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Review: tissue engineering for regeneration of articular cartilage
Johnna S. Temeno! , Antonios G. Mikos *
Department of Bioengineering, Rice University, 6100 Main, Houston, TX 77005-1892, USA Department of Chemical Engineering, Rice University, 6100 Main, Houston, TX 77005-1892, USA Received 27 July 1999; accepted 30 September 1999
Abstract Joint pain due to cartilage degeneration is a serious problem, a!ecting people of all ages. Although many techniques, often surgical, are currently employed to treat this a%iction, none have had complete success. Recent advances in biology and materials science have pushed tissue engineering to the forefront of new cartilage repair techniques. This review seeks to condense information for the biomaterialist interested in developing materials for this application. Articular cartilage anatomy, types of injury, and current repair methods are explained. The need for biomaterials, current commonly used materials for tissue-engineered cartilage, and considerations in scale-up of cell}biomaterial constructs are summarized. 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Cartilage repair; Cartilage regeneration; Cartilage tissue engineering; Cell transplantation; Bioreactors; Chondrogenesis
1. Introduction Joint pain is a major cause of disability in middle-aged and older people . Pain usually results from degeneration of the joint's cartilage due to primary osteoarthritis or from trauma causing loss of cartilage . As many as 36 million Americans su!er from some form of arthritis  and cartilage damage from sports injury is also common. It has been shown that up to 16% of injuries to the knee cause intra-articular bleeding [3,4]. Since cartilage shows very little tendency for self-repair, these injuries are maintained for years and can eventually lead to further degeneration (secondary osteoarthritis) . Given the debilitating nature of severe joint pain, scientists and surgeons have tried for decades to repair or regenerate lost cartilage, but there has been little success due to the complex properties of the tissue and its essential function in the body. Hyaline cartilage, the type of cartilage found in joints, provides stable movement with less friction than any prosthetic replacement, and can alter its properties in response to di!erences in loading. Although appearing to be a simple, avascular matrix, hyaline cartilage possesses properties such as resistance
* Correspondence address: Department of Bioengineering, Rice University, 6100 Main, MS 142, Houston, TX 77005-1892, USA. Tel.: #713-285-5355; fax: #713-285-5353. E-mail address: firstname.lastname@example.org (A.G. Mikos)
to compression and the ability to distribute loads that cannot be fully replaced by any other tissue or device designed to date [5,6]. This review will provide an overview of cartilage anatomy and types of injury, as well as focus on current strategies for cartilage repair in the knee joint, including promising new strategies involving tissue engineering. The knee, with two distinct articulating surfaces is a condylar-type joint. Both bones, the femur and the tibia, that comprise the joint are covered with a layer of hyaline cartilage at the joint surface. Immediately below this is the periosteal covering of the bone. The synovial membrane encircles the joint, thereby forming a barrier to retain the synovial #uid in the knee. This #uid provides lubrication and nutrients for the cartilage since no blood vessels penetrate into the tissue from the subchondral bone . Although only hyaline cartilage is found in the knee, two other kinds, "brocartilage and elastic cartilage, are seen in other areas of the body. All three are composed of chondrocytes and extracellular matrix macromolecules. Elastic cartilage forms the ear and nose and is characterized by the presence of elastin in the extracellular matrix (ECM). Fibrocartilage has a higher proportion of collagen in the ECM than hyaline cartilage and is found at the ends of tendons and ligaments in apposition to bone. Hyaline cartilage has a white, glassy appearance, and unlike "brocartilage, shows no macroscopic evidence of "bers .
