BASIC SCIENCE OF ARTICULAR CARTILAGE REPAIR

OSTEOCHONDRAL INJURIES OF THE KNEE

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BASIC SCIENCE OF ARTICULAR CARTILAGE REPAIR Kyriacos A. Athanasiou, PhD, Amita R. Shah, BS, R. Jason Hernandez, BS, and Richard G. LeBaron, PhD

Although highly desirable, functional restoration of diseased and damaged human articular cartilage continues to remain one of the most challenging orthopaedic problems. The clinical outcomes of treatments that are intended to promote successful and complete repair of full- and partial-thickness articular cartilage defects essentially remain unpredictable. One of the most exciting theories is that replacement of damaged articular cartilage can be achievable with ex vivo produced cartilage. This is a tissue engineering approach that promises functional restoration of cartilage defects using scaffolds, cells, and bioactive agents. Tissue engineering of articular cartilage represents an exciting direction in the efforts to solve the complex problem of cartilage regeneration. The ex vivo fabrication of cartilage constructs is central in addressing this complex problem in a cogent manner. An accurate delivery system for ex vivo cells has generated great interest. Biodegradable polymer scaffolds of polyglycolic acid material, seeded with articular chondrocytes, support ex vivo genesis of a cartilaginous extracellular matrix, especially when scaffolds are maintained in a system that delivers precise medium composition, supplements, and flow rate.23,27 This article discusses selected techniques that have been employed to improve the repair potential of articular cartilage, ranging from conventional cell culture to use of complex bioreactors. A treatment regimen that promises consistently

From the Department of Bioengineering, Rice University, Houston (KAA); the Department of Chemical Engineering, Trinity University (ARS); and the Laboratory of Extracellular Matrix and Cell Adhesion Research, Division of Life Sciences, the University of Texas at San Antonio (RJH, RGL), San Antonio, Texas

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good clinical results would constitute an enormous advancement in musculoskeletal surgery. STRUCTURE-FUNCTION CHARACTERISTICS OF ARTICULAR CARTILAGE

Articular cartilage is essential to normal diarthrodial joint function, because of its ability to reduce joint stress and surface friction. Large forces are absorbed by articular cartilage every day. For example, normal activities such as moderate walking or running result in cyclic forces six to eight times body weight applied to the synovial joints. Articular cartilage has the ability to deform and enlarge its surface contact area to lessen the effect of direct loads by decreasing applied stress. Another remarkable characteristic of this tissue is its exceptional durability. This tissue’s mechanoprotective ability is due largely to its solid-fluid nature. Immediately after cartilage is loaded, most of the applied stress is borne by fluid pressures developed in its interstitial fluid and not directly by the solid matrix. As a result, the extracellular matrix is protected from high stresses. Articular cartilage composition and thickness vary from joint to joint, and topographically in a joint, as a function of age, and among species3, The average thickness of human articular cartilage is at most a few millimeters. The tissue typically is composed of 75% to 80% water and a dense extracellular matrix consisting of 50% to 73% collagen I1 and 15% to 30% proteoglycan macromolecules. The main collagen of articular cartilage (collagen type 11) consists of insoluble tightly woven fibers, resulting in collagen molecules that range from 30 to 200 nm in diameter. Water and proteoglycans are dispersed through the collagen framework as a soluble gel, making the matrix b i p h a s i ~ .Collagen ~~ fibrils, which, like pieces of rope, can withstand tension but not compression, provide the matrix with high tensile strength. Proteoglycans are also important molecules in hyaline cartilage and assist in resistance to compression. This property is primarily due to the hydrophilic nature of their extensive carbohydrates, attracting and entrapping a large amount of water in the intramolecular and intermolecular space.5I As articular cartilage is subjected to functional loading, initially, interstitial water becomes pressurized and supports a significant portion of the load.79Water begins to flow due to a pressure differential through the low permeability extracellular matrix, allowing for energy dissipation through frictional interactions between the solid and fluid phases.50,55 A remarkable characteristic of articular cartilage is its relative acellularity. Chondrocytes, which reside in lacunae, occupy less than 10% of the Chondrocytes interact with extracellular matrix by means of cell surface receptors called integrins. These molecules thus serve as mechanical links between the cell and extracellular matrix and aid in cell homeostasis. Architecturally, articular cartilage has four zones of depth from the articular surface to the subchondral bone. Zone 1, also called the super-

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ficial layer, makes up approximately 10% of the cartilage, determines its load-bearing ability, and serves as a gliding surface for the joint. The top portion of the superficial layer, also called the lamina splendens, is a clear film consisting of a sheet of small fibrils with little polysaccharide and no cells.I4 Deeper in this layer, flat chondrocytes and collagen fibers are arranged tangentially to the articular surface. This tangential orientation of collagen fibers imparts higher tensile strength and stiffness to zone 1.'j6It also affects significantly the tissue's compressive behavior.72 Its removal increases permeability and, indeed, disruption of the superficial layer is an early sign of experimentally induced osteoarthritis.32 Zone 2 is the intermediate or transitional layer and is composed of spherical chondrocytes and randomly oriented collagen fibers. Compared to the superficial zone, the transitional zone has a higher concentration of proteoglycans, lower concentration of collagen, and lower concentration of water.14 In the deep layer (zone 3), the collagen fibers and chondrons (clusters of chondrocytes surrounded by the matrix) are perpendicular to the subchondral plate. The calcified layer, zone 4,joins the deep zone of uncalcified cartilage to the subchondral bone. There are few cells and an abundance of calcium salts, making it a place for growth of underlying bone tissue. The largest and smallest water contents are found in zones 1 and 4, respectively. The highest and lowest proteoglycan contents are in zones 4 and 1, respectively. The sparse number of blood vessels in cartilage indicate a low oxygen and nutrient need. Unlike vascular tissues, articular cartilage exchanges gasses, nutrients, and waste products by way of diffusion through tissue fluid or synovium. Synovial nutrients have to pass through a double barrier (synovial fluid and cartilage matrix) to reach the cell.I4 Under functional loading, the biomechanical interaction between the tissue's solid extracellular matrix and interstitial fluid influences nutrient transport and metabolite removal. Loading affects stress and pressure fields in the tissue, such that pressure differentials may drive nutrient- or metabolite-carrying fluids. Because the tissue's biomechanical properties influence solid-fluid interactions, one can expect that unless cartilage exhibits normal biomechanical properties, nutrient diffusion would not be optimal. Cartilage damage occurs thEough injury to the joint or progressive degeneration of the cartilage, resulting in defects, intra-articular bleeding, osteoarthritis, and other ailments. Symptoms of degenerating cartilage range from locking to pain and swelling. Pain from arthritis is often alleviated in the elderly through joint replacement, but this method is not successful in young and middle-aged patients due to the limited life span of prostheses. Repair of defects with a more permanent result that would be applied to younger patients is an area of active investigation.'jO INTRINSIC INABILITY OF ARTICULAR CARTILAGE TO REGENERATE

