Articular Cartilage

45 Articular Cartilage Francois Ng kee Kwong and Myron Spector

INTRODUCTION Types of Articular Cartilage Defects That Present in the Clinic Cartilage defects are a common source of pain and/or loss of function in patients presenting to the orthopedic clinic. While, any joint can be affected, the joint most commonly affected is by far the knee. A chondral lesion was found in 63% of a large series of over 31,000 arthroscopic procedures performed in patients with a symptomatic knee (Curl et al., 1997). Articular cartilage damage is often associated with meniscal and anterior cruciate ligament injuries (Shelbourne et al., 2003). These defects can be divided according to their etiology or morphology. Focal injuries typically occur as a result of a sporting injury and hence tend to affect the younger population. Focal defects can be further subdivided into chondral or osteochondral lesions, depending on the depth of the defect. Chondral lesions, also known as partial thickness lesions, lie entirely within the cartilage and do not penetrate into the sub-chondral bone. In the adult, defects of this nature do not regenerate because of the lack of cells which could participate in the repair process. Osteochondral defects penetrate through the vascularized sub-chondral bone and some spontaneous repair occurs as mesenchymal chondroprogenitor cells invade the lesion and form cartilage. However, full-thickness defect repair is only transient and the novel tissue formed does not have the functional properties of native hyaline cartilage (Shapiro et al., 1993). On the other hand, degenerative chondral changes typically occur in the older population as a result of arthritic changes. They often involve a large area of the affected joint, but start off as a focal lesion initially. Rationale for Cell Therapy Articular cartilage has a limited capacity for self-regeneration after injury. This was recognized as early as in 1743 by Hunter who stated that cartilage “once destroyed is not repaired.” This is because none of the normal inflammatory and reparative processes of the body are available to repair the tissue. This itself is a result of its isolation from the systemic regulation, lack of blood vessels, and nerve supply (Mankin, 1982). Furthermore, chondrocytes which are surrounded by an extracellular matrix cannot freely migrate to the site of injury from an intact healthy site, unlike most tissues (Buckwalter and Mankin, 1998), and there is no provisional fibrin clot filling the defect into which cells can migrate. Full-thickness defects induce mesenchymal chondroprogenitor cells to differentiate into repair tissue, but this is predominantly fibrous in nature and degenerates with time. The two major problems that need to be addressed in repair of articular cartilage are the filling of the defect void with a tissue that has the same mechanical properties as articular cartilage and the promotion of successful integration between the repair tissue and the native articular cartilage and calcified cartilage. Even

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a small defect caused by mechanical damage will fail to heal and degenerate over time progressing to osteoarthritis (OA). Conventional surgical techniques of cartilage repair are partially successful in alleviating symptoms, but fail to regenerate tissue anywhere similar in nature to native articular cartilage. There was no promising solution to this problem until Brittberg et al. introduced a cell-based therapy in which culture-expanded chondrocytes were transplanted into defects, raising the expectations of a breakthrough in repairing damaged articular cartilage (Brittberg et al., 1994). Current Cell Therapies Available in the Clinic The possible cell-based tissue repair techniques can be broadly classified into three major categories: (1) targeting local connective tissue progenitors where new tissue is desired, (2) transplanting culture-expanded or modified connective tissue progenitors, and (3) transplanting fully formed tissue generated in vitro or in vivo. In current clinical practice, the first two techniques are already in use while the last one is being actively investigated in animal models and pre-clinical trials. These techniques are generally aimed at delivering chondrogenic cells to the cartilage defect, either in the form of tissues containing precursor cells (e.g. the periosteum or perichondrium) or in the form of autologous chondrocytes isolated from a biopsy of healthy cartilage and expanded in number in vitro. Periosteal Transplantation Rubak initially described this technique in a rabbit model of cartilage defect (Rubak, 1982). He used a periosteal flap to cover the defects. The defects were repaired and filled after 4 weeks with a hyaline-like cartilage whereas the empty control defect showed fibrocartilage-like repair tissue. The first clinical study was published by Niedermann et al. who reported successful results in all of their four initially treated patients (Niedermann et al., 1985). Perichondrial Transplantation Autologous perichondrium has also been employed for cartilage repair (Homminga et al., 1989, 1990, 1991). Perichondrium, taken from the cartilaginous covering of the rib, is placed into the chondral defect of the affected joint. The first clinical study of this approach was performed by Homminga et al. (1990). A major shortcoming of perichondrial grafting is the limited availability of large grafts. Graft size is limited to the rib size, so that several rib perichondrial grafts have to be harvested to fill a large defect. Additionally, endochondral ossification and delamination of the cartilage from the sub-chondral bone plate are potentially significant limitations to the long-term efficacy of this repair. Autologous Chondrocyte Implantation Since first published in 1994 (Brittberg et al., 1994), techniques of cell isolation, expansion in culture, and implantation have remained essentially the same. Cartilage (150–300 mg) is harvested arthroscopically from a minimally load-bearing area of the upper aspect or the medial condyle of the affected knee. The biopsy is then transported to a laboratory facility using a transport media. Chondrocytes are isolated using standard techniques. After a certain period of cell expansion (11–21 days (Peterson et al., 2000), depending upon the growth kinetics) a certain number of cells (e.g. minimally 12 million for Genzyme’s Carticel procedure) are provided in a serum-free and gentamycin-free transport medium.

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Using a medial or lateral parapatellar incision, the defect is debrided to the level of normal-appearing surrounding cartilage. The integrity of the tidemark needs to be maintained in order to avoid infiltration of undifferentiated mesenchymal stem cells (MSCs) which could contribute to the formation of fibrocartilagenous repair tissue (Brittberg et al., 1999). A periosteal flap is harvested from the anterior aspect of the proximal tibia or distal femur, formed to the shape of the lesion, and sutured to the rim of the defect. The chondrocyte suspension is subsequently injected under the periosteal flap and the border of periosteal cover sealed using fibrin glue. Post-operative rehabilitation protocols generally involve continuous passive motion and limited weight bearing for an extended time. Cooperation of the patient in this respect is essential for a favorable outcome, hence difficult to control. This contributes to difficulty in evaluating outcome data. In a variation of this technique, porcine type I/III collagen membrane has been used in place of the periosteal membrane (Bartlett et al., 2005). Its outer surface is smooth, giving a low-friction surface. Its inner surface is rough because of large gaps between collagen fibers into which chondrocytes can be seeded.