0142-9612/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 9 9 ) 0 0 2 1 3 - 6
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2. Composition and anatomy of articular cartilage 2.1. Chondrocytes In humans, chondrocytes represent only about 1% of the volume of hyaline cartilage but are essential since it is these cells that replace degraded matrix molecules to maintain the correct size and mechanical properties of the tissue. Thus, microscopically, the cells' endoplasmic reticulum and Golgi apparatus are prominent. In addition, many contain lipid and glycogen stores and secretory vesicles. Some chondrocytes have cilia that extend from the cell into the ECM and are believed to play a role in sensing the mechanical environment of the cell, since chondrocytes are known to modify matrix properties in response to loading . Chondrocytes originate from mesenchymal stem cells (MSCs) found in the bone marrow in mature individuals. During embryogenesis, the MSCs start to di!erentiate into chondrocytes and secrete a cartilaginous matrix. During this time, the cells continue to divide. They pass through various lineage states and eventually the chondrocytes in the central zone, located next to what will soon be bone, enter the "nal stage of development and become hypertrophic chondrocytes, producing proteins that are important in calci"cation of the matrix. Other chondrocytes (on the periphery) secrete collagen and matrix molecules in the right proportions to produce hyaline cartilage. Mature articular chondrocytes, unable to proliferate, appear rounded and are completely encased in matrix [5,9]. 2.2. Extracellular matrix 2.2.1. Collagen Collagen types II, VI, IX, X, and XI are found in articular cartilage, although type II accounts for 90}95% of the collagen in the matrix. Type II collagen has a high amount of bound carbohydrate groups, allowing more interaction with water than some other types. Types IX and XI, along with type II, form "brils that interweave to form a mesh. This organization provides tensile strength as well as physically entrapping other macromolecules. Although the exact function of types IX and XI are unknown, type IX has been observed to bind super"cially to the "bers and extending into the inter-"ber space to interact with other type IX molecules, possibly acting to stabilize the mesh structure. Type X is found only near areas of the matrix that are calci"ed [6,10]. 2.2.2. Proteoglycans Proteoglycans are composed of about 95% polysaccharide and about 5% protein. The protein core is associated with one or more varieties of glycosaminoglycan (GAG) chains. GAG chains are unbranched polysaccharides made from disaccharides of an amino sugar and
another sugar. At least one component of the disaccharide has a negatively charged sulfate or carboxylate group, so the GAGs tend to repel each other and other anions while attracting cations and facilitating interaction with water. Hyaluronic acid, chondroitin sulfate, keratan sulfate, dermatan sulfate and heparan sulfate are some of the GAGs generally found in articular cartilage [5,10,11]. There are both large aggregating monomers and smaller proteoglycans present in articular cartilage. The aggregating proteoglycans, or aggregans, are composed of monomers with keratan sulfate and chondroitin sulfate GAGs attached to the protein core. In most aggregan molecules, link proteins connect many (up to 300) of these monomers to a hyaluronic acid chain. Aggregans "ll most of the inter"brillar space of the ECM and are thought to be responsible for much of the resilience and stress distribution in articular cartilage through their ability to attract water. There are no chemical bonds between the proteoglycans and collagen "bers; aggregation prevents di!usion of the proteoglycans out of the matrix during joint loading [5,6,10]. The smaller proteoglycans include decorin, biglycan and "bromodulin. They have shorter protein cores and fewer GAG chains than their larger counterparts. Unlike aggregans, these molecules do not a!ect physical properties of the tissue, but are thought to play a role in cell function and organization of the collagen matrix . 2.2.3. Noncollagenous proteins In contrast to proteoglycans, glycoproteins have only a small amount of oligosaccharide associated with the protein core. These polypeptides help to stabilize the ECM matrix and aid in chondrocyte}matrix interactions. Both anchorin CII and cartilage oligomeric protein anchor chondrocytes to the surrounding matrix. Other noncollagenous proteins commonly found in most tissues, such as "bronectin and tenascin, are also observed in articular cartilage and are believed to perform similar functions as the glycoproteins . 2.2.4. Tissue yuid Tissue #uid is an essential part of hyaline cartilage, comprising up to 80% of the wet weight of the tissue. In addition to water, the #uid contains gases, metabolites and a large amount of cations to balance the negatively charged GAG's in the ECM. It is the exchange of this #uid with the synovial #uid that provides nutrients and oxygen to the avascular cartilage. In addition, the entrapment of this #uid though interaction with ECM components provides the tissue with its ability to resist compression and return to normal shape after deformation [5,10]. 2.3. Zones of organization Articular cartilage can be divided into four zones (super"cial, transitional, middle and calci"ed) based on