Compared to many other tissues, the cell to matrix ratio, mitotic activity, and turnover rate are very low in articular cartilage. Young

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cartilage has a high concentration of transforming growth factor beta (TGF-P), which accelerates synthesis of extracellular matrix. As cartilage ages, it shows a decrease in the number of marrow mesenchymal stem cells and chondrocyte activity,14thus the potential for spontaneous, quality repair is compromised. Spontaneous repair in mature cartilage usually results in primarily fibrous tissue at the superficial layers and fibrocartilage in the lower zones. These repair tissues are unable to withstand the high compressive loads encountered at the articulating surfaces and eventually deteriorate. Some articular cartilage defects do not penetrate the subchondral bone (chondral or partial thickness defects) and if superficial! then they rarely become di~ab1ing.l~ A more consuming defect can perforate into underlying bone (osteochondral or full-thickness defect), exposing the defect to a supply of blood and eventual formation of a fibrin clot. Spontaneous repair is more likely in osteochondral defects because a response from the mesenchymal stem cells in the marrow can be triggered. Thus, partial-thickness defects are not usually as easily repaired as full-thickness defects, presumably because mesenchymal stem cells are absent and neighboring chondrocytes cannot effectively repair adjacent defects. Repair also can be initiated through an extrinsic mechanism, acquiring nonosseous tissue from the articular surface, although the newly formed tissue differs biochemically and biomechanically from hyaline cartilage. A more recent theory proposes that proteoglycans in the cartilage prevent the adhesion of mesenchymal stem cells in the defect.35Improvement in the movement and adhesion of mesenchymal stem cells might be achieved by application of enzymes that cleave matrix components such as glycosaminoglycans. This effort could liberate cells from the dense extracellular matrix and promote their movement into the defect. More desirable repairs for superficial defects can be obtained using these enzymes in conjunction with growth factors.35 Present research implements biodegradable scaffolds as biocompatible conduits for cartilage repair or regenerati~n.~ The ultimate goal is to provide in the defect the components that generate neocartilage that is indistinguishable from host cartilage. Biodegradable scaffolds provide a synthetic matrix in which chondrocytes are retained and host cells are recruited into the defect. The scaffold allows chondrocytes, growth factors, stem cells, or surface modifiers to be incorporated on or in the structure. Other methods of articular cartilage repair include grafting, transplants, subchondral microfracture, electrical stimulation, and lasers. Although repair is achieved in the defects, these procedures have not proved to be especially successful.6*For example, organization and integration between host and new cartilage is poor.12To summarize, evaluation of the existing articular cartilage repair studies suggest the outcomes are inconsistent and nonuniform. The quality of neocartilage can vary from extremely poor (essentially the defect remains empty) to a complete resurfacing with hyaline cartilage that exhibits morphologic, biochemical, and biomechanical properties comparable to native cartilage.

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EVALUATION OF THE QUALITY OF CARTILAGE REPAIR

Historically, evaluation of repair articular cartilage has been based on gross morphology and histology, however, it is recognized that equally important assessment criteria should include quantification of the tissue’s functional abilities, by measuring its biomechanical and biochemical properties. Gross morphology is a qualitative evaluation method in which the researcher rates the cartilage after visual inspection.2 The appearance of adhesions, tissue color, smoothness, and level of the repair surface in comparison to normal cartilage are inspected. Histologic evaluation is a semiquantitative system. The success of repair for most studies is based on gross observations, such as tissue morphology (e.g., hyaline cartilage, fibrocartilage), matrix staining, surface 94 Each catesmoothness, thickness, and integration with host ~artilage.4~1 gory is rated on a scale, and although the scale can be modified slightly for various studies, this offers a more standard way to rate the success or failure of a particular scaffold. When warranted, more quantitative tests can be used to measure the neotissue’s compositional characteristics and functional abilities. Biochemical evaluation provides quantitative measures of collagen and sulfated glycosaminoglycan. Total collagen content can be estimated by When evaluatquantifying chloramine T oxidation of hydr~xyproline.~~ ing collagen content of articular cartilage, type I1 collagen can be differentiated from type I collagen by cyanogen bromide digestion and comparison of the products to marker cyanogen bromide peptides generated from purified collagen.18An estimation of total sulfated glycosaminoglycan can be obtained by proteolytic digestion (e.g., papain) of the material and staining the liberated glycosaminoglycan with 1,9-dimethylene blue. Quantifying the optical shift of the product against a standard curve of chondroisin sulfate glycosaminoglycan will quantify the Quantification of the biomechanical properties of repair cartilage is significant, because these properties determine if the repair tissue can undertake the functions demanded of articular cartilage. Indentation is the most commonly used method to obtain the mechanical properties of neoti~sue.~,Articular cartilage is biphasic, with a compressible solid phase consisting of proteoglycans, collagen, and chondrocytes, and an incompressible fluid phase.55Under compressive loads, cartilage displays viscoelastic behavior due to the interstitial fluid that flows in the porous solid and across tissue surfaces. Following appropriate mechanical models of cartilage’s stress-strain relationships, such as the biphasic intrinsic properties that describe neotissue quality can be ~btained.~,These properties include the tissue’s aggregate modulus, which indicates the tissue’s ability to respond to compressive loads; Poisson’s ratio, which measures apparent compressibility; and permeability, which provides a measure of the fluid’s ability to move in and out of the porous solid.”, 55 Tests to measure the biomechanical