CELL THERAPIES An optimal cell source should have the following characteristics: no immunorejection, no tumorigenicity, immediate availability, availability in pertinent quantities, controlled cellular proliferation rate, predictable, and consistent chondrogenic potential as well as controlled integration into the surrounding tissues. Autologous versus Allogeneic An autologous source of stem cells is most desirable as cells are collected from each patient, thereby eliminating complications associated with immune rejection of allogeneic tissue. Even with an autologous system, challenges exist in assuring a safe and reproducible product. Genzyme established a quality assurance program based on US FDA Good Manufacturing Practice regulations, which was reviewed recently (Mayhew et al., 1998). Process variables have to be controlled rigorously and sterility testing and endotoxin testing maintained. Moreover, assessments of cell viability and growth kinetics are a crucial part of non-conformance reporting. According to Genzyme data, 1.64% of the cartilage biopsies received were contaminated (Mayhew et al., 1998). Contamination was recorded only for 0.03% during processing and in 0.16% at release. Endotoxin content ranged between less than 0.15 and 0.5 EU/ml (allowable limit 82.5 EU/ml) and cell viability was 90.9
4.06% at release. Measurement of growth kinetics revealed 0.311 doublings per day. Out of 1377 cartilage biopsies, 86 non-conformances were identified related to biopsy quality, only 12 were related to cell processing. Limitations of the autologous approach in obtaining stem cells and the desire to obtain “marketable products” which could benefit as many patients as possible have provided incentives for the development of generic cell lines, which can be taken off the shelf as, and when, needed for patient treatment. These universal cells would have the following advantages: (i) availability through the development of large cell banks; (ii) consistency and efficacy because only cells with desirable characteristics and controlled critical parameters are selected and amplified; and (iii) sterility and assurance of compatibility through extensive safety testing. Until recently, it was difficult to envision utilization of allogeneic generic cells in orthopedics as it was believed that their transplantation would require immunosuppressive drugs to reduce associated risks of rejection. However, cultured MSCs exhibit a poorly immunogenic phenotype (Tse et al., 2003). In vivo, a single intravenous administration of MSCs led to a modest, but significant, prolongation of skin graft survival (Bartholomew et al., 2002). These data have greatly enhanced the therapeutic appeal of MSCs because they raised the possibility of creating universal cell lines. Indeed, allogeneic adult stem cells are already being investigated in patients with meniscal injuries, in a phase 1 FDA approved clinical trial (http://www.osiristx.com/).

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Intra-operative versus Culture Expanded Intra-operative cell-based therapies have the advantage of being less time consuming and less costly than ex vivo therapies. Ex vivo therapies also have the disadvantage of involving an additional harvesting step. The advantage of an ex vivo technique is that the surgeon can select specific cells (i.e. bone marrow cells or stem cells) and the cellular delivery vehicle for specific clinical problems. It is also safer than an in vivo strategy when working with viruses for gene therapy because no viral particles or DNA complexes are injected directly into the body. In addition, ex vivo strategies have a high efficiency of cell transduction. Articular Chondrocytes Methods for Intra-operative Cell Therapy Osteochondral transplantation has been used clinically for more than 25 years. Large osteochondral allografts have been employed for orthopedic tumor surgery and to a lesser extent for repairing degenerative defects. However, for smaller defects these procedures introduced significant morbidity. More recently osteochondral autografting has been introduced into the clinic as an alternative treatment for small and medium sized defects. Promising reports by Matsusue and Bobic have fueled interest in this method (Matsusue et al., 1993; Bobic, 1999). With this technique an osteochondral plug is harvested from a lower weight-bearing area of the knee joint and transferred to the prepared defect, implanted using a press-fit technique. Culture-Expanded Cells The rationale for using articular chondrocytes for a cell-based therapy is that they already possess the desired phenotype. Chondrocytes comprise the single cellular component of adult hyaline cartilage and are considered to be terminally differentiated, thus being highly specialized. Their main function is to maintain the cartilage matrix, synthesizing-types II, IX, and XI collagen; the large aggregating proteoglycan, aggrecan; the smaller proteoglycans, biglycan and decorin; and specific and non-specific matrix proteins that are expressed at defined stages during growth and development. Freshly isolated articular chondrocytes continue to exhibit their specific phenotype in culture for at least several days to weeks. This makes them a suitable cell type for a cell-based treatment of chondral defects. While the steps involved in the isolation and expansion of articular chondrocytes for autologous chondrocyte implantation (ACI) are quite similar among various commercial and academic laboratories, there may be important differences. One such difference is the use of the patient’s own serum for culturing the cells, as described originally by Brittberg et al. (1994). One commercial enterprise, Genzyme Biosurgery (Cambridge, Massachusetts, USA), uses approved and validated fetal bovine serum (FBS), instead of the patient’s serum, in the culture media. Another potentially important difference is that Genzyme needs to freeze and store the isolated cells in order to allow for verification of adequate insurance coverage prior to the implantation procedure. A recent study has indicated that this freeze-thaw cycle may adversely affect the outcome of the procedure (Perka et al., 2000). Cryopreserved chondrocytes seeded into polymer scaffolds yielded an 85% repair of an osteochondral defect in rabbits, whereas 100% of the defects treated with noncryopreserved cells were filled. One of the disadvantages of employing articular chondrocytes is that they do not readily proliferate in vitro. Cells from a younger population have been found to undergo 0.3 doublings per day, using a standardized and validated approach for culturing cells for later implantation (Mayhew et al., 1998). Even lower proliferation rates are obtained in older patients and arthritic cartilage (Peterson et al., 2000). Another report demonstrates the rapid replicative senescence of articular chondrocytes (Martin and Buckwalter, 2001). Once chondrocytes are deprived of their three-dimensional environment, their phenotype switches to a more

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fibroblastic cell form, expressing types I and III collagen, instead of cartilage-specific type II collagen (Goldring et al., 1986, 1988; Saadeh et al., 1999). Decades ago (in 1922) it was reported that the removal of articular cartilage from the joints of rabbits led to the formation of bone around the margins of the joint (Fisher, 1922). Fisher suggested that the results were due to reactive changes brought about by the removal of cartilage from the joint and not due to a response to necrotic cartilage in the joint. This early study was followed up by another in which osteochondral defects were created in the patella surface of the femurs of rabbit knee joints (Key, 1931). In some instances “the operation was followed by a severe chronic, progressive arthritis which involved not only the femur, but also the tibia and patella.” The author noted that “the most interesting changes were the hyperplastic phenomena which occurred in the lower end of the femur. These changes were not continuous with or even adjacent to the defect, but occurred in the non-traumatized portions of the lower end of the femur, and in many instances both the patella and the upper end of the tibia were also involved.” These changes were present to some degree in every joint. The author noted that the experiments prove “that many of the changes which occur in the hypertrophic arthritis can be produced experimentally in the joints of rabbits by simply creating a defect in the cartilage and that these changes are not dependent upon the presence of dead cartilage within the joint.” Our own studies demonstrated in a canine model that the harvesting of articular cartilage predisposes the other cartilage in the same joint to changes associated with early OA (Lee et al., 2000). While the lesion itself in a knee joint may serve to induce such osteoarthritic changes in the joint, the additional surgical procedure of harvesting cartilage may exacerbate the condition. There is, then, a compelling need for an alternative cell source for a cell-based cartilage repair procedure. MSCs MSCs isolated from the bone marrow and other sources can provide an alternative and abundant supply of cells for cartilage repair procedures. Adult marrow stromal cells are being investigated for the treatment of defects in connective tissues using cell and gene therapy and tissue engineering approaches – see for reviews (Caplan, 1991; Prockop, 1997). Differentiation of such cells can be obtained in vitro by changing the culture conditions after their expansion or in vivo as a consequence of the new “physiological” microenvironment in the transplant area. Whole Marrow Implants Safety of Whole Marrow Injected/Implanted in Human Subjects Whole autologous and allogeneic bone marrow has been injected and implanted into human subjects for decades to treat myriad medical problems with no adverse events associated with the MSC sub-population present. Of note, for example, is the procedure in which up to 1 liter of whole bone marrow is routinely infused into the bone marrow transplant patient. This infusion contains a small but significant proportion of MSCs and does not seem to have any significant side-effects. In an example of one such study in which the MSC population of whole marrow was to provide the principal therapeutic effect (Horwitz et al., 1999), non-manipulated bone marrow from HLA-identical or singleantigen-mismatched siblings was intravenously infused into three children with severe deforming osteogenesis imperfecta after they had received ablative conditioning therapy. The nucleated cell doses ranged from 5.7 to 7.5  108 cells/kg. All three showed engraftment with hemopoietic donor cells. Improvements in clinical outcome were associated with increases in growth velocity and reduced frequencies of bone fracture. The authors concluded that “allogeneic bone marrow transplantation can lead to engraftment of functional