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di!erences in matrix morphology and biochemistry. In each zone, there are three distinct regions: the pericellular region, the territorial region and the interterritorial region. The pericellular and territorial regions provide means for chondrocyte attachment to the ECM and protection of the cells during loading. The pericellular region contains almost exclusively noncollagenous binding proteins. Part of the territorial region is composed of collagen "brils that follow the pericellular envelope but may extend to encompass two or more chondrocytes. Farther away from the cells, the territorial collagen is less aligned and "brils cross each other to form a basket structure around the chondrocyte(s). The interterritorial region is marked by an increase in "bril diameter and orientation of "bers in a more parallel fashion. This region is primarily responsible for the mechanical properties of the tissue . 2.3.1. Superxcial zone The super"cial zone is the thinnest zone and is made of two distinct layers. An acellular sheet of predominantly collagen "bers (the lamina splendens) covers the joint. Deep to this, the second layer is composed of #attened chondrocytes with their long axes parallel to the articular surface. The ECM in this area has more collagen and less proteoglycan than the other zones. There are also large amounts of "bronectin and water. This combination of molecules imparts more tensile strength to this area of the matrix, which could be useful in resisting shear from the articulating surfaces. The super"cial zone also is important for the compressive strength of the tissue and possibly in isolation of cartilage from the immune system [5,10]. 2.3.2. Transitional zone Occupying more volume than the super"cial zone, the transitional zone includes chondrocytes that are spherical and contain synthetic organelles such as the endoplasmic reticulum, Golgi bodies and mitochondria. The ECM in this area has larger collagen "brils, more proteoglycan and less collagen and water than in the previous zone. The interterritorial "brils are aligned obliquely or randomly to the articular surface, as opposed to parallel in the super"cial zone [5,10]. 2.3.3. Middle (radial) zone The middle zone is usually the biggest and has the largest diameter collagen "brils, the most proteoglycan, and the least water. The cells are rounded, like in the transitional zone, but are stacked in columns perpendicular to the articulating surface. The territorial region of the matrix encloses each column [5,10]. These cells show high synthetic activity*ten times that of super"cial zone chondrocytes . Also, in this area, the orientation of the interterritorial "bers changes again so that they are
now aligned perpendicular to the joint surface. The "bers extend into the tidemark, a basophilic line of unknown composition that indicates the beginning of calci"ed tissue [5,10]. 2.3.4. Calcixed cartilage zone The thin calci"ed cartilage zone lies closest to the subchondral bone and acts as a transition from soft hyaline cartilage to bone. Signi"cant shear stress can be produced in this area due to the interface between the soft cartilage and much sti!er bone . The chondrocytes here are smaller, with almost no endoplasmic reticulum. In some places, they seem to be completely surrounded by calci"ed ECM, indicating that they have very little metabolic activity [5,10].
3. Articular cartilage injury There are three main types of cartilage injury: matrix disruption, partial thickness defects, and full thickness defects. Matrix disruption occurs from blunt trauma, such as dashboard injuries in automobile accidents. The ECM is damaged, but if the injury is not extreme, the remaining viable chondrocytes will increase their synthetic activity to repair the tissue. Partial thickness defects demonstrate disruption of the cartilage surface ("ssures, etc.) but this does not extend to the subchondral bone. Immediately following the injury, nearby cells begin to proliferate, but for reasons that remain unclear, cellular attempts to "ll the defect cease before it is repaired. Full thickness defects arise from damage that transverses the entire cartilage thickness and penetrates the subchondral bone. In this case, the defect is "lled with a "brin clot and a classic wound healing response ensues. With this type of injury, unlike the others, there is access to a population of progenitor cells from the bone marrow which can migrate to "ll the defect [13,14]. These cells usually cause replacement of the "brin clot with tissue intermediate between hyaline and "brocartilage. This tissue is usually less sti! and more permeable than native cartilage, which could contribute to its eventual degradation over a period of months . Several factors may explain why cartilage demonstrates very little capability for self-repair after injury. Chondrocytes are not required to proliferate to maintain cartilage, as in some other tissues (i.e. skin), and mature chondrocytes have a relatively low metabolic activity . Except in the case of full-thickness defects, there is no direct access to progenitor cells (such as are found in bone marrow) and the resident chondrocytes may be impeded by the ECM from migrating to "ll the defect [13,15]. In addition, the proteoglycans in the ECM can prevent cell adhesion, further undermining the native repair process [15,16].