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characteristics at the neocartilage-cartilage interface from chondrocyteseeded devitalized matrices also have been developed.6z

EX-VIVO MANIPULATIONS OF CARTILAGE'S HEALING POTENTIAL: TISSUE ENGINEERING

Tissue engineering is a rapidly developing field that essentially bridges basic and applied research. This field can be viewed as a technology whereby cells will proliferate and organize their extracellular matrix in a three-dimensional lattice to form ex vivo a clinically functional tissue exhibiting histochemical, biochemical, and biomechanical properties that are similar to native, healthy tissue. Indeed, tissue engineering has been an active area of research in the orthopaedic field and likely will yield novel methods of treating damaged and diseased articular cartilage. Production of ex vivo grown articular cartilage that exhibits histologic, immunologic, biochemical, and biomechanical properties of native articular cartilage has been an active area of research. Cartilaginous material exhibiting these properties appears to be synthesized best in bioreactors, which are growth chambers that regulate the amounts of nutrients and gases in the growth medium while rotating or stirring to ensure each cell is in contact with the culture medium, enhancing the gas exchange and transport of nutrients.u, 27 For example, a rotating bioreactor was used by Freed et alE to subject cell-seeded polyglycolyic acid scaffolds to various stimuli. Dunkelman et alZ3immobilized cell-laden polyglycolyic acid scaffolds inside polycarbonate capsules to direct and control fluid-induced shear throughout the lattice. After several weeks of culture in either bioreactor, analyses revealed that areas in the material and extending to the material's edge exhibited organized extracellular matrix components consistent with articular cartilage. Seeded scaffolds that were not subject to similar stimuli resulted in heterogeneous, less organized material, suggesting that the mechanical stimulus conveyed by the flow of growth medium is a desirable feature that promotes cartilage matrix synthesis. Other reports also concluded that mechanical stimuli promote expression and organization of articular cartilage m o l e c ~ l e s33,. ~47,~93~Another important advantage of using bioreactors is the homogeneous distribution of nutrients. The distribution of nutrients must be adequate to pass the diffusion barrier, created by the synovial fluid and cartilage layer, and reach the chondrocytes. Hindered diffusion can reduce cellular uptake of needed nutrients and gases. This potential problem can be resolved largely by bioreactors. The bioreactor engineered by Dunkelman et aP3 evenly distributes nutrients and controls flow throughout a 2-mm-thick, 1-cm diameter polyglycolyic scaffold. A rotating bioreactor also more evenly distributes nutrients, as evidenced by the good histologic quality of the resultant cartilaginous material.27 Various tissue engineering approaches employed to generate articular cartilage ex vivo are discussed in the following paragraphs.

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NATURAL SCAFFOLDS

Natural biomaterials exhibit useful features that can facilitate repair of articular cartilage, including suitable adhesion properties, greater biocompatibility, and reduced toxicity during scaffold degradation. For these reasons, scaffolds consisting of natural biomaterials have attracted significant attention regarding repair of osteochondral defects. Natural biomaterials used as scaffolds in vivo include collagens, hyaluronan,78 alginategO periosteum,@ and coral." These biomaterials have shown promise in both in vivo and in vitro experiments, when tested as three-dimensional scaffolds to transport cells into an articular cartilage defect. Collagen

In vivo studies frequently use collagen I as a natural biomaterial vehicle for cartilage regeneration.", 59, 95 Certain collagen scaffolds exhibit properties, such as porosity and resilitogy that can be good features to promote healing of a cartilage defect. Collagen scaffold efficacy in tissue regeneration and modeling is improved vastly when used in combination with bioactive agents, however. The combination of collagen scaffolds with chondrocytes, mesenchymal stem cells, growth factors, or surface modifiers has resulted in improved articular cartilage remodeling when compared with collagen alone. Collagen and Cells

Type I collagen has been used as a gel lattice to investigate the possibility that embedded chondrocytes will form articular cartilage. Evidence suggests that collagen gels support chondrocyte pr~liferation.~~ Chondrocytes have been observed, however, to de-differentiate toward a fibroblast phenotype after several days in collagen gels.90Cell-laden collagen gels implanted in articular cartilage defects in the horse resulted in chondrocyte proliferation and synthesis of cartilaginous components; however, the surface layers of collagen grafts appeared poorly organized.68Collagen gels seeded with allograft chondrocytes resulted in synthesis of hyaline cartilage when implanted into defects.95After 48 weeks, defects implanted with collagen-cell composites were filled with hyaline-like cartilage that exhibited "indentation values" similar to normal cartilage; however, no tidemark or subchondral bony plate had formed. The biosynthesized material that filled the control (empty) defects in vivo exhibited inferior histologic and biochemical properties. Thus, the presence of chondrocytes appears to be a desirable feature of collagen scaffolds intended to promote healing of articular defects. It is still not clear, however, if a three-dimensional collagen lattice is an efficient scaffold for cartilage regeneration in vitro or in vivo.