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mesenchymal progenitor cells, indicating the feasibility of this strategy in the treatment of osteogenesis imperfecta and perhaps other MSCs disorders as well.” There were no reports of adverse response to the marrow infusion. For decades an array of “marrow stimulation” techniques, including abrasion arthroplasty, drilling, and micro-fracture, have been used to treat cartilage defects. Each of these methods introduces marrow-derived MSCs into the joint. While these procedures have not yielded lasting symptomatic relief they demonstrate that the presence of endogenous bone marrow-derived MSCs in the joint does not lead to adverse clinical sequelae. Since the early days of bone grafting autogenous marrow has been known to be of value in improving the osteogenic response (Salama et al., 1973). Whole autogenous marrow has been implanted in various sites in the body with no untoward clinical findings. In more recent years bone marrow and bone marrow fractions including the stromal cell population have been injected percutaneously to treat non-unions in human subjects (Connolly et al., 1998). There have been no adverse events reported. An apparatus has become commercially available (Select, DePuy Acromed Inc,) for the intra-operative concentration of MSCs/osteoprogenitor cells from whole marrow.

Efficacy of Whole Marrow for Cartilage Repair in Pre-clinical Animal Studies

The rationale for the benefits to be derived from MSCs also draws from investigations demonstrating the contribution of whole marrow to cartilage repair. In one such study (Solchaga et al., 2002), autologous bone marrow incorporated into a fibronectin-coated hyaluronan-based sponge was implanted into 3-mm diameter osteochondral defects in a rabbit model. Control groups were implanted with the scaffold alone. Except for the 3-week specimens, the histological appearance of the defects was similar in both groups. “Four weeks after surgery, the defects were filled with bone with a top layer of cartilage well integrated with the adjacent cartilage. At each harvest time, the overall histological scores of the specimens did not reveal statistical differences between the treatment groups. However, as revealed by the results of the 3-week sacrifices, bone marrow loading appeared to accelerate the first stages of the repair process.” Coagulated bone marrow aspirates have been used together with gene therapy techniques in a rabbit model of cartilage defect (Pascher et al., 2004). Mixture of an adenoviral suspension with the fluid phase of freshly aspirated bone marrow resulted in uniform dispersion of the vector throughout and levels of transgenic expression in direct proportion to the density of nucleated cells in the ensuing clot. Furthermore, cultures of MSCs previously transduced ex vivo with recombinant adenovirus were readily incorporated into the coagulate when mixed with fresh aspirate. These vector-seeded and cell-seeded bone marrow clots were found to maintain their structural integrity following extensive culture and maintained transgenic expression in this manner for several weeks. These genetically modified bone marrow clots were able to generate similarly high levels of transgenic expression in osteochondral defects with better containment of the vector within the defect. In a rodent pre-clinical model, Gurevitch and colleagues demonstrated that implantation of a composite comprising demineralized bone matrix and a bone marrow cell suspension in a damaged area of a joint resulted in the generation of a new osteochondral complex comprising articular cartilage and sub-chondral bone (Gurevitch et al., 2003). In the same study, the authors implanted the same composite material into an ablated bone marrow cavity and a calvarial defect (Gurevitch et al., 2003). The resulting tissue formed was respectively trabecular bone and stromal microenvironment supporting hematopoiesis and flat bone, respectively. They concluded that the new tissue formation followed differentiation pathways controlled by site-specific physiological conditions, thus developing tissues that precisely met local demands.

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Methods for Culture Expansion Sources: Bone Marrow, Fat, Muscle Use of Bone Marrow as the Source of MSCs: Other Sources of MSCs Human MSCs which have been reported to be present in bone marrow, adipose tissues, dermis, muscles, and peripheral blood (Young et al., 2001) and other connective tissue (Zohar et al., 1997) have the potential to differentiate along different lineages including those forming bone, cartilage, fat, muscle, and nerve. Several studies have compared the characteristics of MSCs from these different sources. One such study (Lee et al., 2004) compared phenotypes and gene expression profile of the human adipose tissue-derived stromal cells (ATSCs) in the undifferentiated states with bone marrow-derived MSCs. Both cell types expressed CD29, CD44, CD90, CD105 and were absent for HLA-DR and c-kit expression. The study confirmed that the marrow-derived MSCs were inducible to differentiate into osteoblasts, adipocytes, and chondrogenic lineages. While the results showed that ATSCs were superior to marrow-derived MSCs with respect to maintenance of proliferating ability, “the proliferating ability and differentiation potential of ATSC were variable according to the culture condition.” That the phenotypes and the gene expression profiles of ATSCs and marrow-derived MSCs were found to be similar may not provide enough of a compelling argument for the use of ATSCs, particularly because of the fact that there are many more safety and efficacy studies of marrow-derived MSCs compared to ATSCs. Culture Procedures

