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Abstract

Articular cartilage damage and osteoarthritis are the most common joint diseases. Joints are prone to damage caused by sports injuries or aging, and such damage regularly progresses to more serious joint disorders, including osteoarthritis, which is a degenerative disease characterized by the thinning and eventual wearing out of articular cartilage, ultimately leading to joint destruction. Osteoarthritis affects millions of people worldwide. Current approaches to repair of articular cartilage damage include mosaicplasty, microfracture, and injection of autologous chondrocytes. These treatments relieve pain and improve joint function, but the long-term results are unsatisfactory. The long-term success of cartilage repair depends on development of regenerative methodologies that restore articular cartilage to a near-native state. Two promising approaches are (i) implantation of engineered constructs of mesenchymal stem cell (MSC)-seeded scaffolds, and (ii) delivery of an appropriate population of MSCs by direct intra-articular injection. MSCs may be used as trophic producers of bioactive factors initiating regenerative activities in a defective joint. Current challenges in MSC therapy are the need to overcome current limitations in cartilage cell purity and to in vitro engineer tissue structures exhibiting the required biomechanical properties. This review outlines the current status of MSCs used in cartilage tissue engineering and in cell therapy seeking to repair articular cartilage defects and related problems. MSC-based technologies show promise when used to repair cartilage defects in joints.

Keywords

Articular cartilage Mesenchymal stem cells (MSCs)Tissue engineering Osteoarthritis

Introduction

Articular cartilage damage is usually caused by sports injuries, accidental trauma, and aging and regularly progresses to more serious joint disorders including osteoarthritis (OA), necrosis of subchondral bone, or arthritis. After traumatic or pathological injury, hyaline articular cartilage, the load-bearing tissue of joints, has a very limited or no intrinsic capacity for repair, and even minor lesions or injuries may trigger progressive damage and joint degeneration. An estimated 15% of the world's population has joint disease. More than 39 million people in the European Union and over 20 million Americans have OA; by the year 2020, these numbers are projected to double (23,34).

OA is a chronic degenerative joint disease characterized by progressive destruction of articular cartilage and thinning (with eventual wearing out) of that cartilage, resulting in painful, limited joint movement.

Degeneration of articular cartilage is attributable mainly to changes in the activities of chondrocytes involved in catabolic activities and also involves other joint tissues, including the meniscus. Also, sclerosis and edema may develop in underlying subchondral bone, and intermittent inflammation may be noted in the synovium. Cartilage defects attributable to underlying disease in an OA patient are distinct from focal cartilage lesions caused by acute injury. Patients with OA are likely to be older, and often the entire articulating surface is affected. Generally, the cartilage lesions of OA are large, unconfined, and present in more than one location.

Current Treatments Are Less Than Satisfactory

Current treatments for articular cartilage damage, including surgical interventions (microfracture; placement of osteochondral auto- or allografts) seeking to repair articular cartilage rarely restore full function. Indeed, fibrocartilage may develop in the long term, rather than the desired hyaline articular cartilage. Repair and regeneration of cartilage defects remain challenges in orthopedic surgery. The long-term success of cartilage repair depends on the development of regenerative methodologies resulting in restoration of articular cartilage to a form closely resembling native tissue.

Tissue engineering-based cartilage repair is a major research thrust of efforts to create more biologically functional cartilage tissue. Cartilage engineered via autologous chondrocyte implantation (ACI) was first reported by Brittberg et al., in 1994 (14). The chondrocytes were taken from non-weight-bearing parts of intact joint regions, expanded in culture, and next transplanted into defective joint areas. Initial ACI clinical trials were promising (92); however, the treatment required direct extraction of chondrocytes from the patient, thus inducing additional donor site morbidity of healthy articular cartilage. Other disadvantages included loss of the chondrocytic phenotype in cells expanded in monolayer culture, lack of applicability of the technique to large lesions, the limited proliferative capacity of chondrocytes, poor functionality and quality of the extracellular matrix (ECM) synthesized, and limited technical efficacy in patients older than 40 years (because of poor cellular activation). Additionally, newly regenerated cartilage within a joint space often consisted of fibrocartilage rather than the desired hyaline cartilage (46). A second-generation ACI procedure incorporated various biomaterials, including gels made of collagen type I, hyaluronin, and collagen type I/III membrane scaffolds, to recreate the three-dimensional environment (30). Prospective clinical studies showed that the clinical outcomes were the same as those afforded by the first-generation procedure (83). A possible way of overcoming the limited supply of primary chondrocytes is to rather use multipotent mesenchymal stem cells (MSCs).

Current treatments for OA aim primarily to alleviate symptoms, reduce pain, and control inflammation. All of the anti-inflammatory drugs, steroids, and hyaluronic acid (HA) are used to these ends, but the treatments do not control the progressive degeneration of joint tissues (123). Surgical treatments, including osteochondral graft transplantation (mosaicplasty) and microfracture, seeking to repair the cartilage of OA patients, relieve pain temporarily, but are unsatisfactory in the long term, and eventually fail (52,93). Tissue-engineering efforts such as ACI or matrix-induced ACI (MACI) are potential longterm modalities for the biological repair or regeneration of degenerated joint tissues. However, a major limitation of both ACI and MACI is that these approaches cannot be used to treat large cartilage defects (13,131), such as those found in OA patients.

Mesenchymal Stem Cells (MSCs) May Be A Source of Progenitor Cells for Cartilage Repair

Multipotent adult MSCs can differentiate into cells of the chondrogenic lineage. Thus, a number of experimental strategies have been developed to investigate whether MSCs are candidates for use in regeneration and maintenance of articular cartilage. Can MSCs replace chondrocytes in this context? MSCs can be isolated from a wide variety of tissues, thus allowing further damage to diseased or injured tissues to be avoided. MSCs are readily available in large quantities, are easy to isolate in the absence of significant donor site morbidity, and are easy to expand in vitro (unlike chondrocytes). MSCs differentiate into chondrocytes in response to chondrogenic signals. In addition, MSCs synthesize an ECM with properties very similar to those of healthy hyaline joint cartilage. Further, because of their excellent proliferative capacity, MSCs can be cultured in large numbers (66,91,121). Finally, because the cells exhibit trophic and regenerative potentials, MSCs may also be valuable in treatment of chronic degenerative disorders, and may prevent cartilage degradation in OA patients.