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Although collagen type I is used commonly as a carrier, other collagen types can may exhibit properties that facilitate the overall repair process. For example, the effects of collagen types I and I1 as scaffolds seeded with autologous chondrocytes were evaluated in adult canine articular defects.57After a healing period of 15 weeks, the defects were evaluated histomorphometrically. Although the material in all of the defects contained fibrous tissue, defects that were implanted with a type I1 collagen-chondrocyte composite contained the greatest total amount of reparative tissue. This outcome suggests that type I1 collagen has properties that are conducive forward cartilage remodeling. Collagen and Hyaluronan

Hyaluronan is a naturally occurring nonsulfated glycosaminoglycan consisting of alternating N-acetylglucosamine and glucuronic acid residues to form an extended linear polymer. It is also a component of healthy articular cartilage. Hyaluronan is used commonly in surgical procedures by direct application onto tissues, providing a viscous solution that helps prevent tissue dehydration and can reduce improper healing of surgical wounds. Hyaluronan is upregulated during embryonic development and wound healing. It is localized to pericellular matrices surrounding moving and proliferating cells, suggesting that it plays a significant role in cell movement.86,87 When hyaluronan was combined with collagen scaffolds, this construct promoted greater cell proliferation and synthesis of chondroitin sulfate glycosaminoglycans in vitro when compared to control collagen scaff0lds.4~Thus, as a carrier of cells, hyaluronan could have desirable effects on tissue repair and remodeling, promoting cell proliferation and biosynthesis of extracellular matrix molecules. The physiochemical properties of native hyaluronan, however, are not conducive to formation of a three-dimensional scaffold. If employed as a solution in vivo, it would be lost rapidly. Nonetheless, because hyaluronan is a naturally occurring polymer with chemical features highly conserved across species, it remains an attractive biomaterial as a cell implantation vehicle. Hyaluronan can be modified to form a more robust material; osterifications introduced in native hyaluronan provide chemical groups that allow hyaluronan to exhibit enhanced structural integrity.1° Derivatives of hyaluronan have been used to form fibers, three-dimensional scaffolds, films of various thicknesses, tubes, and sponges. Modified hyaluronan polymer has been reported to support cell attachment and the formation of extracellular matrix molecules consistent with matrix of osteochondral tissue.13,78 A hyaluronan derivative seeded with mesenchymal progenitor cells and implanted subcutaneously resulted in composites that contained more cartilage and bone when compared to ceramic controls. Further investigation regarding the value of hyaluronan in tissue engineering of articular cartilage is anticipated. Commercial sources of hyaluronan are available, both as a general research item

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and as a clinical reagent. Additionally, hyaluronan synthases of various organisms have been cloned.19,36 Recombinant hyaluronan synthase could provide sources for this polymer in the future. Chitosan

Chitosan is a derivative of chitin, a naturally occurring polymer consisting of N-acetylglucosamine. Chitin is a component of the cell walls of fungi, some protists, and skeletons of arthropods. Hydrolysis of the aminoacetyl groups of chitin generates chitosan. Polymers of chitosan are biocompatible, and hold some promise as scaffold materials.48The gross shape of chitosan vehicles can be controlled and pore diameters in the material can be regulated. Although the use of chitosan as a vehicle to transport cells and affect articular cartilage defects remains to be well characterized, studies examining this material as a potential delivery agent for small peptides could be useful in cartilage synthesis in a three-dimensional scaffold.53 Fibrin Glue

A mixture of fibrinogen with thrombin results in a compound that essentially solidifies, creating a biologic substance that has served as a "glue" in some clinical procedure^.^^ Fibrin glue already is used as a reagent that helps control bleeding and facilitates adherence of tissues. An attractive feature of fibrin glue is its ability to act as a solution that can be mixed with cells and subsequently injected in a defect or molded in vitro to form a specific size and shape. Chondrocytes embedded in a fibrin glue will maintain their morphology, proliferate, and produce an extracellular matrix.34Thus, as a vehicle, the soluble phase of a fibrin glue-cell composite allows the formation of precise three-dimensional shapes that could serve as templates for cartilage synthesis, regulating the final size and shape of the cartilage product. Molding fibrin glue-chondrocyte composites into specific shapes has been demonstrated as a technique that might facilitate reconstructive surgery. Ting et als5polymerized a human nasal shape containing human costal chondrocytes. Following culture in vitro the structure was implanted subcutaneously into a nude mouse. After several weeks the structure was excised and shown to retain some of its original features. Extracellular matrix and viable chondrocytes were present, suggesting a possible use of this approach to create human cartilage from a fibrin glue template. A similar result was demonstrated by injecting into subcutaneous tissue of nude mice a chondrocyte-fibrin glue mixture. Subsequent analyses revealed the chondrocyte-fibrin glue composites exhibited a homogenous cartilaginous material that contained glycosaminoglycan and type I1 collagen. A fibrin glue control (no cells) did not support cartilage synthesis. The ability of a fibrin glue-cell composite

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to enhance repair when applied to a weightbearing articular defect could be problematic, however. A study testing fibrin glue in articular defects suggested that the fibrin glue did not exhibit sufficient biomechanical strength to support formation of a three-dimensional repair tissue in V~VO.~~ Agarose

Alginate and agarose gels have been investigated for their use as vehicles to support synthesis and organization of articular cartilage extracellular matrix components. Alginate gels (as opposed to collagen gels) were shown to support the differentiation phenotype of chondrocytes. Although cell numbers in alginate gels exhibited an initial loss, the chondrocyte phenotype was maintained?O a finding consistent with the report that rabbit articular chondrocytes de-differentiated in monolayer cultures will re-express a cartilage phenotype when maintained in anchorage-independent culture in agarose gels.” Implantation of cellladen agarose gels holds some promise as an approach to enhance deposition and organization of osteochondral components. In another study, agarose gels containing allograft chondrocytes were implanted into rabbit full-thickness defects, and exhibited formation of new subchondral bone. The newly synthesized components appeared to integrate with host articular cartilage when compared with controls.65 SALIENT COMPONENTS OF SUCCESSFUL STRATEGIES TO ACHIEVE SATISFACTORY HEALING Cells and Bioactive Agents

Evidence suggests that remodeled osteochondral defects contain new chondrocytes in the repaired defect. The new cells are believed to have originated from host mesenchymal stem cells.74This finding suggests that migration of endogenous chondrocytes into an implant material in an articular defect does not contribute to cartilage repair. Because the presence of few endogenous chondrocytes will not promote significant remodeling, the quality of the repair process should be facilitated by using a scaffold containing a high cell density. The addition of cells onto scaffolds indeed makes a significant difference in the outcome of cartilage genesis.64Cells that have been added to scaffolds include primary chondrocytes and mesenchymal progenitor cells. Chondrocytes