MSCs represent a minor fraction of the total nucleated cell population in the marrow. They can be plated and enriched using standard cell culture techniques. Frequently, the whole marrow sample is subjected to fractionation on a density gradient solution such as Ficoll, after which the cells are plated at densities ranging from 1  104 cells/cm2 to 0.4  106 cells/cm2 (Pittenger et al., 1999; Lodie et al., 2002; McBride et al., 2003). Cells are generally cultured in basal medium such as Dulbecco’s modified Eagle’s medium (low glucose) in the presence of 10% FBS (Pittenger et al., 1999). MSCs in culture have a fibroblastic morphology and adhere to the tissue culture substrate. Primary cultures are usually maintained for 12–16 days, during which time the non-adherent hematopoietic cell fraction is depleted. Optimal expansion of MSCs from marrow requires the pre-selection of FBS. As MSCs are expanded in large-scale culture for human applications it will be important to identify defined growth media, without or with reduced FBS, to ensure more reproducible culture techniques and enhanced safety. Safety of MSCs in Animal Models The use of culture-expanded MSCs in animal models has recently been reviewed (Barry, 2003, #14598). Several studies have focused on the use of monolayer-expanded bone marrowderived MSCs as a renewable and readily accessible source for the treatment of infarcted cardiac tissue. Studies that have injected MSCs in mouse models of myocardial infarcts have not reported adverse effects (Orlic et al., 2001). In other work (Murphy et al., 2003) autologous culture-expanded MSCs were injected into the knee joints of goats in which OA was induced by complete excision of the medial meniscus and resection of the anterior cruciate ligament. Six weeks after induction of OA, a single dose of 10 million MSCs, suspended in a dilute solution of sodium hyaluronan, was delivered to the injured knee by direct intra-articular injection. Control animals received sodium hyaluronan alone. “In cell-treated joints, there was evidence of marked regeneration of the medial meniscus, and implanted cells were detected in the newly formed tissue. Degeneration of the articular cartilage, osteophytic remodeling, and sub-chondral sclerosis were reduced in cell-treated joints compared with joints treated with vehicle alone without cells.” “Animals tolerated the cell injection well, and there was no evidence of local inflammation, immobilization, or unloading of the joint resulting from the cell treatment.”

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Efficacy of MSCs for Cartilage Repair in Pre-clinical Animal Studies That MSCs may yield results comparable to autologous chondrocytes was supported by in vitro studies (Kavalkovich et al., 2002) that have demonstrated that MSC cultures undergoing chondrogenesis synthesize glycosaminoglycan (GAG) at levels significantly higher than explant cultures or primary chondrocyte cultures. Numerous studies in vivo (Caplan, 1991) have supported the supposition that these bone marrow-derived MSCs offer advantages over committed cells (i.e. differentiated cells such as articular chondrocytes) for cellseeded implants developed to facilitate tissue regeneration (e.g. articular cartilage) (Wakitani et al., 1994; Ponticiello et al., 2000). This strategic approach holds that regeneration can be facilitated by the recapitulation of certain phases of embryonic development, and that these stem cells will allow for such, whereas fully differentiated cells will not. Presumably the endogenous regulators in the implant site will serve to induce the implanted undifferentiated stem cells to differentiate along the desired pathway. In one study (Im et al., 2001) using mature rabbits, bone marrow-derived MSCs expanded in culture in monolayer were implanted into a full-thickness osteochondral defect artificially made on the patellar groove of the same rabbit. The semiquantitative histological scores were significantly higher in the experimental group than in the non-cell-treated control group (p  0.05). “In the experimental group immunohistochemical staining on newly formed cartilage was more intense for type II collagen in the matrix and reverse transcriptase-polymerase chain reaction (RT-PCR) from regenerated cartilage detected mRNA for type II collagen in mature chondrocytes. These findings suggest that repair of cartilage defects can be enhanced by the implantation of cultured MSCs.” In another animal study (Wakitani et al., 1994), autologous culture-expanded MSCs incorporated into type I collagen gels were transplanted into 3  6 mm full-thickness (3 mm in depth) defects in the weightbearing surfaces of the medial femoral condyles of rabbit knees. In the contralateral knee, the defect was filled with collagen gels without cells or the defect was left empty. The defect composed 40–50% of the weight-bearing surface of the condyle, “among the largest ever reported in repair studies in rabbits.” “Two weeks after the transplantation of the mesenchymal cells, the whole area of the original defect was occupied by cartilage. … Twelve weeks after the transplantation, the repair cartilage in the defect became a little thinner than the adjacent normal cartilage, which became a little thinner 24 weeks after the transplantation (the longest observation period in the study).” The authors concluded that large, full-thickness defects of the weight-bearing region of the articular cartilage could be repaired with hyaline-like cartilage after implantation of autologous mesenchymal cells. There were no untoward responses reported. In an animal study Zhou et al. implanted autologous culture-expanded MSCs into osteochondral defects in pigs (Zhou et al., 2004). The amount and make-up of the reparative tissue compared favorably to their prior ACI results using the same animal model. No untoward tissue reactions to the implantation of the MSCs were reported. Zhou et al. employed MSCs that were grown in a chondroinductive environment prior to implantation and their defects extended into sub-chondral bone. The fate (survival) of allogeneic marrow-derived and culture-expanded MSCs implanted in osteochondral defects was determined using transgenic rats (Oshima et al., 2005). An autologous transplantation model was simulated using transgenic rats – whose transgenes produce no foreign proteins – as donors, and wildtype rats as recipients. MSC masses were transplanted into osteochondral defects created in the medial femoral condyle of wild-type rats; the cell aggregates were fixed with fibrin glue. “Twenty-four weeks after transplantation, the defects were repaired with hyaline-like cartilage, which was thicker than normal, and with sub-chondral bone. Using the in situ hybridization technique, cells derived from the transplanted ones were detected within both the cartilaginous and the sub-chondral bone layers. … The findings indicate that the transplanted mesenchymal cells contributed to the repair of the osteochondral defects.”

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In another study (Yanai et al., 2005) bone marrow-derived culture-expanded MSCs were implanted into large full-thickness articular cartilage defects in rabbits that underwent joint distraction. The final cell density was adjusted to 5.0  106 cells/ml in a type I collagen gel. The histological scores were significantly higher in the groups with MSC–collagen gel implants. The authors concluded that the repair of large defects of cartilage can be enhanced by joint distraction, collagen gel, and MSCs. Characterization of Phenotype

Identification and Therapeutic Use of the Adherent Cell Population from Bone Marrow Numerous studies have investigated characteristics of the stromal cell population of marrow that includes the MSC (Barry and Murphy, 2004). Many of these studies have characterized the MSC on the basis of selected surface proteins (Barry et al., 1999, 2001; Reyes et al., 2001; Young et al., 2001; Gronthos et al., 2003). Related studies have attempted to isolate more purified sub-populations of MSCs using cell sorting for selected surface markers, including: positive CD105(+)/negative (CD45(–)GlyA(–) (Reyes et al., 2001); endoglin (Majumdar et al., 2003 and Stro-1) (Gronthos and Simmons, 1995). Related studies have focused on the effects of supplementation of the medium with selected growth factors on the characteristics of isolated sub-populations of MSCs (Gronthos and Simmons, 1995). In one recent study (Lodie et al., 2002), the properties of selected MSC sub-populations were compared: positive or negative selection with antibody to CD105 or CD45/GlyA. The results indicated that “in the initial stages of culture, each cell population proliferated slowly, reaching confluence in 10–14 days. Adherent cells proliferated at similar rates whether cultured in serum-free medium supplemented with basic fibroblast growth factor (Solchaga et al., 2005), medium containing 2% FBS supplemented with epidermal growth factor and plateletderived growth factor, or medium containing 10% FBS alone. Cell surface marker analysis revealed that more than 95% of the cells were positive for CD105/endoglin, a putative MSCs marker, and negative for CD34, CD31, and CD133, markers of hematopoietic, endothelial, and neural stem cells, respectively, regardless of cell isolation and propagation method. CD44 expression was variable, apparently dependent on serum concentration.” Of importance was the fact that this study found that there was similarity in the function of the various cell populations with each “expressing the cell type-specific markers beta-tubulin, type II collagen, and desmin, and demonstrating endothelial tube formation when cultured under conditions favoring neural, cartilage, muscle, and endothelial cell differentiation, respectively. On the basis of these data, adult human bone marrow-derived stem cells cultured in adherent monolayer are virtually indistinguishable, both physically and functionally, regardless of the method of isolation or proliferative expansion.” For the purpose of a cartilage repair therapeutic agent there are no data that indicate that any specific sub-population of MSCs would be safer and more efficacious than the entire adherent cell population. Other Cell Types: Synovial Cells Another tissue in which MSCs have been demonstrated is the synovial tissue (De Bari et al., 2001, 2003). De Bari and colleagues have demonstrated that stem cells isolated from periosteum can be expanded in vitro to over at least 15 passages without loss of their phenotypic traits and that the chondrogenic potential of these progenitor cells was independent of donor age.