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4. Treatments for articular cartilage defects 4.1. Symptom management Due to the di$culties in natural repair of cartilage, clinicians have turned to other methods to return functionality of the joint. If cartilage damage is severe, symptoms are managed using resection or other surgical techniques. Resection of articular cartilage is often followed by implantation of a prosthesis in the joint . Relief of pain can also be provided by an osteotomy, which removes part of one of the bones in an articulating surface to decrease loading of the joint. This also gives the surgeon an opportunity to bring regions of the joint with remaining cartilage in contact with each other or to correct misalignments due to previous degeneration . These techniques are often employed after other methods have failed . 4.2. Cartilage restoration In patients with smaller lesions, attempts are made to restore cartilage to the joint surface through a variety of methods. These include implanting replacement tissue grafts, or employing techniques that encourage the native repair process . 4.2.1. Tissue grafts Cartilage can be replaced either with small plugs from a less weight bearing region of the joint (autografts) or with a full allograft. Autografts are limited by the small amount of cartilage available in the body for transplantation to other sites. Usually, this cartilage is taken from the patella, the femoral condyle or the proximal "bula [1,4,17]. While this procedure does seem to decrease pain in many patients (70% good results at two to "ve years ), the e!ects of damage to the donor site must be considered. There is also concern that tissue from a less weight bearing region will not be able to withstand the forces imparted at the joint surface . Allografts have resulted in a 95% survival rate at "ve years, making them a useful treatment. However, fresh grafts have been seen to induce an immune response once implanted [1,4,17]. Frozen grafts may reduce this response as well as provide more time for screening of the donor tissue for pathogens, but freezing can reduce tissue viability . Another option is the use of periosteal or perichondrial grafts. For the former, periosteal tissue is taken from an adjacent area and placed in the cartilage defect with the belief that the undi!erentiated periosteal cells will be induced to form chondrocytes in their new environment. This technique has been used for more than a decade in the United States with somewhat encouraging results, depending on exact placement of the periosteal tissue in the defect and other factors [4,8]. A similar procedure can be done with perichondrium from the rib, but more
chondrogenesis was observed using periosteal grafts than perichondrial grafts . Furthermore, there was up to 70% failure of these grafts at "ve years due to ossi"cation of the perichondrial-derived tissue [3,18]. Although these procedures have proven to be e!ective for some patients, and remove the problem faced in other autografts of tissue damage due to alteration of position in the joint, they do result in donor site morbidity and can require a second incision to obtain the graft tissue . 4.2.2. Cartilage regeneration In an e!ort to eliminate the need for a donor site, and because of concerns associated with allogeneic and autogeneic implants, many attempts have been made to heal or regenerate existing cartilage, rather than replace it. The proposed techniques have focused on either enhancing the intrinsic regenerative properties of the tissue or transplanting extra chondrocytes to form more tissue. Unfortunately, neither of these has been completely successful, especially in older populations [1,4]. 126.96.36.199. Regeneration enhancement. The most common treatment of cartilage degeneration is to penetrate the subchondral bone, either by abrasion or drilling. This, in essence, creates a full-thickness defect. A clot forms over the bone surface which, if left unloaded, can provide a sca!old for migration of MSCs and their eventual di!erentiation into chondrocytes and osteocytes . However, results of this method are mixed: the tissue formed ranges from no cartilage to "brocartilage to hyaline cartilage, depending on the patient. Even when hyaline-like cartilage is created, the mechanical properties and durability are less than that of the original tissue. Due to the large variability in outcomes, the best that can be concluded is that this method has some chance of helping and little chance of harming the patient [1,3,4]. Less invasive techniques to restore cartilage include treatment with lasers or electrical stimulation, decreased joint loading in conjunction with continuous passive motion, or the use of pharmacological agents to stimulate chondrocytic activity (see  for a more complete review of each technique). Passive motion shows considerable regeneration for small defects (less than 3 mm), and has been found to enhance other procedures for cartilage healing [1,4,14]. Injections of various pharmacologics, such as growth factors (transforming growth factor- 1, insulin-like growth factor-1 and bone morphogenic proteins) and corticosteroids, have been attempted as a means to increase chondrocyte proliferation and matrix deposition. However, corticosteroids have produced ambiguous results and growth factors can also stimulate the formation of osteophytes that can be quite painful. Therefore, more research should be done to determine which pharmacologics should be administered and in what doses before this treatment will be e!ective .