Because chondrocytes from cartilage adjacent to a defect do not migrate into the defects area, both primary chondrocytes and passaged

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cells have been seeded onto scaffolds and tested for their ability to make cartilaginous 26 The use of chondrocytes derived directly from donated cartilage is limited, however, because overharvesting of cartilage chondrocytes contributes to problems at the harvest site. Thus, serial passage of chondrocytes as monolayers is a convenient means to acquire a large number of cells that generally retain the chondrocyte phenotype throughout the first few passages. The large number of cells then can be seeded easily onto three-dimensional material. A desirable seeding protocol will result in a homogenous distribution of chondrocytes and cartilage extracellular matrix material throughout the original scaffold dimension. Progenitor Cells

Mesenchymal-derived progenitor cells are becoming increasingly popular as a potential cell source for repair of osteochondral defects. Because of their ability to differentiate into cells that synthesis and organize subchondral bone and articular cartilage tissue, progenitor cells could become important constituents of composites to repair osteochondral defects. At present, the outcomes of experiments testing for the potential use of mesenchymal-derived progenitor cells in repair of osteochondral defects are promising. Mesenchymal progenitor cells can be expanded in vitro, and retain the ability to differentiate and produce cartilage and bone This finding has held true for progenitor cells derived from human,= mouse,3l and rabbit.*l,39 The ability of progenitor cells to exhibit differentiation after expansion in vitro is a significant incentive to understand further the precise molecular mechanisms that regulate differentiation pathways. Grande et a130cultured mesenchymal stem cells in polyglycolic acid polymer matrices. After cell culture in vitro, the matrices were implanted into full-thickness defects in rabbit articular cartilage. At 12 weeks, histology of the defects that contained polyglycolic acid polymer matrices seeded with mesenchymal stem cells revealed a material that was similar to normal cartilage. The surface layer of repair tissue-exhibited a cartilaginous material with a similar thickness compared to normal articular cartilage. The defect area also contained subchondral bone. This outcome suggested the mesenchymal stem cells differentiated into osteoblasts and chondrocytes in the matrix and recreated the threedimensional orientation of the osteochondral tissue. As progenitor cells are isolated and characterized, their potential as an increasingly valuable resource for the engineering of articular cartilage should be realized. There are also possibilities of using cartilage from areas that have regenerative properties, such as mandibular condylar cartilage. This cartilage has a layer of prechondroblastic mesenchymal stem cells that likely contribute to chondrogenesis and maturation of hyaline cartilage.29 Embryonic stem cells also have been considered as a potential cell source for orthopaedic research. These cells are pluripotent, giving rise

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to different cell types when the appropriate differentiation factors are introduced. The potential for young cells to effect considerable repair is demonstrated by Namba et a156when a superficial defect was introduced in fetal cartilage. After 28 days, the defect had repaired itself without fibrous scar tissue, suggesting that fetal chondrocytes have the capacity to repair cartilage defects. It is also suggested that this ability is suppressed as the progenitors differentiate and age. Another example of the use of progenitor cells is reported by Martin et a1.5*When chick embryo bone marrow stromal cells were seeded in a polyglycolic scaffold along with fibroblast growth factor-2, the cells successfully differentiated into epiphyseal cartilage. The number of reports suggesting that progenitors are a potential cell source for basic orthopaedic research are increasing, and it can be anticipated that our understanding of these cells and their application to applied orthopaedic research will move forward. The age of the cells, regulation of their differentiation pathway, and ethical concerns about the source and means of collection of these embryonic cells are current issues. Seeding Density

It is increasingly evident that the presence of cells is a necessary element in our approach to tissue engineering of articular cartilage, which is known to exhibit one of the lowest cellular densities of any tissue. Cells can be added onto scaffolds that subsequently can be implanted in cartilage defects, or cells can be incorporated into matrices for ex vivo production of cartilage constructs. A key aspect of cell seeding is the initial number of cells that one should use per scaffold or per defect volume. Despite its importance, systematic studies have not been performed to examine the effects of the initial cell density on cartilage formation and determine the optimal cell numbers. In the authors’ experience, seeding polylactide-polyglycolide scaffolds with a density of less than 10 million articular chondrocytes per milliliter does not result in an acceptable cartilage construct. The amount and integrity of the construct increase with increasing initial cell density. These observations concur with a report in the literature, in which scaffolds were seeded at a density ranging from 20 to 100 million cells per milliliter; higher seeding densities resulted in increased cartilaginous formation.64 Media Composition

In addition to cell density, equally important in the quest to generate fully functional articular cartilage ex vivo is the use of appropriate culture media. The growth medium for chondrocyte cultures should contain inorganic salts, amino acids, vitamins, a buffering component (such as a bicarbonate buffer or HEPES), D-glucose and sodium pyruvate as a source of energy, and glutamine, which is used as an energy and

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carbon source. Other supplements useful for culturing chondrocytes also are added. Serum, typically at 10% of the volume of the growth medium, is added to provide growth factors and attachment factors. For the synthesis of cartilage, supplements including ascorbic acids, additional proline, and a cocktail of nonessential amino acids can be included. Ascorbic acid is a water-soluble vitamin (vitamin C ) that can aid in the folding and stabilization of newly synthesized collagen chains, and formation of intra- and intermolecular collagen crosslinks. The use of 50 pg/mL fresh ascorbic acid, added every 72 hours, in cell-seeded scaffolds is important for the synthesis of cartilage.23Dulbecco's Modified Eagle Medium (DMEM) is a growth medium appropriate for chondrocyte cultures and cartilage synthesis.22A suitable cartilage-specificmedia formulation may consist of DMEM containing 10% fetal bovine serum, 2 mM L-glutamine, nonessential amino acids, 50 pg/mL proline, and 1 mM sodium pyruvate. Growth and Differentiation Factors

