND, not done; 1,25, 1,25 dihydroxyvitamin D3; 24,25, 24,25 dihydroxyvitamin D3.
proliferation of the hyaline-like resting zone cells. Of those factors that stimulate proliferation, some also stimulate proteoglycan production based on the incorporation of radiolabeled sulfate into glycosaminoglycans, which indicates that cartilage matrix synthesis is enhanced. Most of the factors also promote endochondral differentiation of the cells, however. Alkaline phosphatase specific activity is increased, and this enzyme, which is associated with extracellular organelles called matrix vesicles, is involved in calcification. Resting zone cells normally respond primarily to the vitamin D metabolite 24R,25(OH)2D3, whereas the more mature growth zone cells respond primarily to 1a,25(OH)2D3. When resting zone cells are treated with TGF-b1, bone morphogenetic protein2, or 24R,25(OH)2D3 for extended periods of time, however, responsiveness to1a,25(OH)2D3 is upregulated and responsiveness to 24R,25(OH)2D3 is lost, which indicates that these cells not only have become more differentiated but also have acquired a growth zone chondrocyte phenotype [58 – 60]. The factors that show the most promise for pretreatment of chondrocytes are factors that stimulate proliferation and cartilage matrix production but retard or inhibit endochondral differentiation. Of all of the factors that the authors have tested to date, the best candidates seem to be Emdogain (EMD) (Biora, Inc., Malmo, Sweden), which increases the pool of chondroprogenitor cells [12,57], TP508 (Chrysalin, Chrysalis, BioTechnology, Inc, Galveston, TX), which enhances matrix synthesis but not differentiation, and platelet-derived growth factor-BB (PDGF-BB), which stimulates proliferation and matrix synthesis, but not endochondral maturation [42]. To test whether these in vitro assays are relevant to in vivo behavior, the authors pretreated resting zone chondrocytes for 4 hours or 24 hours with PDGF-BB
before implantation in nude mouse muscle. For these experiments, they used scaffolds provided by OsteoBiologics, Inc. (San Antonio, TX). After treatment, the cells were loaded onto the scaffolds and implanted bilaterally in the calf muscles. Implanted tissue was examined histomorphometrically for the presence of new cartilage and the degree of chondrocyte hypertrophy at 4 and 8 weeks. The results showed that PDGF-BB pretreatment increased the amount of neocartilage at 8 weeks and prevented chondrocyte hypertrophy; however, the shorter exposure to the growth factor was more effective than the longer exposure [43]. This result shows that the type of factor used is important, and it indicates that the pretreatment regimen may be critical. Finally, several other cell-based strategies are in various states of development. Mesenchymal stem cells (MSCs) were first used by Wakitani et al. [70] to repair full-thickness defects in rabbits. These multipotent cells differentiated into chondrocytes and formed neocartilage within the experimental defects that was comparable to neocartilage formed by committed chondrocytes. The effectiveness of the strategy was improved through the use of gels to create MSCbased constructs [54,64]. The use of MSCs is a powerful technology because of their high proliferative capacity and their ability to differentiate into tissues. There are some caveats, however. For example, recent studies suggest that the phenotype of MSCs can vary, depending on the physiology of the donor [47]. Even taking this into consideration, MSCs hold great promise. One of the most innovative uses is the use of fat as a source of MSCs [25,76]. MSCs in fat possess the ability to differentiate into chondrocytes when treated with growth factors that induced chondrogenesis, such as TGF-b. Similarly, MSCs isolated from perichondrium can be used if pretreated
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Fig. 2. Mosaic arthroplasty showing the placement of osteochondral cores within a defect. (Courtesy of Smith and Nephew, Endoscopy Division, Andover, MA.)
with TGF-b or when transfected with plasmids that contain TGF-b1 DNA [32]. Osteochondral defects In instances in which the subchondral bone is damaged, a core of cartilage and underlying bone is removed. If the defect is small enough, healing occurs by a process similar to that described for microfracture. In larger defects, the repair of the subchondral bone is inadequate to support the repair cartilage, and the result is poor. For these types of defects, mosaic arthroplasty is preferred. Mosaic arthroplasty. Mosaic arthroplasty relies on the use of an osteochondral plug from a region of the articular cartilage that is normally not required for weight bearing [36,37]. The surgeon may remove several cores of cartilage and bone from this site and then place them in a mosaic-like fashion in the defect site on the weight-bearing region of the joint (Fig. 2). This method works well because the cartilage in each plug remains viable, but problems arise because the transplanted cartilage does not fuse with the surrounding host tissue. Just as the edges of the cartilage defect site seal, so do the edges of the cartilage plugs. The defect site is osteochondral and permits clot formation and osteochondral repair around and between each plug. This repair tissue is fibrocartilage as would be the case for microfracture. The final result is an improvement over simply allowing the larger osteochondral defect to heal via clot formation and mesenchymal cell defferentiation, but the me-
chanical instabilities created by the variation in tissue type may predispose this strategy to failure. Cell-based therapies. Cell-based therapies also are used for osteochondral repair, but they depend on a suitable delivery device because the subchondral plate is not present to serve as a substrate. All of the same cell strategies as described for full-thickness defects with respect to cell source are applicable. The physical properties of the scaffold are critical to ensure appropriate healing of the defect with bone and cartilage, however. Simple biodegradable felts work well for growing cartilage allografts ex vivo for use in full-thickness defects [27,29,36], because all that is required is a structural support for the cells. In contrast, the scaffolds used in osteochondral defects also must possess mechanical properties. In small defects, a singlephase implant is possible, because the marrow stromal cells that populate the scaffold quickly form new bone and provide a substrate for the overlying neocartilage. In larger defects, however, two-phase implants are preferred, with each phase mimicking the structural and mechanical properties of the tissue with which it interfaces. Various versions of a two-phase implant are in development. Frenkel et al. [30] used type I collagen as the structural base of the implant, varying the density of the collagen fiber mesh, with looser fibers in the cartilage phase and more compacted fibers in the bone phase. At the interface the network of fibers is so dense that it serves as a barrier for migration of
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Fig. 3. Placement of a two-phase polylactic acid/polyglycolic acid osteochondral implant in the condyle. The stiffer bone phase interfaces with the subchondral bone, whereas the cartilage interfaces with a scaffold that mimics its mechanical properties. In this particular implant, the cartilage phase is covered with a surface that is designed to reduce shear stress. (Courtesy of OsteoBiologics, Inc., San Antonio, TX.)