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188.8.131.52. Cell transplantation Cell suspensions. Another option is transplantation of chondrocytes or undi!erentiated cells to restore lost tissue mass. An optimal approach would be to take a small biopsy of cells (progenitor or di!erentiated chondrocytes) expand the number of cells, and then return them to the defect once the proper mass has been generated. Implantation of individual chondrocytes has been researched since 1968, but the success rate has been less than 40%, due to the problem of retaining the cells in the defect for a length of time that would allow them to begin to produce matrix [20,21]. However, recently, a surgical method has been developed which uses a periosteal #ap sutured over the defect as a barrier beneath which cultured autologous chondrocytes are injected. The cells remain in the defect and this technique has shown promise in human trials conducted over the past 12 years in Sweden and the United States. A small biopsy of autologous tissue is obtained during arthroscopic evaluation of the joint and is cultured for approximately three weeks, resulting in a 10}12-fold increase in cell number. During the surgical procedure, the periosteum (cambium layer toward the defect) is attached with sutures and "brin glue to healthy cartilage near the lesion and the cultured cells are injected beneath it [18,22,23]. In the initial study, 14 of 16 patients with defects in the femoral condyle had good results after 2 yr, while healing in patellar defects was less encouraging (two of seven had good outcomes) . Since that time, further studies have corroborated these results. A US study begun in 1995 reports graft failure in only 7% of 70 patients and indicates that this procedure may hold promise to repair joints with multiple lesions . In a long-term study, 30 of 31 patients maintained good results over 5}10 yr in Sweden . Despite these encouraging results, this procedure, although clinically available in the US and other countries, should not be regarded as a panacea. As indicated above, the best results are found for healing lesions in the femoral condyle  and the need for a periosteal #ap requires a donor site. Interestingly, although short-term (three months) studies with rabbits reported healing similar to that found in humans , long-term results in canines have not been as promising. Although initially the procedure described above caused cartilage regeneration in canine knees, this was not permanent, and 12}18 months after surgery, the cartilage began to degrade . In another study , at 12}18 months, no di!erence was observed between the healing of control defects, defects with addition of a periosteal #ap alone, or with a #ap and autologous chondrocytes. Because the canine cartilage repair process seems very similar to that of the human , explanations for these discrepancies are yet to be determined.
Cells in scawolds. Alternatively, instead of tissue #aps, highly porous sca!olds may be used to maintain di!erentiated cells in a given area. This design is optimal because it signi"cantly reduces donor site morbidity and, in addition to simply providing a boundary for retention of cells, the sca!old also acts as a substrate to which the anchorage-dependent chondrocytes can adhere . When cultured two-dimensionally, such as in Petri dishes, these cells dedi!erentiate, assuming a more #attened appearance and producing Type I instead of Type II collagen. However, when grown in three dimensions, chondrocytes maintain their di!erentiated phenotype and function. In this way, sca!olds can encourage the proliferation of chondrocytes without sacri"cing important functions to dedi!erentiation . Sca!olds have also been used without seeded chondrocytes as a technique for regeneration enhancement by encouraging cell migration [30,31], but these studies are not reviewed here. Please refer to the recent article by Hunziker  for further treatment of this subject. Various sca!old materials have been tested, including both naturally derived and synthetic polymers. (For more complete listings, see [11,14,15]). This review will not discuss all of these materials, but rather focus on those that have been most extensively used in tissueengineered constructs. Although recent work with hyaluronic acid-based carriers is promising [32,33], the natural polymer that has received the most attention is collagen. In 1983, it was found that chondrocytes maintain di!erentiated phenotype and GAG production for six weeks in collagen gels . Since that time, researchers have continued to show encouraging results from use of these sca!olds. A key advantage is that, because collagen can be recognized by cellular enzymes, it can be remodeled and degraded to provide space for the growing tissue . Collagen matrices have also been found to have the proper molecular cues to stimulate new collagen production by transplanted cells as compared with other sca!old types . Caplan et al. reported that when MSCs were seeded into collagen gels implanted in osteochondral defects in rabbits, embryogenesis was recapitulated and both bone and hyaline cartilage were formed, although mechanical properties of the regenerated tissue were signi"cantly less than normal values, and there was some evidence of degeneration after 24 weeks [37,38]. Although preliminary results are promising for naturally derived polymers, concerns about the feasibility of "nding large amounts of these materials for clinical applications, as well as assurance of pathogen removal has prompted other researchers to investigate the use of synthetic polymers. These materials can be easily massproduced and their properties can be tailored for speci"c applications. This includes creating degradable polymers, which like collagen, allow room for tissue growth in the construct and eliminate the need for a second surgery