Bioactive molecules, such as the family of fibroblast growth factors (FGF) and bone morphogenetic proteins (BMP), influence numerous cellular functions. In articular cartilage, chondrogenesis and cell metabolism are affected by the presence of such factors. For example, cells can differentiate into the correct phenotype aided by factors such as TGF-P and BMP at appropriate concentration^^^; however, TGF-P can have toxic effects at high Athanasiou et a14 used a biodegradable osteochondral 50:50 poly-DL-lactide-co-glycolide implant with human recombinant TGF-P1 to treat 7-mm defects in goats. This study demonstrated that there is potential for TGF-P delivered by biodegradable scaffolds to improve the quality of repair cartilage. In a different study, recombinant human BMP-2 in conjunction with a collagen carrier had positive effects on the healing of full-thickness osteochondral defects in rabbits. It greatly accelerated the formation of new subchondral bone and improved the histologic appearance of ~ a r t i l a g e .As ~ ~basic issues related to the effects of these growth factors or cytokines are elucidated, combinations of these factors could be incorporated into scaffolds to affect different cell functions and to boost the synthesis of articular cartilage. Biodegradable scaffolds designed to release bioactive agents at controlled doses and temporal increments likely will play an important role in cartilage repair in the near future. Scaffolds

To engineer articular cartilage in the laboratory, scaffolds should be compatible with the unique functional environment of articular cartilage, including composition, lack of vascularity, and concomitant inability to respond to injury and disease. The scaffold should be porous and perme-

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able to allow for the ingress of nutrients and removal of waste products, while temporarily supporting cells and synthesis and organization of extracellular matrix components. The scaffold should be biodegradable, allowing for extracellular matrix to occupy the increasing void space. The scaffold should also exhibit biomechanical characteristics compatible with those of native cartilage. This is important because cartilage metabolism, synthesis, and organization of extracellular matrix are affected to a large extent by the mechanical environment experienced by chondrocytes. Athanasiou et a19designed cartilage-specific implants using 50 :50 poly-D,L-lactide-co-glycolide, which were used subsequently in animal s t ~ d i e sThe . ~ in vitro degradation characteristics of various versions of this scaffold also were studied to determine the effects of design variables on these implants' ability to carry functional loads and undergo degradation.', 9, 76 For example, a blend of three copolymers of 50:50 poly-D,L-lactide-co-glycolide can be used to extend the in vitro useful life of a cartilage scaffold.s0Furthermore, the original porosity designed into the scaffold can have profound effects on its functional behavior.8 Although most cartilage scaffold degradation studies are performed under quasi-static or completely static conditions, a dynamic functional environment of the scaffold can have a significant effect on its in vitro degradation and release of bioactive agents.s4Another critical issue in determining the degradation characteristics of alpha-hydroxypolyesters is the lowering of the pH around the scaffold due to the release of polylactic acid and polyglycolic acid as the material is hydrolyzed. To address this issue, incorporation of basic salts into the design of the biodegradable scaffold has been proposed.' Variations of scaffolds made of alpha-hydroxypolyesters supported ex vivo synthesis of cartilage.=, 2 m 3 , 93 These scaffolds were made of fibrous polyglycolic acid material and seeded with chondrocytes. Although this design lacks any biomechanical similarity to articular cartilage, the scaffolds supported cell proliferation, viability, and synthesis of a cartilaginous material that retained the three-dimensional shape of the original material. These equipments suggest that polyglycolic acid fibers can function as scaffolds 93 that support generation of cartilaginous material.23,*w8, Surface Modification of Scaffolds

In tissue engineering applications of articular cartilage, the ability of chondrocytes to attach to polylactides-polyglycolides or other scaffolds in the absence of serum is not clear, because serum contains large quantities of fibronectin and vitronectin, both powerful mediators of cell adhesion. It could be desirable to eliminate serum from such applications, because of health risks associated with human- and animalderived proteins. Although other reagents, such as polylysine, cause cells to stick, presumably through charge-mediated interactions, cells on a substratum of polylysine do not exhibit a well-organized spreading, formation of an actin cytoskeleton, or formation of focal adhesions, all

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believed to be important cellular a t t r i b ~ t e sIf. ~serum ~ is eliminated, then it could be useful to apply cell adhesion synthetic peptides onto surfaces that are intended for cell attachment. Synthetic peptides that are known to interact with cell surface adhesion molecules, including i n t e g r i n ~ ~ ~ and proteoglycans,2° are currently under development and likely will play a significant role in tissue engineering of ~artilage.4~ Mechanical Loading

By design and function, articular cartilage is a biomechanical structure that routinely gets exposed to large and complex stress fields. Chondrocytes are expected to experience significant mechanical stimuli, which could affect the synthesis and organization of articular cartilage. Chondrocytes and numerous other cell types are directly connected to their microenvironment by focal adhesions, which can be defined loosely as discrete regions of the cell's plasma membrane that bind to extracellular material, connecting the cell to material in the microenvironment. In addition to providing structural attachment between cells and the extracellular matrix, focal adhesions also serve as mediators of signal transduction, propagating extracellular stimuli. Although the precise signaling pathways that are involved in mediation of mechanotransductional stimuli are not completely understood, it is evident that mechanical stimuli are desirable for articular cartilage synthesis and modeling. In particular, externally applied mechanical forces appear to be necessary in tissue engineering studies of articular cartilage. For example, increased deposition of a mechanically better matrix is observed in polyglycolic acid scaffolds seeded with bovine chondrocytes under turbulent flow, or in rotating vessels, than under static condition^.^^ Static conditions decrease proteoglycan synthesis, as shown in several previous studies.31,40, 67 Mechanical stimuli also affect proteoglycan synthesis in native cartilage, for example, with increasing load magnitude or loading duration proteoglycan synthesis was reduced in bovine articular cartilage disks.81Thus the metabolic activity of chondrocytes can be affected significantly by varying mechanical parameters, such as load duration and magnitude. Indeed, metabolic activity of cells appears to be affected differentially by varying loading regimens. Over short periods, cyclic loading inhibits cell divisions8,91 and increases the synthesis of sulfated glycosaminoglycans and other components.61,88, 91 In a recent significant study, in vivo applied loading was shown to have a significant effect on cartilage synthesis.82Small titanium chambers, one with a mobile piston used to apply external compressive loads, were implanted into rat tibiae and allowed to harbor infiltrated host mesenchymal cells for 3 weeks. Thereafter, twice a day for 7 weeks, 20 cycles of load stimuli were applied to the mesenchymal tissue in the load chamber. Histologic analysis revealed that both chambers contained bone; however, the load chamber contained cartilage next to the piston, whereas the control chamber (no load) contained bone only, suggesting that the cyclic load