Tissue Distribution and Sources of Human MSCs

MSCs were first identified in bone marrow (BM) (35) but are now known to reside in connective tissues, notably adipose tissue (AT) (165), the placenta (37), the umbilical cord (120), dental pulp (42), tendons (6), trabecular bone (106), the perichondrium (2), the periosteum, the synovium (25), and the infrapatellar fat pads (57). MSCs may occur in virtually all postnatal organs and tissues (88). MSCs have also been reported to occur in perivascular locations and to express markers specific for pericytes (22,87). However, BM and AT are the two main sources of MSCs used in cell therapy because of the high expansive potentials of such cells and the availability of reproducible isolation procedures. Historically, the first-characterized MSCs derived from BM (BM-MSCs) remain the most intensively studied and still serve as a reference cell type. AT-derived MSCs (AT-MSCs) are easier to isolate in high numbers than are BM-MSCs (65). Nevertheless, although AT-MSCs are similar to BM-MSCs, the transcriptomic and proteomic profiles of AT-MSCs show specificities characteristic of the tissue of origin (105).

MSCs are defined using three criteria proposed by the International Society for Cellular Therapy (27). First, MSCs are a heterogeneous cell population isolated by exploiting the fact that the cells adhere to plastic. In culture, MSCs can develop into fibroblastic colony-forming units. Second, MSCs are distinguished by phenotype. MSCs express the cell-surface marker cluster of differentiation 73 (CD73), CD90, and CD105 and are negative for CD11b, CD14, CD34, CD45, and human leukocyte antigen (HLA)-DR. More recently, the CD271 marker has been used to isolate highly enriched BM-MSC populations (15). BM-MSCs are negative for the CD34 marker, but native AT-MSCs can be isolated by purifying cells that do in fact express CD34, although such expression rapidly disappears upon cell proliferation in vitro (86,125). Third, MSCs can differentiate into at least three mesenchymal cell lineages, namely bone, fat, and cartilage.

AT-MSCs are generating growing interest (166). Adipose tissue is easy to access in all individuals and contains a large proportion of MSCs, accounting for ~5% of all stromal cells; the MSC density is ~100-fold that of BM (61). AT can be easily harvested via lipoaspiration, which is a minimally invasive technique, and large numbers of MSCs can be obtained. Differentiation of AT-MSCs into chondrocytes is crucial in terms of successful cartilage regeneration (82).

MSCs from human umbilical cords (Wharton's jelly MSCs; WJ-MSCs and cord blood may have the potential to be used in cartilage tissue engineering and cell-based therapy for OA. These tissues are easily obtained, and the cells are readily extracted, proliferate very well, and are of high purity compared with AT- and BM-MSCs. Valiyaveettil et al. reported that Wharton's jelly was abundant and contained significant amounts of HA, and some glycosoaminoglycans (GAGs), immobilized in an insoluble matrix of collagen fibrils (144). Liu et al. reported that umbilical cord WJ-MSCs stained weakly with toluidine blue and safranin O and were (immunohistochemically) weakly positive for type II collagen. RT-PCR showed that chondrogenic transcription factor sex-determining region Y box 9 (SOX9) and cartilage-specific collagen type 2 a 1 were expressed in WJ-MSCs (79). These are all features of prechondrocytes, showing that the cells had the innate potential to differentiate into chondrocytes. This is not the case with either BM- (124) or AT-MSCs (165). Wang et al. found that WJ-MSCs secreted more GAGs than did BM-MSCs during chondrogenic differentiation (151).

Properties and Immunomodulatory Features of MSCs

MSCs have an immunomodulatory property. This is a capacity to inhibit the proliferation and function of several types of immune cells and is evident both in vitro and in vivo (38). Induction of MSC immunomodulation is mediated principally by soluble materials. Indoleamine 2,3-dioxygenase is a major player in human MSC immunomodulation. Nitric oxide is expressed at low levels in human MSCs upon stimulation with interferon-g (73,119). All of the following factors, transforming growth factor b1 (TGF-b1), hepatocyte growth factor (HGF), heme oxygenase 1, interleukin-6 (IL-6), leukemia inhibitory factor, HLA-G5, the IL-10 and IL-1 receptor antagonist, and prostaglandin E₂, have been proposed to mediate MSC immunomodulation [reviewed in Djouad et al. (26)]. MSCs suppress B- and T-cell proliferation and alter the functions of these cells, inhibit the proliferation of activated natural killer cells, interfere with generation of mature dendritic cells from monocytes or CD34⁺ progenitor cells, and induce an immature dendritic cell phenotype (38,100). Finally, MSCs inhibit Th17 cell differentiation and induce fully differentiated Th17 cells to develop a T-cell regulatory phenotype (39). Although soluble mediators are the principal actors in MSC immunosuppression, cell–cell interactions are also involved. Stimulation of the toll-like receptor (TLR) can modulate the actions of MSCs on the immune system. Indeed, TLR4-primed MSCs, or MSC1, elaborate principally proinflammatory mediators, whereas TLR3-primed MSCs and MSC2 express mostly immunosuppressive mediators (153). Human WJ-MSCs subcutaneously implanted into the rat and rabbit only sparsely promoted CD4⁺ and CD8⁺ T-cell infiltration, and the WJ-MSCs remained alive 4 weeks after implantation, showing that immune rejection was weak or absent (80).

MSCs can also attenuate tissue injury, inhibit fibrotic remodeling and apoptosis, promote angiogenesis, stimulate stem cell recruitment and proliferation, and reduce oxidative stress (127). Two prominent factors in these processes are HGF and vascular endothelial growth factor (VEGF). Activation of the “Janus kinase-signal transducer and activator” of the transcription 3 axis of myocytes increases expression of the target genes HGF and VEGF (126). The other mediators important during tissue remodeling are matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). MSC-secreted TIMPs protect implanted cells via TIMP-mediated inhibition of MMP activity (81).