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to remove the implant. There is also the possibility that growth factors or other pharmacologics could be included in the sca!old to encourage certain cellular responses, such as proliferation or di!erentiation. However, few of these polymers have been FDA approved for use in humans . Much current research has focused on chondrocyte interaction with those biodegradable polymers that are FDA approved: poly(glycolic acid) (PGA), poly(L-lactic acid) (PLLA) and their copolymer, poly(DL-lactic-coglycolic acid) (PLGA) [36,39}46]. They are poly( -hydroxy esters) and are degraded by hydrolysis. PLLA is more hydrophobic and less crystalline than PGA and degrades at a slower rate . Firm hyaline-like cartilage has been observed after six weeks when undi!erentiated perichondrial cells were seeded onto PLLA meshes and implanted in the femoral condyles of rabbits [42,43]. Similar cartilage morphology was found using PGA porous non-woven sca!olds seeded with bovine chondrocytes and cultured in vitro for 12 weeks. In this case, mechanical properties such as compressive modulus and aggregate modulus were similar to that of normal bovine cartilage . Both types of degradable polyester tended to increase proteoglycan synthesis compared to a collagen sca!old . Cell growth was approximately twice as high initially (less than two months) on PGA than PLLA matrices when seeded with bovine chondrocytes, but after six months, total cellularity was found to be similar. Initial di!erences were attributed to the fact that PLLA degrades much more slowly and so left less space for cell proliferation . PLLA has been found to be less toxic to human chondrocytes than PGA in studies maintaining a constant pH over 12 days . A main disadvantage of all of these sca!old materials is that they require an operation for implantation. This has led to the development of polymers that can be injected along with cells and then cross-linked in situ to form matrices. Several investigators are exploring the option of combining "brinogen and thrombin to form a degradable "brin mesh that can be used to support chondrocytes [47}49]. When the cell}"brinogen}thrombin mixture was injected into defects in horses, more GAGs, aggregan and type II collagen were present in the new tissue at eight months than in defects that were left untreated . Alginate and bovine articular chondrocytes, injected into nude mice and crosslinked with calcium, also formed cartilage, but studies longer than 12 weeks have not been conducted to determine if there is any long-term tissue degradation [29,50,51]. Some forms of alginate have been found to be immunogenic, as evidenced by increased lymphocyte number and presence of anti-alginate antibodies . A further concern about the biocompatibility of the material is that, according to Cao et al., there is more in#ammatory response to alginate than to injectable synthetic materials . Synthetic polymers, such as poly(ethylene oxide) (PEO) or
copolymers of ethylene and propylene oxide P(EO-coPO), have also been tested as injectable matrices for chondrocyte transplantation. Sims et al. reported that 12 weeks after subcutaneous delivery of bovine chondrocytes in a PEO gel into nude mice, cartilage that was biochemically similar to natural bovine cartilage had been produced . In a comparative study, when seeded with autologous chondrocytes and injected in pigs, the P(EO-co-PO) copolymer was found to produce more cartilage-like tissue than either PGA sca!olds or alginate hydrogels. In this case, however, it must be noted that the chondrocytes were of aural rather than articular origin . Types of cells used. Certain polymer properties, such as degradation time, may be dictated by the type of cells to be used in the sca!olds. As evidenced in the above examples, both fully di!erentiated chondrocytes and progenitor cells have been employed to produce hyaline-like cartilage. Chondrocytes are very easy to obtain, but it has been found that as the cells are cultured for longer periods before implantation, the cartilage formed is increasingly "brous in nature . Also, according to Caplan et al., chondrocytes implanted in osteochondral defects in rabbits did generate cartilage, but a subchondral plate was only observed if undi!erentiated MSCs were used [20,38]. MSCs can be expanded many-fold with little e!ect on the tissue that is eventually formed and have been shown to di!erentiate into both bone and cartilage cells, making them prime candidates for transplantation in tissue-engineered constructs [38,55}57]. With either type of cell chosen, good sca!old seeding is required for the formation of functional cartilage. Seeding density is important since it has been found that cells seeded too sparsely result in incomplete "lling of the sca!old, leading to the possibility of "brous ingrowth which would adversely a!ect the properties of the tissue generated [58,59]. In addition, surface tension can prevent liquid media with cells from entering some synthetic sca!olds, such as PGA and PLLA. Therefore, these meshes must be pre-wet with alcohol which is then displaced by media to obtain seeding deep within the construct . Other techniques to improve seeding include dipping the sca!olds in poly(L-lysine) or type II collagen to improve initial cell attachment . If materials that set in situ are used, in addition to good cell seeding, the cytotoxicity of the reagents and temperature change during setting must be minimized to promote cartilage formation in the construct [28,60].