CELL–SCAFFOLD IMPLANTS Owing to the now well established phenomenon of dedifferentiation of chondrocytes in monolayer culture, there has been increasing interest in three-dimensional systems of culture and delivery of cells to the

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chondral defect. These systems can provide an environment for growth more similar to native tissue and hence contribute to the phenotypic stability of the chondrocytes. A scaffold also provides an increased surface area for cell attachment. Choosing the right scaffold in cartilage repair requires consideration of a number of factors. Bell described the ideal scaffold for tissue engineering as one that provides a transitional framework whereby the cells populating it create a replacement tissue as the scaffolding material disappears (Bell, 1995). Ideally this scaffold should be degraded at the same rate that the cells produce their own framework. The following requirements are necessary for cartilage tissue engineering. The scaffold should: 1. support cartilage-specific matrix production (collagen type II and aggrecan). Our previous studies showed

that there is a considerable difference in performance among scaffolds, even if only changing the collagen type, pore size, or method of cross-linking. 2. provide enough mechanical support for early mobilization of the treated joint. 3. allow for cell migration of cells to achieve bonding to the adjacent host tissue. In a comparison of several matrix materials (polylactic acid, collagen gel, porous collagen), Grande et al. showed a marked variability of the chondrocyte response (Grande et al., 1997). Bioabsorbable polymers such as polyglycolic acid (PGA) enhanced proteoglycan synthesis, whereas collagen matrices stimulated synthesis of collagen. Not only is there a lack of clinical data on matrix applications for cartilage repair, there are only a few preclinical studies in larger animals. Most of the in vivo work has been done in rabbits and has shown comparatively favorable results (Grande et al., 1989; Kawamura et al., 1998; Ponticiello et al., 2000). However, few studies have systematically compared different methods in a larger animal model. Breinan et al. compared the effects of three different treatments on the healing of articular cartilage defects in a canine model previously developed for ACI (Breinan et al., 2000). In the articular surface of the trochlear grooves of 12 adult mongrel dogs, two 4-mm diameter defects were made to the depth of the tidemark. Four dogs were assigned to each treatment group: (i) micro-fracture treatment, (ii) micro-fracture with a type II collagen scaffold placed in the defect, and (iii) a type II collagen scaffold seeded with cultured autologous chondrocytes. After 15 weeks, the defects were studied histologically. Data quantified on histological cross sections included area or linear percentages of specific tissue types filling the defect, integration of reparative tissue with the calcified and the adjacent cartilage, and integrity of the sub-chondral plate. Total defect filling averaged 56–86%, with the greatest amount found in the dogs in the micro-fracture group implanted with a type II collagen matrix. The profiles of tissue types for the dogs in each treatment group were similar: the tissue filling the defect was predominantly fibrocartilage, with the balance being fibrous tissue. There were no significant differences in the percentages of the various tissue types among the three groups. Taking the results of these dog experiments together and comparing the different repair methods 15 weeks post-operatively, there was a significant correlation between the degree to which the calcified cartilage layer and sub-chondral bone were disrupted and the amount of tissue filling the defect. Moreover, when it formed, hyaline cartilage most frequently occurred superficial to intact calcified cartilage. Ochi et al. investigated the clinical, arthroscopic, and biomechanical outcome of transplanting autologous chondrocytes, cultured in atelocollagen gel, for the treatment of full-thickness defects of cartilage in 28 human knees over a minimum period of 25 months (Ochi et al., 2002). Symptomatically, all patients improved over the follow-up period. There were few side-effects, except for hypertrophy of the graft in three knees and partial detachment of the periosteum in three. Biomechanical test revealed that the transplants had acquired hardness similar to that of the surrounding cartilage.

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In a recent human study (Wakitani et al., 2002), autologous culture-expanded MSCs were implanted in patients in a cartilage repair procedure. The study population was 24 knees of 24 OA patients (average age 63 years; range 49–70 years) undergoing high tibial osteotomy. Ten milliliters of heparinized bone marrow blood was aspirated from both sides of the iliac crest. After approximately 10 days in culture when the attached cells became subconfluent, they were detached and subcultured for and additional 20 days 1.3  107 cells were embedded in type I acid soluble collagen from porcine tendon, put onto a collagen sheet, and gelated. This gelcell composite, which was then cultured overnight, was implanted into 12 knees. The other 12 subjects served as cell-free controls.“In the cell-transplanted group, as early as 6.3 weeks after transplantation the defects were covered with white to pink soft tissue, in which metachromasia was partially observed. Forty-two weeks after transplantation, the defects were covered with white soft tissue, in which metachromasia was observed in almost all areas of the sampled tissue and hyaline cartilage-like tissue was partially observed. Although the clinical improvement was not significantly different, the arthroscopic and histological grading score was better in the cell-transplanted group than in the cell-free control group.” There were no adverse responses reported in the study. This study demonstrated the safety and feasibility of autologous culture-expanded bone marrow-derived MSC transplantation for the repair of articular cartilage defects in humans.

SCAFFOLD-FREE CONSTRUCTS A number of animal studies, using chondrocytes without any scaffold as a method of cell-based therapy, preceded the introduction of ACI. There have been fewer studies where stem cells, without any scaffold, have been used as a cell-based therapy for cartilage repair. One such study involved 16 mature white rabbits from which MSCs were aspirated from the bone marrow (Im et al., 2001). These stem cells were then cultured in monolayer and implanted on to a full-thickness osteochondral defect artificially made on the patellar groove of the same rabbit. Another group of 13 rabbits served as a control group and the animals were sacrificed after 14 weeks. The semiquantitative histological scores were significantly higher in the experimental group than in the control group. In the experimental group immunohistochemical staining of newly formed cartilage was more intense for type II collagen in the matrix and RT-PCR from regenerated cartilage detected mRNA for type II collagen in mature chondrocytes. These findings suggest that repair of cartilage defects can be enhanced by the implantation of cultured MSCs.