The trophic properties of MSCs are linked to tissue regenerative processes via bioactive factors. Such factors may act directly, triggering intracellular processes in injured cells, or indirectly, inducing neighboring cells to secrete functionally active mediators.

MSCs as Seed Cells in Tissue Engineering to Repair Cartilage Defects

The chondrogenic potential of MSCs was first reported by Ashton et al. in 1980 (4). Standard methods for inducing MSC chondrogenesis have been reported. Under appropriate culture conditions, thus in the presence of defined exogenous factors, MSCs can be directed toward chondrogenic differentiation. Growth factors that promote chondrogenesis, or that demonstrate a chondrogenic effect, both in vivo and in vitro, include TGF-β, ascorbate, dexamethasone, and insulin-like growth factors. Several types of combined scaffolds have been evaluated for use in cartilage engineering employing MSCs [reviewed in Vinatier et al. (146)]. More recently, many researchers have focused on the use of BM-MSCs, combined with many types of scaffolds, to engineer cartilage tissue in animal models, including the rabbit (16,32,33,117,152,161,164) and the miniature pig (18). AT-MSCs have also been used in such work (63,162).

In addition to BM- and AT-MSCs, MSCs from other sources, including synovium, muscle (90), the periosteums, and the human placenta (76), are being explored in the context of cartilage tissue engineering. Synovium stem cells exhibit the greatest chondrogenic potential (47,70,77, 99,113,135).

Clinical Applications of Msc Tissue Engineering in Repair of Cartilage Defects

Treatment of animals with MSCs has yielded ambiguous results, and few clinical trials exploring the utility of MSC tissue engineering in the repair of human articular cartilage have been performed. The first use of transplanted MSCs, seeded within collagen type I hydrogels, to repair isolated, full-thickness cartilage defects in humans was reported by Wakitani et al.; patients enjoyed significantly improved clinical outcomes after 1, 4, and 5 years (148). In all, 24 patients with knee OA underwent high tibial osteotomy; 12 received BM-MSCs embedded in a collagen gel, and 12 (controls) a cell-free scaffold. The implants were placed in cartilage defects of the medial femoral condyle. Two years later, the treated group exhibited a significantly greater extent of hyaline cartilage formation compared to the control group (148). Furthermore, in a later study, the group investigated the efficacy of autologous BM-MSCs for repair of cartilage defects and demonstrated clinical and histological improvement in treated compared with control patients (149). Further, in a later study, the same group investigated the efficacy of autologous BM-MSCs for repair of cartilage defects and demonstrated clinical and histological improvements in treated compared to control patients (150). Kuroda et al. described a 31-year-old male who received autologous BM stromal cells to repair a 20×30-mm full-thickness cartilage defect. One year after surgery, clinical symptoms had improved substantially. The patient had resumed his activities at the predefect level and experienced no pain or other complication (74). Giannini et al. developed a one-step arthroscopic technique for cartilage repair; the approach featured a device used to concentrate BM-derived cells, and the use of collagen powder or an HA membrane (as a scaffold for cell support) in a platelet-containing gel. The authors performed an in vitro preclinical study to verify the capability of BM-derived cells to differentiate into chondrogenic and osteogenic lineages and to be supported on scaffolds. Next, in a prospective clinical study, the authors used the technique to repair talar osteochondral lesions in 48 patients. The minimum follow-up time was 24 months. The American Orthopedic Foot and Ankle Society score improved from 64.4± 14.5 to 91.4±7.7. Histological evaluation revealed regenerated tissue at various stages of remodeling, but no treated joint had exclusively hyaline cartilage (40,41). The use of stem cells from umbilical cord blood to treat cartilage defects was approved by the US Food and Drug Administration after secondary clinical trials were conducted. However, no report has yet tracked implanted MSCs over a long period. It is thus unclear how long MSCs survive, and their fate is unknown.

Major Biological Obstacles to Persistent Cell-Based Regeneration of Articular Cartilage

Successfully reconstructed cartilage tissue would ideally be structurally reunited with peripheral cartilage and exhibit the biomechanical properties required for permanence. Attainment of such characteristics will ultimately drive the clinical use of MSCs in repair of weight-bearing articular joint cartilage; efficacy and mechanical properties will be the critical metrics. However, some problems remain when MSCs are used for articular cartilage repair.

Mechanical Properties of the Constructs

The mechanical properties of constructs populated by MSCs are inferior to those of both native tissue and tissue-engineered constructs formed by fully differentiated chondrocytes (31). The tensile properties of MSCs are also inferior to those of native cartilage, attributable to low collagen content (21,51,85,140).

Heterogeneity and Inefficient Differentiation of MSCs

Heterogeneity of initial cell populations and poor matrix deposition was thought to contribute to functional limitations of MSCs (50). Vogel et al. reported that the adherent MSC population was heterogeneous and consisted of fibroblastoid cells, small single cells, and polygonal cells of different sizes (147). Colter et al. demonstrated that the smaller cells were more “naive” and capable of trilineage differentiation, whereas the larger cells were more mature and restricted in differentiation potential (20). Ylostalo et al. observed that smaller cells in clonal cultures of BM-MSCs tended to reside at the periphery, whereas larger cells were prevalent in the colony center (159). Hardingham et al. analyzed cartilage tissue formation from individual clonal populations of MSCs and showed that both the matrix formation capacity and phenotype retention ability of individual clones varied greatly (96). Huang et al. demonstrated that MSCs from the same population showed markedly different extents of proteoglycan deposition in the pericellular space at early stages of culture (50).