5. Bioreactors for chondrocyte and cartilage tissue growth In the past ten years, there have been signi"cant advances in growing cartilage with properties similar to those found in vivo, but to transfer this technology to a clinical setting, scale-up remains a large problem. Both
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methods to mass-produce the needed biomaterials, and large-scale production of cells must be developed. Because synthesis is very speci"c to the material chosen (natural vs. synthetic), only large-scale expansion of cells is reviewed here. It is estimated that to resurface an entire joint would require an engineered cartilage implant of the size 5 cm in diameter (1}5 mm thick), while current laboratory techniques test constructs only approximately 5 mm in diameter . High-density cell cultures can be used to prevent chondrocytic dedi!erentiation that often occurs in two-dimensional cultures [62,63], but bioreactors present numerous advantages for growing large amounts of tissue quickly . These advantages include that bioreactors provide uniform mixing and precise control over mass transfer rates, facilitating maintenance of nutrient levels and pH. In addition, shear stress can be easily monitored in many types of reaction vessels . It has been found that shear stress can have a signi"cant impact on the morphology and thus the mechanical properties of cartilage, so regulation of this parameter is essential for quality control of the product . Using process control techniques, the #ow rates in the reactor can be modulated to maintain important parameters (i.e. nutrient concentration) over time as the construct becomes less permeable due to matrix deposition and increased chondrocyte number . 5.1. Spinner yasks
ance of the di!erentiated chondrocytic phenotype [64,66]. 5.3. Perfusion culture While the above types of bioreactors show favorable results, there is concern that the mechanical mixing may induce unwanted shear gradients in the reactor vessel. To eliminate this possibility, microcarriers can be placed in #uidized bed or air-lift reactors where mixing is achieved by #uid recirculation rather than impeller motion . Another simple design to improve mass transfer while eliminating mechanical mixing is the perfusion culture system. Sca!olds are seeded with cells statically and subsequently hooked to a perfusion system that uses a peristaltic pump to deliver a constant #ow of media to the culture chamber containing the constructs. The media then exits through another port that empties into a waste vessel. Good matrix formation and mature chondrocytic phenotype was observed after two weeks in culture  and, after nine days, the cellularity in test constructs was higher than static controls . In a long-term study, tissue with histological and mechanical characteristics similar to native tissue was evident after 50 days of perfusion . Similar experiments using marrow stromal cells indicate that perfusion enhances growth and function over two weeks . However, currently, this system is only useful for three-dimensional expansion of cells in sca!olds. 5.4. Rotating-wall bioreactors
One of the simplest bioreactor designs is the spinner #ask. Typically, sca!olds seeded with chondrocytes are attached to needles hanging from the stopper of the #ask. Media is added to cover the sca!olds and mixing is maintained with a magnetic stir bar in the bottom of the #ask. Media is changed every few days to insure high nutrient concentration. Studies by Freed et al. showed that constructs cultured in spinner #asks were larger and contained more cells after "ve weeks than those grown in Petri dishes . 5.2. Microcarriers To culture individual cells (not seeded in sca!olds) in a bioreactor, microcarrier suspensions can be used. Various types of microcarrier beads, such as collagen or dextran, have been tested. These carriers are suspended in a reactor with continuously stirred media and an inoculum of chondrocytes is added. As in the spinner #asks, the media is exchanged every few days. Cell doubling times were shown to be around two to three days in these bioreactors as compared up to "ve days in Petri dishes, suggesting that the increased mass transfer in the bioreactor encouraged cell growth. In addition, the microcarrier suspension appeared to enhance mainten-