CURRENT CLINICAL OUTCOMES By far, the most commonly used cell therapy for cartilage repair is ACI, first reported by Brittberg et al. (1994). This was a case series of 23 patients treated in Sweden for symptomatic cartilage defects. Thirteen patients had femoral condylar defects, ranging in size from 1.6 to 6.5 cm2, due to trauma or osteochondritis dissecans. Seven patients had patellar defects. Ten patients had previously been treated with shaving and debridement of unstable cartilage. Cartilage was harvested arthroscopically from a minimally load-bearing area of the upper aspect or the medial condyle of the affected knee. Chondrocytes were isolated and culture expanded in a cell culture laboratory. In a second procedure, following a medial or lateral parapatellar incision, the defect was debrided and a periosteal flap was harvested and sutured to the rim of the defect. Finally, the chondrocyte suspension was injected under the periosteal flap. Follow-up of the patients was over 16–66 months, with a mean of 39 months. Initially, the transplants eliminated knee locking and reduced pain and swelling in all patients. After 3 months, a repeat arthroscopy showed that the transplants were level with the surrounding tissue and spongy when probed, with visible

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borders. A repeat arthroscopic examination showed that in many instances the transplants had the same macroscopic appearance as they had earlier but were firmer when probed and similar in appearance to the surrounding cartilage. Two years after transplantation, 14 of the 16 patients with femoral condylar transplants had good-to-excellent results. Two patients required a second operation, because of severe central wear in the transplants, with locking and pain. A mean of 36 months after transplantation, the results were excellent or good in two of the seven patients with patellar transplants, fair in three and poor in two; two patients required a second operation because of severe chondromalacia. Biopsies showed that 11 of the 15 femoral transplants and 1 of the 7 patellar transplants had the appearance of “hyaline-like” cartilage. These results and the fact that a commercial service for culturing autologous chondrocytes was established led to a dramatic increase in the use of this cell-based therapy for cartilage repair. Recently, there have been a number of randomized trials comparing ACI with the conventional methods of cartilage repair. Knutsen et al. randomized 80 patients with a single symptomatic cartilage defect on the femoral condyle to either ACI or micro-fracture (Knutsen et al., 2004). Two years post-operatively, arthroscopy with biopsy for histological evaluation was carried out. Both methods had acceptable short-term clinical results. There was no significant difference in macroscopic or histological results between the two treatment groups and no association between the histological findings and the clinical outcome at the 2-year time-point. Bentley et al. reported on a prospective, randomized comparison of ACI versus mosaicplasty for osteochondral defects in the knee (Bentley et al., 2003). One hundred patients with a symptomatic lesion of the articular cartilage in the knee were randomized to undergo either ACI or mosaicplasty. The mean followup period was 19 months and involved a clinical examination. The results demonstrated a significant superiority of ACI over mosaicplasty. The 1 year arthroscopic assessment demonstrated excellent or good repairs in 82% of ACIs and only 34% of mosaicplasties. Browne et al. recently reported on a multicenter cohort study to assess the clinical outcomes of patients treated with ACI for lesions of the distal femur (Browne et al., 2005). A modified Cincinnati knee rating system was used to measure outcomes at baseline and at 5 years. Overall, patients reported a statistically significant improvement in their overall score. Additional analysis of the data showed that 62 patients improved, 6 reported no change, and 19 worsened. In recent years, biological (including tissue engineering) therapies for the treatment of cartilage defects have progressed significantly and are becoming important modalities of treatment in orthopedic surgery. However, for all these therapies long-term outcome is unknown, and there is a lack of controlled studies comparing the different treatment options.

SUMMARY Regenerating cartilage tissue in vivo is likely to remain challenging over the next few years. However, cellbased therapies have already shown a great promise in being better at regenerating the damaged tissue than conventional surgical techniques. These techniques can be further improved upon by investigating the role of scaffolds in trials in repairing cartilage defects. Alternative cell sources, such as stem cells derived from bone marrow, may also provide an improvement in the quality of tissue regenerated.

ACKNOWLEDGMENT This work was supported by the US Department of Veterans Affairs.

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REFERENCES Barry, F.P. (2003). Biology and clinical applications of mesenchymal stem cells. Birth Defects Res. C. Embryo Today 69(3): 250–256. Barry, F.P. and Murphy, J.M. (2004). Mesenchymal stem cells: clinical applications and biological characterization. Int. J. Biochem. Cell Biol. 36(4): 568–584. Barry, F.P., Boynton, R.E., Haynesworth, S., Murphy, J.M. and Zaia, J. (1999). The monoclonal antibody SH-2, raised against human mesenchymal stem cells, recognizes an epitope on endoglin (CD105). Biochem. Biophys. Res. Comm.. 265(1): 134–139. Barry, F., Boynton, R., Murphy, M., Haynesworth, S. and Zaia, J. (2001). The SH-3 and SH-4 antibodies recognize distinct epitopes on CD73 from human mesenchymal stem cells. Biochem. Biophys. Res. Comm.. 289(2): 519–524. Bartholomew, A., Sturgeon, C., Siatskas, M., Ferrer, K., McIntosh, K., Patil, S., Hardy, W., Devine, S., Ucker, D., Deans, R., et al. (2002). Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp. Hematol. 30(1): 42–48. Bartlett, W., Skinner, J.A., Gooding, C.R., Carrington, R.W., Flanagan, A.M., Briggs, T.W. and Bentley, G. (2005). Autologous chondrocyte implantation versus matrix-induced autologous chondrocyte implantation for osteochondral defects of the knee: a prospective, randomised study. J Bone Joint Surg. Br. 87(5): 640–645. Bell, E. (1995). Strategy for the selection of scaffolds for tissue engineering. Tissue Eng. 1: 163–179. Bentley, G., Biant, L.C., Carrington, R.W., Akmal, M., Goldberg, A., Williams, A.M., Skinner, J.A. and Pringle, J. (2003). A prospective, randomised comparison of autologous chondrocyte implantation versus mosaicplasty for osteochondral defects in the knee. J. Bone Joint Surg. Br. 85(2): 223–230. Bobic, V. (1999). Autologous osteochondral grafts in the management of articular cartilage lesions. Orthopade 28(1): 19–25. Breinan, H.A., Martin, S.D., Hsu, H.P. and Spector, M. (2000). Healing of canine articular cartilage defects treated with microfracture, a type-II collagen matrix, or cultured autologous chondrocytes. J. Orthop. Res. 18(5): 781–789. Brittberg, M. (1999). Autologous chondrocyte transplantation. Clin. Orthop. Relat. Res. 367(Suppl 367): S147–S155. Brittberg, M., Lindahl, A., Nilsson, A., Ohlsson, C., Isaksson, O. and Peterson, L. (1994). Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. New Engl. J. Med. 331(14): 889–895. Browne, J.E., Anderson, A.F., Arciero, R., Mandelbaum, B., Moseley Jr., J.B., Micheli, L.J., Fu, F. and Erggelet, C. (2005). Clinical outcome of autologous chondrocyte implantation at 5 years in US subjects. Clin. Orthop. Relat. Res. 436: 237–245. Buckwalter, J.A. and Mankin, H.J. (1998). Articular cartilage: tissue design and chondrocyte–matrix interactions. Instr. Course Lect. 47: 477–486. Caplan, A.I. (1991). Mesenchymal stem cells. J. Orthop. Res. 9(5): 641–650. Connolly, J.F. (1998). Clinical use of marrow osteoprogenitor cells to stimulate osteogenesis. Clin. Orthop. Relat. Res. 355(Suppl 355): S257–S266. Curl, W.W., Krome, J., Gordon, E.S., Rushing, J., Smith, B.P. and Poehling, G.G. (1997). Cartilage injuries: a review of 31,516 knee arthroscopies. Arthroscopy 13(4): 456–460. De Bari, C., Dell’Accio, F. and Luyten, F.P. (2001). Human periosteum-derived cells maintain phenotypic stability and chondrogenic potential throughout expansion regardless of donor age. Arthritis Rheum. 44(1): 85–95. De Bari, C., Dell’Accio, F., Vandenabeele, F., Vermeesch, J.R., Raymackers, J.M. and Luyten, F.P. (2003). Skeletal muscle repair by adult human mesenchymal stem cells from synovial membrane. J. Cell Biol. 160(6): 909–918. Fisher, A. (1922). A contribution to the pathology and etiology of osteo-arthritis: with observations upon the principles underlying its surgical treatment. Br. J. Surg. 10: 52. Goldring, M.B., Sandell, L.J., Stephenson, M.L. and Krane, S.M. (1986). Immune interferon suppresses levels of procollagen mRNA and type II collagen synthesis in cultured human articular and costal chondrocytes. J. Biol. Chem. 261(19): 9049–9055. Goldring, M.B., Birkhead, J., Sandell, L.J., Kimura, T. and Krane, S.M. (1988). Interleukin 1 suppresses expression of cartilage-specific types II and IX collagens and increases types I and III collagens in human chondrocytes. J. Clin. Invest. 82(6): 2026–2037. Grande, D.A., Pitman, M.I., Peterson, L., Menche, D. and Klein, M. (1989). The repair of experimentally produced defects in rabbit articular cartilage by autologous chondrocyte transplantation. J. Orthop. Res. 7(2): 208–218.