“Transient” or “Permanent” Chondrocytes

During in vitro chondrogenesis, MSCs upregulate expression of hyaline cartilage-specific markers (including collagen type II), cartilage-specific proteins including aggrecan, and the expression levels of markers typical of hypertrophic chondrocytes, thus collagen type X and alkaline phosphatase (56). Collagen type X constitutes 45% of all collagen produced in hypertrophic chondrocytes and is therefore considered an important marker of enchondral bone formation (128). Collagen type X expression is considerably upregulated in three-dimensional cultures of chondrogenically differentiated MSCs; the encoding mRNA is detectable at about day 7 by RT-PCR (5,104) and the protein by about day 14 by immunohistochemistry (137). In contrast, collagen type X is either not expressed at all or is expressed only marginally, in healthy mature chondrocytes and in engineered cartilage synthesized by mature chondrocytes (163). Pelttari et al. showed that subcutaneous implantation of in vitro differentiated MSC pellets resulted in extensive calcification and vascular invasion in vivo, in contrast to the observations with chondrocyte-derived pellets, which form stable ectopic cartilage (114). Jukes et al. found that chondrogenically differentiated embryonic stem cells growing on ceramic particles formed bone tissue after implantation in vivo; the chondrocyte on ceramic particles maintained stable cartilage phenotype and formed new cartilage matrix with no signs of hypertrophy and calcification (59). These data highlight the differences between differentiated MSCs and native articular chondrocytes. Thus, MSC chondrogenesis may approximate the mutable and undesired phenotype termed “transient,” rather than the “permanent” phenotype of chondrocytes (156). Transient chondrocytes reside in the growth plates of developing joints, which undergo hypertrophy and eventual ossification, whereas permanent chondrocytes reside in the articular cartilage and maintain a fixed chondrocytic phenotype throughout their lifetime.

Improving the Structure and Functional Utilities of Msc-Based Tissue-Engineered Constructs

Identification of the Markers of Permanent Chondrocytes and Elimination of Heterogeneity

In most studies, differentiation of MSCs toward the chondrogenic phenotype is demonstrated by measuring induction of major ECM components specific to hyaline cartilage, including aggrecan and type II collagen. Sometimes, SOX9, a transcriptional mediator, is responsible for initiating expression of components of the cartilaginous ECM, including aggrecan, collagen types II and XI, fibronectin, and tenascin. To guard against injection of transient chondrocytes, the levels of collagen type X and alkaline phosphatase should be monitored during chondrogenesis, as should the extent of mineralization. Monitoring of such markers may help to purify chondrogenic MSCs, thus overcoming cell heterogeneity.

Recently, several promising reports have suggested that it is feasible to inhibit expression of collagens type I and X to control the chondrogenic differentiation pathway of MSCs (7,115,118,154). Bian et al. showed that coculture of human MSCs with human articular chondrocytes in HA hydrogels enhanced the mechanical properties and cartilage-specific ECM content of tissue-engineered cartilage. Coculture decreased expression of collagen type X by MSCs; again, collagen type X is a major marker of (undesirable) MSC hypertrophy (7). Petit et al. showed that the extent of expression of type X collagen was reduced, at least transiently, upon growth on nitrogen-rich surfaces of both plated chondrocytes and MSCs from OA patients. This would enhance chondrogenesis (115). Rampersad et al. found that growth on nitrogen-rich plasma-polymerized ethylene surfaces downregulated type X collagen expression in vitro when MSCs from OA patients were precultured on such surfaces before formation of pellet cultures. This is a key feature of any agent that might be used to suppress hypertrophy and promote cartilage repair (118). Weiss et al. reported that parathyroid hormone-like peptide and basic fibroblast growth factor play critical roles in regulating the terminal differentiation of BM-MSCs; the factors suppress inhibited the TGF-β-responsive collagen X synthesis and alkaline phosphatase induction, thus preventing hypertrophic differentiation of BM-MSCs during pellet culture in vitro (154).

Mechanical Stimulation

MSCs have been shown to be sensitive to mechanical stimuli. Several studies have confirmed that MSC chondrogenesis can be modulated by mechanical stimulation, which improves the expression levels of key cartilage markers. Takahashi et al. reported that application of a static compressive force promoted chondrogenesis during differentiation of embryonic limb bud mesenchymal cells. Compressive forces have been shown to induce expression of type II collagen, aggrecan, and SOX9 (136). Henrionnet et al. explored the effects of calibrated cyclic mechanical loading on chondrogenesis by human BM-MSCs in a three-dimensional alginate culture, and on the maintenance of the chondrogenic phenotype. Biomechanical stimulation of MSCs increased formation of collagen of types I and II, and the expression levels of cartilage oligomeric matrix protein and SOX9. Such results suggest that mechanical stimulation may efficiently induce chondrogenic differentiation of MSCs in vitro, when such cells are to be used for cartilage tissue engineering within a three-dimensional environment (45). Huang et al. applied long-term dynamic compression to MSC-seeded constructs and assessed whether variations in preculture loading duration, the loading regimen, and the presence/ absence of TGF-β3 during loading, influenced functional outcomes and phenotypic transitions. Loading initiated prior to chondrogenesis decreased functional maturation, although chondrogenic gene expression levels increased. Loading initiated after both chondrogenesis and matrix elaboration further improved the mechanical properties of MSC-based constructs, but only when TGF-β3 levels were maintained, and only when specific loading parameters were chosen (49). These results show that dynamic compressive loading initiated after a sufficient period of chondroinduction and in the continual presence of TGF-β enhances matrix distribution and the mechanical properties of MSC-seeded constructs.

Kock et al. found that the major shortcoming of tissue-engineered cartilage was a lack of collagen, compromising tensile properties (67). Huang et al. measured the tensile properties of MSC-seeded hydrogels in free-swelling culture and found that although such properties initially increased with culture time (as matrix was deposited), the tensile properties plateaued to levels far below those of native tissue within a few weeks. Biochemical evaluation revealed poor collagen deposition and organization (51). Huang et al. designed and characterized a novel sliding contact bioreactor to recapitulate mechanical stimuli arising from physiological joint loading (when two distinct cartilage layers are in contact) and evaluated the short-term mechanosensitivity and the long-term development of functional properties using MSC-seeded agarose constructs. Finite element modeling of sliding contacts on agarose showed that both the tensile strain (parallel to the direction of sliding) and fluid efflux/influx were depth-dependent, being highest in regions closest to the surface of the construct. Short-term sliding contact of MSCs seeded in agarose improved the expression levels of chondrogenic genes, which were dependent on both the axial strain applied and TGF-β supplementation. Long-term sliding contact significantly improved the tensile properties of MSC-seeded constructs and elicited alterations in type II collagen and proteoglycan accumulation, both as functions of depth (48).