A promising bioreactor design that can be used with either microcarriers or sca!olds without the concern of mechanical mixing is the rotating-wall vessel reactor originally designed by NASA to simulate the e!ects of microgravity. The most common type is the slow turning lateral vessel (STLV). The reactor is comprised of two concentric cylinders. The stationary inner cylinder has a membrane that allows gas exchange while the outer cylinder, made from a non-permeable material, rotates. The space between the two cylinders is perfused continuously with media. Pre-seeded polymer constructs or chondrocytes and microcarriers are placed inside the reactor and are kept in a state of constant free-fall by adjusting the speed of rotation of the outer cylinder so that the centrifugal force just balances the force of gravity and the #uid drag on the objects inside. Mixing occurs due to the small amount of unavoidable settling which creates movement of the sca!old/microcarrier relative to the media [61,65]. With microcarrier suspensions, the STLV provided an environment that encourages aggregation of chondrocytes to form tissue. This may be a means to grow larger sections of cartilage that could be placed directly into a defect without the use of an implanted matrix to
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retain the cells . Using sca!olds, when grown in the STLV for "ve weeks, cell}polymer constructs showed more GAG production than those placed in spinner #asks . In further experiments, the STLV constructs were found to have 68% as much GAG, 33% as much collagen and similar cellularity to natural cartilage . However, while these constructs demonstrated better mechanical properties than those grown in other conditions (static and modi"ed spinnner #ask), the equilibrium modulus, dynamic sti!ness and streaming potential were only 20}25% as high as fresh cartilage explants . In order to improve the quality of cartilage produced, researchers have begun to determine optimal operating parameters for the STLV bioreactor. When rates of oxygen exchange were varied, it was discovered that, although cartilage is generally regarded as a hypoxic tissue, higher rates of oxygen tension resulted in larger tissue constructs from pre-seeded polymer sca!olds. These constructs also showed higher amounts of ECM synthesis than those grown at a lower oxygen concentration . However, the STLV may be replaced by a larger rotating-wall vessel, the high aspect ratio vessel (HARV), to create the large constructs needed for joint resurfacing. Because of the size and design di!erences, the HARV has "ve times as much surface area for gas exchange per unit of reactor volume as the STLV. This increase in surface area may be especially important given the previous "ndings relating construct size to oxygen tension. This vessel has been used to culture constructs up to 10 cm in diameter . 5.5. Further development
an optimal polymer for use in the sca!old. Also, the type of cells (di!erentiated or progenitor) that are best for this application has yet to be decided. It is the interaction of these two parameters (material and cells), along with the possible addition of growth factors and/or mechanical stimulation to in#uence cellular development, which will determine the ultimate success of the implant. As these issues are considered, long-term results are of utmost concern: no current experimental strategy has succeeded in consistently producing cartilage that shows no signs of degeneration after one year. Structure at these late time points is also extremely important. None of the studies reviewed here have created a tissue that fully regenerates the natural zonal organization of articular cartilage, even after several months in vivo. A "nal hurdle to full cartilage regeneration is the integration of any newly formed tissue with the existing cartilage. Without this integration, forces that occur at the interface could have deleterious long-term e!ects on the replacement tissue. Despite considerable work that must be done to optimize parameters for formation of tissue-engineered cartilage, because this tissue is not vascularized and incorporates only one cell type, it is likely the next tissue after skin to be successfully regenerated. Further development of techniques for chondrocyte transplantation would be an incredible advance in the "eld, providing hope for many patients that must endure chronic joint pain due to failure of conventional methods to restore cartilage function. Acknowledgements
Many in vitro studies have shown that mechanical forces greatly in#uence the development of cartilage (see  for a review of the e!ects of mechanical forces) . Recently, researchers have begun to combine the above culture systems with external mechanical stimuli to improve the quality of the cartilage produced. Carver et al. recently described a perfusion system which applies intermittent hydrostatic pressure to chondrocytes imbedded in polymer sca!olds. The pressurized sca!olds demonstrated cartilage development with higher GAG content after "ve weeks than in samples that were not exposed to pressure .
The work on skeletal tissue engineering has been supported by the National Institutes of Health (R29AR42639, R01-AR44381, and R01-DE13031). J.S. Temeno! acknowledges the "nancial support of a Whitaker Foundation Graduate Fellowship. References
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