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caused the mesenchymal tissue to differentiate into cartilage. Thus, application of mechanical stimuli, whether hydrostatic pressure, fluid flow-induced shear, or interaction with a solid object, generally promotes expression of cartilage molecules and synthesis of a cartilaginous material. A means to convey stimuli should be a serious consideration when designing bioreactors intended to facilitate cartilage genesis in vitro. FUTURE POSSIBILITIES Identification of Appropriate Cell Types As research moves inexorably toward the use of cells to engineer articular cartilage, identification of the appropriate cell type has become a critical issue. Certain cell types, such as articular chondrocytes and progenitor cells, appear to be of particular prominence. Articular chondrocytes are isolated easily from mature articular cartilage by enzymatic digestion and can be expanded in culture. Progenitor cells are another possible cell source, because they can expand and differentiate into chondrocytes under suitable conditions. It has been demonstrated that osteochondral progenitor cells isolated from periosteum and bone marrow and expanded in vitro can serve as a cell source for synthesis of articular cartilage and bone.94Such experiments suggest osteochondral progenitor cells expanded in vitro retain the signaling pathways leading toward differentiation into cartilage and bone cell phenotypes. Other progenitor cells isolated from mouse,2l and rabbit16,39 have been demonstrated to differentiate and synthesize cartilage. With increasing understanding of cell behaviors and the mechanisms allowing for reproducible ex vivo synthesis of clinically acceptable articular cartilage, there is a clear indication that progenitor cells and articular chondrocytes will become valuable resources for the engineering of articular cartilage.

Optimization of the Use of Growth Factors

It is well accepted that protein growth factors, such as TGF-P and BMP, affect and control differentiation of mesenchymal stem cells or chondrocytes, and also can stimulate chondrogenesis and cell metabol i ~ mTGF-P . ~ ~ delivered by biodegradable scaffolds is believed to improve the quality of repair cartilage? Recombinant human BMP-2 delivered by means of a collagen carrier also has been shown to have positive effects.71Because different growth factors or other bioactive agents exert distinctly different influences, one can expect that, in the future, a combination of growth factors or cytokines can be incorporated into scaffolds to affect different cell functions, such as differentiation, metabolism, and synthesis of extracellular matrix. Appropriate doses of these bioactive agents could be added so that suitable factors are released at temporally distinct points to boost the entire sequence of articular chondrogenesis.

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Bioreactors: The Mechanical Environment

Central in the efforts to produce articular cartilage ex vivo is modulation of the biomechanical environment experienced by cells. Mechanical forces, which appear to influence numerous cell types, appear to be essential in tissue engineering of articular cartilage because they have a profound effect on chondrocyte function and cartilage regeneration. As a result, bioreactors are used routinely to modulate fluid flow-induced shear. In general, static conditions have negative effects, whereas mechanical forces due to agitation or stirring can have positive influences. Microgravity also can be beneficial, but shear stress appears to be the most potent biomechanical stimulator. Shear appears to have a direct effect on cell metabolism and extracellular matrix synthesis, whereas hydrostatic pressure affects cytoskeletal organization without influencing matrix synthesis significantly. It also could be beneficial to incorporate an intermittently applied compressive stress regimen in cartilage bioreactor. To optimize the use of mechanical forces in bioreactors, media flow in bioreactors needs to be well characterized because stress fields experienced by cells on scaffolds are not fully characterized. It is essential to quantify the stress-strain fields experienced by individual cells attached on scaffolds and how these mechanical stimuli affect their mechanotransductional abilities. It is envisioned that, in the future, bioreactors will employ complex loading regimens to affect positively the synthesis of articular cartilage. Surface Modification

Scaffolds designed to promote healing of articular cartilage defects only now are beginning to be considered. For example, scaffolds can be designed such that their surfaces are modified with cell adhesion peptides. In bone cells, it was demonstrated recently that the amino acid sequence Lys-Arg-Ser-Arg promoted preferential adhesion of neonatal rat calvarial osteoblasts when compared to an integrin-binding sequence Arg-Gly-Asp-Ser.20Findings such as these suggest that different cell types, such as chondrocytes or mesenchymal stem cells, can recognize selectively specific surface modifiers. Future work using molecular approaches, such as phage display technology,7° could identify specific amino acid sequences that are preferentially recognized by chondrocytes and progenitor cells. In addition to adding appropriate modifiers on the external surface of a scaffold, it could be beneficial to modify scaffold internal surfaces to achieve preferred chemotaxis. For example, it could be desirable to attract more cells to the central core of the scaffold or develop a scaffold with polarized adhesion characteristics such that chondrocytes will attach to one region and bone cells to a different region of the same scaffold. This would allow for engineering of polarized complex composites that potentially can be used to repair osteochondral defects. Another promising avenue is the identification and

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characterization of novel extracellular matrix molecules in cartilage development. The authors are studying the biologic activity and developmental expression of a protein with a molecular mass of 68,000 named p68Bi6-h3.47 Although this protein is found in numerous adult tissues and promotes cell in situ hybridization experiments suggest that in and near developing bone and cartilage p68Big-h3 is transcribed extensively. The authors’ pilot data suggest that p68Big-h3 can enhance production of cartilage and bone. Use of Intermediate Constructs