Natural Cartilage-Derived ECM Scaffolds

Although establishment of MSCs with a chondrogenic phenotype is important during loading, the preexisting matrix environment is also crucial (50). Yang et al. found that scaffolds derived from naturally decellularized cartilage ECM were devoid of allogeneic or xenogeneic cellular antigens, but most ECM structural and functional proteins were preserved, affording good biocompatibility and providing a natural microenvironment supporting BM-MSC attachment, proliferation, and differentiation into chondrocytes (158). Xue et al. decellularized cut cartilage to prepare acellular cartilage sheets (ACSs). ACSs preserved the natural cartilage matrix, including GAG and type II collagen, and contained several growth factors, including TGF-β1, IGF-1, and bone morphogenetic protein-2. The cited authors found that ACSs exhibited a chondrogenic induction activity promoting BM-MSC differentiation and an antiangiogenic activity stabilizing the engineered cartilage in vivo; the induced BM-MSC-poly(glycolic acid)/poly(lactic acid) group formed bone tissue in vivo (157). Further, to mimic natural cartilage ECM not only biochemically, but also structurally, Jia et al. fabricated an oriented scaffold made of cartilage-derived ECM; microtubules were arranged vertically in parallel. The compressive moduli of oriented and nonoriented scaffolds were 89.35 ± 7.96 and 36.20 ± 4.67 kPa, respectively; thus the modulus of the oriented scaffold was 2.5-fold greater than that of the nonoriented scaffold. Both types of scaffolds were seeded with chondrogenically induced BM-MSCs, and the cell–scaffold constructs were subcutaneously implanted in the dorsa of nude mice. Tissue-engineered constructs formed on oriented scaffolds exhibited superior mechanical properties (E values of 0.196 ± 0.019 and 0.285 ± 0.021 MPa) compared to those formed on nonoriented scaffolds (E values of 0.091 ± 0.007 and 0.153 ± 0.013 MPa), at both 2 and 4 weeks (p < 0.05) (55).

Zonal Organization of Constructs

Anatomically and functionally, articular cartilage has four spatially distinct regions; these are the superficial, transitional, deep, and calcified zones. Each zone is characterized by a specific composition of the ECM, unique mechanical properties, and a particular form of cellular organization. The superficial zone contains a high level of collagen II and a low level of GAG. The transitional zone has a lower collagen II content, but the GAG concentration is higher. The deep zone contains the highest concentration of GAG and the lowest level of collagen II fibers. The calcified cartilage zone has high levels of collagen X, and integrates the cartilage into the subchondral bone. Nguyen et al. developed a three-layer polyethylene glycol-based hydrogel in which chondroitin sulfate (CS) and MMP-sensitive peptides were incorporated into the top layer (the superficial zone), CS into the middle layer (the transitional zone), and HA into the bottom layer (the deep zone); to create near-native articular cartilage that varied spatially in both mechanical and biochemical properties. The cited authors evaluated the ability of spatially varied features within multilayered hydrogels to simultaneously differentiate a single bone marrow stromal cell (BM-SC) population into near-native articular cartilage (thus, also spatially varying in terms of mechanical and biochemical properties). The results showed that collagen II levels decreased gradually from the superficial to the deep zone, whereas collagen X and proteoglycan levels increased, upon steepening of the gradient of the compressive modulus from the superficial to the deep zone (102,103).

Kock et al. developed a dedicated loading regime termed sliding indentation, in which dynamic tension and a strain field varying with depth were applied to cell-seeded agarose constructs (68). The depth-dependent strain field delivered the highest strains in the superficial and middle zone and the lowest in the deep zone. Sliding indentation at 10% of depth, at 1 Hz, was applied to constructs for 4 h/day over 28 days; unloaded constructs served as controls. Sliding indentation increased the amount of collagen in the cartilage layers produced. Further, sliding indentation for 7 days increased the aggrecan and collagen type II expression levels in the top and middle layers compared to the bottom layer and the unloaded control, indicating a depth-dependent response at the level of gene expression, with the highest response being evident in regions placed under the greatest strain. Protein expression analysis after 28 days showed that the depth-dependent staining intensities of both GAG and collagen were similar in all constructs and had been enhanced by use of sliding indentation.

Mscs for Treatment of Chronic Degenerative Disorders, OA

The pathogenesis of OA is characterized by mechanical and oxidative stress, severe inflammation, recruitment of inflammatory cells, and proinflammatory cytokine production. The articular chondrocytes within diseased cartilage synthesize and secrete proteolytic enzymes, such as MMPs and aggrecanases, which degrade the cartilaginous ECM and ultimately trigger apoptotic death of differentiated chondrocytes. The proinflammatory interleukins (IL-1 and IL-6), and tumor necrosis factor (TNF)-α, are the most powerful inducers of such enzymes. The principal pathological and biochemical processes that ultimately lead to development of OA form a complex molecular mechanism. Cartilage defects arising from an underlying disease process in an OA patient are distinct from focal cartilage lesions caused by acute injury or osteochondrosis dissecans. Also, patients with OA are likely to be elderly, and often the entire articulating surface requires treatment. Considerable efforts have been made to identify inhibitors of undesirable molecules and other mediators of OA (Fig. 1). Currently, lesion repair may afford symptomatic relief and delay progression of OA symptoms, but, without effective treatment of the underlying disease, any improvement of a cartilage defect is likely to be short-lived.

Figure 1.