Articular Cartilage 779

Grande, D.A., Halberstadt, C., Naughton, G., Schwartz, R. and Manji, R. (1997). Evaluation of matrix scaffolds for tissue engineering of articular cartilage grafts. J. Biomed. Mater. Res. 34(2): 211–220. Gronthos, S. and Simmons, P.J. (1995). The growth factor requirements of STRO-1-positive human bone marrow stromal precursors under serum-deprived conditions in vitro. Blood 85(4): 929–940. Gronthos, S., Zannettino, A.C., Hay, S.J., Shi, S., Graves, S.E., Kortesidis, A. and Simmons, P.J. (2003). Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow. J. Cell Sci. 116(Pt 9): 1827–1835. Gurevitch, O., Kurkalli, B.G., Prigozhina, T., Kasir, J., Gaft, A. and Slavin, S. (2003). Reconstruction of cartilage, bone, and hematopoietic microenvironment with demineralized bone matrix and bone marrow cells. Stem Cells 21(5): 588–597. Homminga, G.N., van der Linden, T.J., Terwindt-Rouwenhorst, E.A. and Drukker, J. (1989). Repair of articular defects by perichondrial grafts. Experiments in the rabbit. Acta Orthop. Scand. 60(3): 326–329. Homminga, G.N., Bulstra, S.K., Bouwmeester, P.S. and van der Linden, A.J. (1990). Perichondral grafting for cartilage lesions of the knee. J. Bone Joint Surg. Br. 72(6): 1003–1007. Homminga, G.N., Bulstra, S.K., Kuijer, R. and van der Linden, A.J. (1991). Repair of sheep articular cartilage defects with a rabbit costal perichondrial graft. Acta Orthop. Scand. 62(5): 415–418. Horwitz, E.M., Prockop, D.J., Fitzpatrick, L.A., Koo, W.W., Gordon, P.L., Neel, M., Sussman, M., Orchard, P., Marx, J.C., Pyeritz, R.E., et al. (1999). Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat. Med. 5(3): 309–313. Im, G.I., Kim, D.Y., Shin, J.H., Hyun, C.W. and Cho, W.H. (2001). Repair of cartilage defect in the rabbit with cultured mesenchymal stem cells from bone marrow. J. Bone Joint Surg. Br. 83(2): 289–294. Kavalkovich, K.W., Boynton, R.E., Murphy, J.M. and Barry, F. (2002). Chondrogenic differentiation of human mesenchymal stem cells within an alginate layer culture system. In Vitro Cell Dev. Biol. Anim. 38(8): 457–466. Kawamura, S., Wakitani, S., Kimura, T., Maeda, A., Caplan, A.I., Shino, K. and Ochi, T. (1998). Articular cartilage repair. Rabbit experiments with a collagen gel–biomatrix and chondrocytes cultured in it. Acta Orthop. Scand. 69(1): 56–62. Key, J. (1931). Experimental arthritis: the changes in joints produced by creating defects in the articular cartilage. J. Bone Joint Surg. 23: 725–739. Knutsen, G., Engebretsen, L., Ludvigsen, T.C., Drogset, J.O., Grontvedt, T., Solheim, E., Strand, T., Roberts, S., Isaksen, V. and Johansen, O. (2004). Autologous chondrocyte implantation compared with microfracture in the knee. A randomized trial. J. Bone Joint Surg. Am. 86-A(3): 455–464. Lee, C.R., Grodzinsky, A.J., Hsu, H.P., Martin, S.D. and Spector, M. (2000). Effects of harvest and selected cartilage repair procedures on the physical and biochemical properties of articular cartilage in the canine knee. J. Orthop. Res. 18(5): 790–799. Lee, R.H., Kim, B., Choi, I., Kim, H., Choi, H.S., Suh, K., Bae, Y.C. and Jung, J.S. (2004). Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol. Biochem. 14(4–6): 311–324. Lodie, T.A., Blickarz, C.E., Devarakonda, T.J., He, C., Dash, A.B., Clarke, J., Gleneck, K., Shihabuddin, L. and Tubo, R. (2002). Systematic analysis of reportedly distinct populations of multipotent bone marrow-derived stem cells reveals a lack of distinction. Tissue Eng. 8(5): 739–751. Majumdar, M.K., Keane-Moore, M., Buyaner, D., Hardy, W.B., Moorman, M.A., McIntosh, K.R. and Mosca, J.D. (2003). Characterization and functionality of cell surface molecules on human mesenchymal stem cells. J. Biomed. Sci. 10(2): 228–241. Mankin, H.J. (1982). The response of articular cartilage to mechanical injury. J. Bone Joint Surg. Am. 64(3): 460–466. Martin, J.A. and Buckwalter, J.A. (2001). Roles of articular cartilage aging and chondrocyte senescence in the pathogenesis of osteoarthritis. Iowa Orthop. J. 21: 1–7. Matsusue, Y., Yamamuro, T. and Hama, H. (1993). Arthroscopic multiple osteochondral transplantation to the chondral defect in the knee associated with anterior cruciate ligament disruption. Arthroscopy 9(3): 318–321. Mayhew, T.A., Williams, G.R., Senica, M.A., Kuniholm, G. and Du Moulin, G.C. (1998). Validation of a quality assurance program for autologous cultured chondrocyte implantation. Tissue Eng. 4(3): 325–334. McBride, C., Gaupp, D. and Phinney, D.G. (2003). Quantifying levels of transplanted murine and human mesenchymal stem cells in vivo by real-time PCR. Cytotherapy 5(1): 7–18.