chondrocytes down into the bone phase or for migration of marrow stromal cells up into the carti-
lage phase. This design is intended to mimic the barrier provided by the subchondral bony plate. When articular chondrocytes are precultured on the implant before its placement in an osteochondral defect, healing is excellent in animal models. This design also has been produced as a biodegradable implant constructed from polylactic acid fibers [33]. The implant is anchored in the site with small polylactic acid pins. The authors have designed a two-phase implant that not only varies pore size, as would be achieved by modulating the packing density of collagen fibers, but also varies mechanical properties of each phase [4]. This implant is constructed using polylactic acid and polyglycolic acid to create a foam that is stiffer in the bone phase than it is in the cartilage phase. Studies that use rabbits as the animal model show that the material properties of the neocartilage are comparable to those of the surrounding host cartilage when implants of this design are used, particularly when TGF-b1 is incorporated into the cartilage phase. The importance of retaining the mechanical properties of such an implant over the initial healing period was demonstrated in a study using goats as the animal model [3]. Compression of the scaffold also led to premature compression of the neocartilage and failure of the repair strategy, however. In vitro studies were conducted to show that fiber reinforcement can be used to tailor the mech-
Fig. 4. Typical histological appearance near the center of an osteochondral defect that was treated using a two-phase implant to which chondrocytes had been added prior to implantation. The type of experimental defect used in this study was ‘‘critical sized’’ so complete healing across the entire defect space was not anticipated. Subchondral bone is restored and there is excellent integration of neocartilage with the surrounding host tissue. Defects treated with implants that were not preloaded with cells also had a similar appearance. (Courtesy of OsteoBiologics, Inc., San Antonio, TX.)
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anical properties of porous, resorbable scaffolds for optimal performance in an articular cartilage environment [63]. A study using a goat femur model to test these newly developed synthetic, resorbable multiphase implants designed specifically for osteochondral cartilage repair showed the value of this approach [48]. In this screening study, four different multiphase implants with varying mechanical and physical properties were randomly implanted in weight-bearing (high load environment) (Fig. 3) and non – weight-bearing (low load environment), 3-mm diameter defects (n = 64) of goat distal femurs. The implants were assessed for their effectiveness as cartilage repair scaffolds after 16 weeks of healing using gross, biomechanical, and histologic evaluations. All implants were tested as scaffold alone (cell-free) and loaded with autologous costochondral chondrocytes. Qualitative histologic evaluations showed that all groups had a high percentage of hyaline cartilage and good bony restoration, with new tissue integrating well with the native cartilage (Fig. 4). Defect healing in the condyle was significantly higher than in the patellar groove, but there were no differences in healing because of implant type. In weight-bearing sites, the quality of the neocartilage was equally good regardless of whether cells were added to the scaffolds before implantation. This observation suggests that cells resident in the host cartilage may contribute to the formation of neocartilage at the interface of the scaffold and the native tissue when the mechanical environment is favorable, thereby achieving the goal of integration. Marrow stromal cells also can differentiate into hyaline and hypertrophic chondrocytes in an appropriate manner, again when attaching to a physical substrate that is like that of the natural tissue.
Summary This article has shown the problems and challenges of tissue engineering cartilage and has presented the current strategies that are under investigation. The specific characteristics of the tissue are advantages and disadvantages. Surgeries can be performed arthroscopically, but the lack of a robust intrinsic healing response hampers the effectiveness of the therapy. We have not yet solved the problem of the choice of cells, nor have we identified all of the requisites for optimal scaffold design. Efforts thus far have focused on small defects in relatively healthy patients. How aging, disease, and pharmacologic intervention will modify the effectiveness of tissue-engineered cartilage,
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whether it is produced in vivo or ex vivo, is still unknown. Despite the problems, the advances made over the past 5 years suggest that the challenges will be met.
Acknowledgements The authors thank Sandra Messier for her assistance in the preparation of the manuscript, and they acknowledge the support of the National Institutes of Health (US PHS grants DE-08603 and DE-05937). The authors also thank Dr. Frank Barry, Osiris Therapeutics, Inc. (Baltimore, MD) for his assistance in the preparation of the manuscript; Smith and Nephew (Andover, MA) for their contribution of Fig. 2; and OsteoBiologics, Inc. (San Antonio, TX) for their contribution of Figs. 3 and 4.
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