The potential therapeutic mechanisms of mesenchymal stem cells (MSCs) for osteoarthritis (OA). The pathology of OA (shown in the left semicircle) includes inflammation, recruitment of inflammatory cells, and production of the proinflammatory cytokines interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α. Articular chondrocytes within diseased cartilage synthesize and secrete proteolytic enzymes [matrix metalloproteinases (MMPs) and aggrecanases] that degrade the cartilaginous extracellular matrix (ECM) and ultimately trigger apoptotic cell death of differentiated chondrocytes. Repair of OA by MSCs (the right semicircle) is based on the fact that MSCs secrete a broad spectrum of bioactive molecules. These include the anti-inflammatory cytokines IL-6, transforming growth factor (TGF), indoleamine 2, 3-dioxygenase (IDO), and TNF-stimulated gene 6. Also, M1 macrophages are transformed into M2 anti-inflammatory macrophages. MSCs also secrete antiproteinase cytokines [tissue inhibitors of metalloproteinase-1 (TIMP-1) and TIMP-2] and antiapoptosis cytokines, [vascular endothelial growth factor (VEGF) and insulin-like growth factor 1 (IGF)]. MSCs enhance neocartilage formation by secreting bone morphogenetic protein (BMP), fibroblast growth factor (FGF), IGF-1, sex determining region Y box 9 (SOX9), signal transducer and activator of transcription 1 (STAT-1), and TGF-β. Bax, B-cell CLL/lymphoma 2 (BCL2)-associated X protein.

Self-donated MSCs may be the most appropriate source for treatment of OA because of the availability of large quantities of cells and the chondrogenic differentiation property of such cells. The proliferative and chondrogenic capacities of MSCs obtained from patients with OA may be lower than those of cells of healthy individuals (97). However, some previous studies found that sufficient numbers of MSCs of adequate chondrogenic differentiation potential could be isolated from patients with OA, regardless of patient age or disease etiology (53,62,122). Dudics et al. (28) showed that the chondrogenic potential of MSCs from OA patients was similar to that of cells from healthy donors. Moreover, OA is associated with progressive and often severe inflammation. MSCs not only contribute structurally to tissue repair but also possess potent immunomodulatory and antiinflammatory effects (58). MSCs secrete a broad spectrum of bioactive molecules with immunoregulatory (19,143) and/or regenerative activities (17). Via direct cell-cell interaction or by secretion of various factors, MSCs exert powerful effects on local tissue repair by modulating the local environment and activating endogenous progenitor cells (69). These properties mean that MSCs are promising candidates for cell therapy of OA.

Delivery Modes for MSCs

Cells can be delivered to a defective site by two methods. A crucial requirement of MSC-based OA therapy is the ability to deliver cells to the site of interest. Direct intraarticular injection may be possible at early stages of disease, when a defect is restricted to the cartilage layer, but a scaffold or a matrix of some kind is required to support MSCs when the subchondral bone is exposed over a large area. Direct intra-articular injection of MSCs is a minimally invasive alternative to open arthrotomy but has been performed only seldom. Compared to direct intra-articular injection, MSC delivery via a scaffold affords more control of proliferation, matrix production, and self-renewal. The ideal scaffold should be biocompatible, biodegradable upon tissue healing, highly porous (to permit cell penetration and tissue impregnation), sufficiently permeable to allow nutrient delivery and gas exchange, and adaptable to the mechanical environment. Also, the scaffold should have a surface that is conducive to cell attachment and migration, and should permit appropriate ECM formation and transmission of signaling molecules. However, few currently available scaffolds fulfill all of these requirements, and further developments in the field of biomaterial design are clearly needed before optimal neocartilage formation can be attained using cell-scaffold constructs.

Msc-Based Treatment of OA in Animal Models

In animal models, OA is induced primarily by surgical procedures, such as anterior cruciate ligament transaction (ACLT) or ACLT combined with complete medial meniscectomy. Lee et al. injected BM-MSCs (suspended in HA) intra-articularly into cartilage defects in the medial femoral condyles of adult minipigs. At 6 and 12 weeks postoperatively, MSC treatment had enhanced cartilage healing both histologically and morphologically, compared with use of either HA alone or saline (75). Murphy et al. injected autologous BM-MSCs (in a dilute HA solution) directly into the knee joints of goats with OA induced by total medial meniscectomy and resection of the ACL. Joints receiving MSCs showed evidence of marked regeneration of the medial meniscus, and implanted cells were detected in newly formed tissue. All of articular cartilage degeneration, osteophytic remodeling, and subchondral sclerosis were reduced in treated joints (98). Frisbie et al. (36) observed greater improvement with intra-articular injection of BM-MSCs to treat OA induced arthroscopically in the middle carpal joint of a horse model. Single MSC injections have produced exciting results in advanced or late OA. Black et al. (11) reported the results of the first randomized, blinded placebo-controlled trial exploring the effectiveness of stem cell therapy in dogs with chronic hip OA. Animals treated with AT-MSCs showed significant improvement in lameness scores, and in combined scores measuring lameness, pain, and range of motion, compared with control dogs. Black et al. (10) next evaluated the effectiveness of AT-MSC therapy in dogs with chronic OA of the humeroradial (elbow) joint. Lameness, pain upon manipulation, range of motion, and functional disability, were all significantly ameliorated 180 days after stem cell treatment.

Clinical Trials of Msc-Based Treatments for Human OA

Currently, 20 clinical trials are recruiting patients to test the efficacy of MSC injections in treatment of OA (see www.clinicaltrials.gov). These include trials run by the Cairo University School of Medicine in Egypt, the Royan Institute of Tehran University of Medicine, the Postgraduate Institute of Medical Education and Research, in Chandigarh, Ullevaal University Hospital in Oslo (Norway), the National University of Malaysia, the Red De Terapia Celular in Barcelona, the Pontifícia Universidade Católica do Paraná in Brazil, Nantes University Hospital, Stempeutics Research in India and Malaysia, Mesoblast in Australia, the University of Marseille in France, and Osiris Therapeutics in the US. However, the clinical evidence that the technique affords therapeutic benefits in humans is limited. The first data on the use of transplanted MSCs (seeded within collagen type I hydrogels) to repair isolated, full-thickness cartilage defects in humans were reported by Wakitani et al. (148). To date, the literature contains information on 15 clinical trials or case reports with follow-up durations of at least 6 months (Table 1). Reporting and evaluation included pain measurements made using visual analog scales, assessment of range of motion, and MRI. All reports describe reduced pain levels and improved function. MRI revealed significant growth of articular cartilage and regeneration of the meniscus. The longest term treatment and follow-up was conducted by Osiris Therapeutics Inc. (Columbia, MD). Reports on these phase I/II trials include results from seven institutions and 55 patients receiving single injections of BM-MSCs at doses of 50 million or 150 million cells. Cells, in a HA carrier, were injected into the knee joint 7-10 days after operative meniscectomy. All patients were followed-up for 2 years. Those who received MSCs experienced significant reductions in pain and reduced OA progression. Subchondral sclerosis and osteophyte formation were also reduced, compared to placebo controls. MRI revealed regrowth of the meniscus, suggesting that knee OA progression had been reduced (145).