780 THERAPEUTIC APPLICATIONS: CELL THERAPY

Murphy, J.M., Fink, D.J., Hunziker, E.B. and Barry, F.P. (2003). Stem cell therapy in a caprine model of osteoarthritis. Arthritis. Rheum. 48(12): 3464–3474. Niedermann, B., Boe, S., Lauritzen, J. and Rubak, J.M. (1985). Glued periosteal grafts in the knee. Acta Orthop. Scand. 56(6): 457–460. Ochi, M., Uchio, Y., Kawasaki, K., Wakitani, S. and Iwasa, J. (2002). Transplantation of cartilage-like tissue made by tissue engineering in the treatment of cartilage defects of the knee. J. Bone Joint Surg. Br. 84(4): 571–578. Orlic, D., Kajstura, J., Chimenti, S., Jakoniuk, I., Anderson, S.M., Li, B., Pickel, J., McKay, R., Nadal-Ginard, B., Bodine, D.M., et al. (2001). Bone marrow cells regenerate infarcted myocardium. Nature 410(6829): 701–705. Oshima, Y., Watanabe, N., Matsuda, K., Takai, S., Kawata, M. and Kubo, T. (2005). Behavior of transplanted bone marrow-derived GFP mesenchymal cells in osteochondral defect as a simulation of autologous transplantation. J. Histochem. Cytochem. 53(2): 207–216. Pascher, A., Palmer, G.D., Steinert, A., Oligino, T., Gouze, E., Gouze, J.N., Betz, O., Spector, M., Robbins, P.D., Evans, C.H., et al. (2004). Gene delivery to cartilage defects using coagulated bone marrow aspirate. Gene Ther. 11(2): 133–141. Perka, C., Sittinger, M., Schultz, O., Spitzer, R.S., Schlenzka, D. and Burmester, G.R. (2000). Tissue engineered cartilage repair using cryopreserved and noncryopreserved chondrocytes. Clin. Orthop. Relat. Res. 378: 245–254. Peterson, L., Minas, T., Brittberg, M., Nilsson, A., Sjogren-Jansson, E. and Lindahl, A. (2000). Two- to 9-year outcome after autologous chondrocyte transplantation of the knee. Clin. Orthop. Relat. Res. 374: 212–234. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S. and Marshak, D.R. (1999). Multilineage potential of adult human mesenchymal stem cells. Science 284(5411): 143–147. Ponticiello, M.S., Schinagl, R.M., Kadiyala, S. and Barry, F.P. (2000). Gelatin-based resorbable sponge as a carrier matrix for human mesenchymal stem cells in cartilage regeneration therapy. J. Biomed. Mater. Res. 52(2): 246–255. Prockop, D.J. (1997). Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276(5309): 71–74. Reyes, M. and Verfaillie, C.M. (2001). Characterization of multipotent adult progenitor cells, a subpopulation of mesenchymal stem cells. Ann. NY Acad. Sci. 938: 231–233; discussion 233–235. Rubak, J.M. (1982). Reconstruction of articular cartilage defects with free periosteal grafts. An experimental study. Acta Orthop. Scand. 53(2): 175–180. Saadeh, P.B., Brent, B., Mehrara, B.J., Steinbrech, D.S., Ting, V., Gittes, G.K. and Longaker, M.T. (1999). Human cartilage engineering: chondrocyte extraction, proliferation, and characterization for construct development. Ann. Plast. Surg. 42(5): 509–513. Salama, R., Burwell, R.D. and Dickson, I.R. (1973). Recombined grafts of bone and marrow. The beneficial effect upon osteogenesis of impregnating xenograft (heterograft) bone with autologous red marrow. J Bone Joint Surg. Br. 55(2): 402–417. Shapiro, F., Koide, S. and Glimcher, M.J. (1993). Cell origin and differentiation in the repair of full-thickness defects of articular cartilage. J. Bone Joint Surg. Am. 75(4): 532–553. Shelbourne, K.D., Jari, S. and Gray, T. (2003). Outcome of untreated traumatic articular cartilage defects of the knee: a natural history study. J. Bone Joint Surg. Am. 85-A (Suppl 2): 8–16. Solchaga, L.A., Gao, J., Dennis, J.E., Awadallah, A., Lundberg, M., Caplan, A.I. and Goldberg, V.M. (2002). Treatment of osteochondral defects with autologous bone marrow in a hyaluronan-based delivery vehicle. Tissue Eng. 8(2): 333–347. Solchaga, L.A., Penick, K., Porter, J.D., Goldberg, V.M., Caplan, A.I. and Welter, J.F. (2005). FGF-2 enhances the mitotic and chondrogenic potentials of human adult bone marrow-derived mesenchymal stem cells. J. Cell Physiol. 203(2): 398–409. Tse, W.T., Pendleton, J.D., Beyer, W.M., Egalka, M.C. and Guinan, E.C. (2003). Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation 75(3): 389–397. Wakitani, S., Goto, T., Pineda, S.J., Young, R.G., Mansour, J.M., Caplan, A.I. and Goldberg, V.M. (1994). Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J. Bone Joint Surg. Am. 76(4): 579–592. Wakitani, S., Imoto, K., Yamamoto, T., Saito, M., Murata, N. and Yoneda, M. (2002). Human autologous culture expanded bone marrow mesenchymal cell transplantation for repair of cartilage defects in osteoarthritic knees. Osteoarthritis Cartilage 10(3): 199–206. Yanai, T., Ishii, T., Chang, F. and Ochiai, N. (2005). Repair of large full-thickness articular cartilage defects in the rabbit: the effects of joint distraction and autologous bone-marrow-derived mesenchymal cell transplantation. J. Bone Joint Surg. Br. 87(5): 721–729.

Articular Cartilage 781

Young, H.E., Steele, T.A., Bray, R.A., Hudson, J., Floyd, J.A., Hawkins, K., Thomas, K., Austin, T., Edwards, C., Cuzzourt, J., et al. (2001). Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors. Anat. Rec. 264(1): 51–62. Zhou, G.D., Wang, X.Y., Miao, C.L., Liu, T.Y., Zhu, L., Liu, D.L., Cui, L., Liu, W. and Cao, Y.L. (2004). Repairing porcine knee joint osteochondral defects at non-weight bearing area by autologous BMSC. Zhonghua Yi Xue Za Zhi 84(11): 925–931. Zohar, R., Sodek, J. and McCulloch, C.A. (1997). Characterization of stromal progenitor cells enriched by flow cytometry. Blood 90(9): 3471–3481.