It has been the practice to consider the ex vivo formation of neocartilage successful only when the construct’s histochemical, immunochemical, biochemical, and biomechanical properties fall in certain desirable ranges. It turns out, however, that subsequent use of such ”good” cartilage constructs in animals has not been consistently acceptable. The reason is that even though the implanted construct retains its cartilage composition, integration with host tissue is unacceptable. Based on these studies, it is reasonable to hypothesize that a partially developed cartilage construct (defined as an intermediate) could enhance integration of the implant with host tissue. Compared with mature cartilage constructs, intermediates can be more porous, more permeable, and have more metabolically active cells. A systematic study testing intermediates at various levels of development needs to be undertaken in cartilage defects. Design of Cartilage-specific Scaffolds

Biomechanical design is a critical issue when considering the use of scaffolds to affect articular cartilage repair. The capability of a scaffold to adapt and function in various environments (e.g., in vitro and in vivo) depends largely on the actual material composing the scaffold and its biomechanical properties. Both material and biomechanical properties influence scaffold biocompatibility, and hence are consequential to the outcome of the repair process. Most scaffolds used for ex vivo engineering of articular cartilage are made of polylactic acid, polyglycolic acid, or their copolymers. For this reason lactic-glycolic acid polymers will be considered as examples. Byproducts generated by hydrolysis, which can be accelerated by an acidic en~ironment,8~, 92 eventually enter the tricarboxylic acid cycle. Therefore, by controlling the mechanical properties and thus affecting hydrolysis, the rate of polymer hydrolysis can be regulated. For example, porosity can directly influence degradative autocatalysis.8The authors believe that scaffold biocompatibility is affected by a biomechanical design that incorporates both elastic and viscoelastic properties. Scaffold compressive stiffness will modulate the stress-strain environment at the cellular, tissue, and scaffold levels. Thus,

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altered compressive stiffness will influence mechanotransduction and can even promote cell apoptosis due to direct compression. Similarly, scaffold viscoelastic properties influence fluid pumping action, which can be consequential for mechanotransduction and cell apoptosis because of hydrostatic pressure and flow-induced shear. Another property affecting scaffold physiology is permeability, defined as degree of ease or difficulty of fluid flow through a scaffold. Permeability influences the apparent viscoelastic response of scaffold, and can control cell nutrition and evacuation of waste byproducts. Increased scaffold permeability is expected to permit more rapid byproduct evacuation. This potentially can affect pH, reduce autocatalysis, and decrease the rate of degradation, influencing mechanotransduction and cell apoptosis due to fluid flow (pressure, shear). Clearly, scaffolds are important in successful outcomes of tissueengineered articular cartilage. Thus, designing a scaffold with optimal characteristics is a significant endeavor. The scaffold should be biocompatible and bioabsorbable, exhibiting engineered material properties including porosity, degradation rate, and mass loss. It should undergo temporal degradation that is harmonious with the remodeling of newly made extracellular matrix and should provide a framework that facilitates new tissue ingrowth. Surface modifications that introduce chemically reactive moieties, such as hydroxyl, carboxyl, and amine groups that would facilitate attachment of cell adhesion pep tide^^^ or biologic molecules, can increase significantly the effectiveness of the scaffold (Fig. 1).Furthermore, a scaffold should manifest engineered mechanical characteristics including stiffness, compressibility, creep, stress relaxation, and permeability. These features are collectively consequential for successful tissue engineering efforts because material biocompatibility alone without an appropriate biomechanical design is not a guarantee that the scaffold will behave as desired in applications involving tissue engineering of articular cartilage. It is desirable to match initial scaffold elastic and viscoelastic properties to those of the native tissue, increasing the chances that the repair process is compatible with the normal physiology of the host tissue. Furthermore, the design needs to render the scaffold sufficiently permeable and porous. The objective should be to create a biomechanical environment that is conducive to enhanced cellular mechanotransduction. Standardization of Assessment Standards

Cartilage researchers are often faced with the great difficulty of comparing their results from cartilage healing studies to other investigators’ work. This task becomes an exceedingly complex process, because standard protocols do not exist. As a result, different investigators employ or develop assessment techniques that are neither universally accepted nor easily reproduced by others. Of course, when assessing such complex biologic systems, numerous variables affect the experimental

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Figure 1. In the future, scaffolds may be used to engineer articular cartilage ex vivo, using various growth factors, differentiation factors, and cells. These scaffolds should be specifically designed to account for the biological and biomechanical peculiarities of articular cartilage. Cell functions may be further modulated by way of the use of surface modifiers incorporated into and onto the scaffold. MSC = Mesenchymal stem cells.

outcomes. The authors feel that sufficient data have been accumulated regarding cartilage healing studies, to the point where standard protocols need to be established to allow reproducible and meaningful comparisons among studies related to cartilage repair, and functional articular cartilage composites. Because scaffolds are pivotal in both in vivo and ex vivo production of articular cartilage, their evaluation needs to be standardized with regard to mechanical properties, permeability, porosity, material properties, degradation rate, and biocompatibility. Furthermore, the in vivo approaches also need to be standardized concerning the animal models used, surgical approach, defect size, location, defect creation, postoperative management, and duration of healing. Standardization of protocols should remove the vexing inability to compare results from different studies. SUMMARY

As the ability to understand the peculiarities of successful healing of articular cartilage defects moves forward, it becomes clear that this complex orthopaedic problem soon will be successfully addressed. A multidisciplinary approach, combining clinical experience, cogent bio-

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material designs, new cell biologic processes, biomechanical assessment, and modern molecular biology, clearly is leading toward clinically acceptable, viable, and consistent articular cartilage regeneration.

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Address reprint requests to Kyriacos A. Athanasiou, PhD Rice University Department of Bioengineering, MS-142 P.O. Box 1892 Houston, TX 77251-1892