Table 1.

Examples of Therapeutic Applications of Stem Cells in Osteoarthritis

Cell Origin	No. of Recipients	Method of Cell Delivery	Outcome	Follow-Up Duration	Ref.
BMSCs/type 1 collagen from porcine tendon	12 patients; range 49-70 years	MSCs embedded in collagen gel transplanted into the articular cartilage defect	Improved arthroscopic and histological grades	42 weeks	(148)
BMSCs/ceramic ankle prosthesis	Three patients; range 62-71 years	Transplanted into ankle osteoarthritis patients	Improved clinical scores, no inflammatory reaction	2 months-2 years	(107)
BMSCs/collagen gel	A 31-year-old male judo competitor	MSCs embedded within a collagen gel, transferred to the articular cartilage defect	Defects became filled with hyaline-like cartilage tissue	1 year	(74)
BMSCs	36 patients (20 men, 16 women); age: 16 patients <45 years; 20 patients ≥45 years	BMSCs were implanted beneath a patch	Improvement in physical function	2 years	(101)
BMSCs/platelet-rich fibrin glue	Five patients (fourmen, onewoman); range 21-37 years	MSCs placed on platelet-rich fibrin glue and transplanted into full-thickness cartilage defects	Improvement in physical function and symptoms, improvement in RHSSK score, MRI showed filling of cartilage defects and complete surface congruity with native cartilage	1 year	(43)
ADSCs/HA, platelet-rich plasma, calcium chloride, dexamethasone	Two patients: a 29-year-old woman and a 47-year-old man	Intra-articular injection	Increased meniscus cartilage volume on MRI, less pain	12 weeks	(110)
BMSCs	Four patients; range 54-65 years	Intra-articular injection	Walking time before pain onset improved in three patients but unchanged in one, VAS pain scores improved in all patients	6 months	(24)
BMSCs/collagen scaffold	Two patients	BMSCs were implanted into defects in combination with a three-dimensional collagen scaffold	Improvement in KOOS and IKDC scores, arthroscopic assessment showed good filling of defects, tissue stiffness was good, tissue was incorporated into adjacent cartilage	30-31 months	(64)
Infrapatellar fat pad-derived MSC/ platelet-rich plasma	25 patients (eight men, 17 women); range 34–69 years	Intra-articular injection	Improvement in the mean Lysholm score, the Tegner activity scale, and the VAS score	12-18 months	(71)
BMSCs	Six females: range 40-64 years	Intra-articular injection	Less pain and improved walking distance up to 6 months postinjection, continued slight improvement to 12 months postinjection, MRI showed an increase in cartilage thickness in three patients	1 year	(29)
Allogeneic BMSCs/HA	55 patients; range 18–60 years	Intra-articular injection	VAS scoring showed that patients with osteoarthritic changes who received MSCs experienced significant reductions in pain	2 years	(145)
Autologous activated peripheral blood stem cells with growth factor addition/ preservation, along with HA, in conjunction with arthroscopic microdrilling and mesenchymal cell stimulation	Five patients (four women, one man); range 52–59 years	Intra-articular injection	Improved WOMAC and KOO scores, electron microscopy revealed cell attachment and proliferation, histological analysis demonstrated the presence of hyaline cartilage	6 months	(142)
Infrapatellar fat pad-derived MSC/ platelet-rich plasma	18 patients (six men, 12 women); range 41–69 years	Intra-articular injection	Decreased Western Ontario and McMaster Universities Osteoarthritis Index scores, improved Lysholm scores, decreased VAS scores	24–26 months	(72)
BMSCs	12 patients (six men, six women); range 49±5 years	Intra-articular injection	Rapid and progressive improvement of algofunctional, quantitative MRI T2 mapping showed that diseased cartilage area decreased, improved cartilage quality in 11/12 patients	1 year	(108)
BMSCs	Three patients; range 12–21 years	Intra-articular injection	Improved function	2 years	(139)

BMSCs, bone marrow stromal cells; MSCs, mesenchymal stem cells; ADSCs, adipose-derived stem cells.

The benefits and disadvantages of allogeneic versus autologous cell-based clinical therapies continue to be debated. Some animal studies have shown that allogenic and autologous stem cells are similarly effective when used to repair cartilage defects (99,113,138). Nakamura et al. used allogenic synovial MSCs to repair cartilage defects in the pig. Histologically, no lymphocytic infiltration was noted and no antibodies against allogeneic cells were formed. No adverse immune response was detected (99). The transplanted cells adhered within the defects. Some transplanted cells were living at 26 weeks after transplantation. Transplanted cells have contributed to the regeneration of osteochondral defects (76,160), the repair of meniscal lesions (95), and the repair of tendon (129) and bone (3,116). Even xenografts showed promise (54,111,141). Based on animal studies, a clinical trial of allogeneic transplantation to treat severe osteoporosis (No. NCT00775931) was performed at the Masonic Cancer Center of the University of Minnesota in October 2012. The only clinical result reported so far (by Vangsness et al.) was that single MSC injections did not cause any problem up to 2 years later; no abnormal tissue growth was apparent (145). MSCs have also been used to treat conditions other than OA and cartilage defects. Allo-MSC treatment of systemic lupus erythematosus was safe (78,133,134) at a mean follow-up time of 29 months (range, 6 months to 7.5 years). Recent phase I clinical trials of allogenic MSC therapies in patients with myocardial infarction have validated the safety of allogenic cells (44). However, the long-term survival, engraftment, and mechanisms of action of such cells in humans remain unclear (1). Side effects also require review. Moll et al. have commented that administration of allogeneic MSCs can trigger immediate blood-mediated inflammatory reactions, leading to activation of the coagulation and complement cascades and the death of infused cells (94).

Potential problems have emerged in current clinical trials. The appropriate MSC dose for OA therapy needs clarification. MSC functionality and potency vary, and the best subtype of MSCs for use as OA therapy must be determined. Also, it remains unclear whether implanted MSCs survive and whether they become integrated into newly formed tissue. Thus, the ability to monitor the in vivo behavior of implanted MSCs in host tissue and to understand the fate of such MSCs is important for development of successful cell therapies. An effective, noninvasive, nontoxic technique, coupled with long-term cell tracking, is required. Further work is needed to determine whether MSCs should be chondrogenically induced prior to implantation in vivo. Some studies (60,161) have shown that noninduced stem cells repaired cartilage defects even better than did cells that had been chondrogenically induced (18) because stress, and the tissue fluid microenvironment, affected allogenic cells in vivo (84). However, a long-term study found that preinduced cells were superior to noninduced cells (155). Finally, the choice of OA patients is important. Patients who receive early treatment via intra-articular injection of MSCs enjoy better outcomes than do those treated later (1).

Safety of Msc Treatment

MSCs used to treat cartilage diseases are locally transplantable, but not necessarily systemically transplantable. The safety of such therapy was reviewed by Peeters et al., who analyzed 3,039 potentially eligible articles (112). In total, 844 procedures, with a mean follow-up duration of 21 months, were evaluated. Autologous BM-MSCs were used for cartilage repair and to treat osteoarthritis. Two serious adverse events were recorded. One was an infection following bone marrow aspiration (BMA); this was treated with antibiotics. One pulmonary embolism occurred 2 weeks after BMA. Twenty-two other instances of possible procedure-related adverse events (AEs) and seven possible stem cell product-related AEs were documented. The principal procedural AEs were increased pain/swelling, and dehydration after BMA. It was concluded that the use of cultured stem cells for intraarticular joint therapy appeared to be safe. It is reasonable to continue development of articular stem cell therapies. Apart from MSCs used to treat cartilage defects, local injection of MSCs to treat intervertebral disc degeneration has also been reported (89,109,130). Also, impressive case reports on bone regeneration using BM-MSCs, corneal resurfacing using limbal stem cells, and skin regeneration with cell populations containing epidermal stem cells, have appeared. These are applications in regenerative medicine (8). Turning to systemic infusion, such as intravenous injection of MSCs to treat heart failure, side effects have been reported. MSCs embolized in the lungs and damaged the local microcirculation or generated bone or fat in inappropriate organs if transplanted in sufficient numbers (8,12). Data from another clinical trial of MSCs to treat systemic lupus erythematosus have been reported; more than 200 cases were treated worldwide. Upon autologous stem cell transplantation, with a median follow-up duration of 38 months (range, 1 to 110 months), the 5-year overall survival rate was 81 ± 8%. In allogeneic MSC transplantation patients, with a mean follow-up period of 27 months, the 4-year overall survival rate was 94% (82/87), and no side effects were reported (132).

A total of 3,404 clinical trials worldwide, including 107 clinical trials of BM-MSCs used to treat a wide range of diseases, are registered with ClinicalTrials.gov. Further results from large-scale multicenter clinical trials are needed. It is necessary to standardize both the study entry criteria and the clinical response criteria and to focus more on safety (9).

Conclusions and Future Directions

Promising experimental and clinical data are beginning to emerge to support the use of MSCs for repair of articular cartilage defects and OA. Further efforts are needed to solve the remaining problems. Effective methods are required to maintain the articular cartilage phenotype (thus, to eliminate hypertrophy, ossification, and fibrinogenesis) and to construct an engineering method yielding tissue-mimicking native cartilage. A delivery system is needed that effectively localizes cells within a lesion in a manner encouraging chondrogenic differentiation with maintenance of the integrity of repaired tissue. Undoubtedly, a great deal of effort is required at both the basic and clinical research fronts. The promise is that MSCs used in cartilage tissue engineering or cell therapy will ultimately yield replacement cartilage for repair of damaged or diseased joints and will be employed routinely in the clinic to treat patients with OA.

Footnotes

Acknowledgments

This work was supported by National Science Foundation of China (NSFC, 8107458, 31240048, 81472092), National High Technology Research and Development Program of China (2012AA020502), Beijing Metropolis Beijing Nova program (2011115) and People's Liberation Army 12th five-year plan period (BWS11J025). The authors declare no conflicts of interest.

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Mesenchymal Stem Cells for Treating Articular Cartilage Defects and Osteoarthritis

Abstract

Keywords

Introduction

Current Treatments Are Less Than Satisfactory

Mesenchymal Stem Cells (MSCs) May Be A Source of Progenitor Cells for Cartilage Repair

Tissue Distribution and Sources of Human MSCs

Properties and Immunomodulatory Features of MSCs

MSCs as Seed Cells in Tissue Engineering to Repair Cartilage Defects

Clinical Applications of Msc Tissue Engineering in Repair of Cartilage Defects

Major Biological Obstacles to Persistent Cell-Based Regeneration of Articular Cartilage

Mechanical Properties of the Constructs

Heterogeneity and Inefficient Differentiation of MSCs

“Transient” or “Permanent” Chondrocytes

Improving the Structure and Functional Utilities of Msc-Based Tissue-Engineered Constructs

Identification of the Markers of Permanent Chondrocytes and Elimination of Heterogeneity

Mechanical Stimulation

Natural Cartilage-Derived ECM Scaffolds

Zonal Organization of Constructs

Mscs for Treatment of Chronic Degenerative Disorders, OA

Delivery Modes for MSCs

Msc-Based Treatment of OA in Animal Models

Clinical Trials of Msc-Based Treatments for Human OA

Safety of Msc Treatment

Conclusions and Future Directions

Footnotes

Acknowledgments

References