Damage to articular cartilage frequently results from injury or disease. Because of its limited intrinsic healing capacity, articular cartilage cannot fully regenerate; thus, articular cartilage injuries eventually lead to secondary degenerative disease of the involved joint (1, 2). Researchers have designed clinical methods and proposed various experimental approaches by which to achieve better repair of injured articular cartilage, including abrasion arthroplasty (3, 4), microfracture (5), and transplantation of chondrocytes (6–9), perichondrium (10), meniscal allografts (11), periosteum (12), or osteochondral grafts (13). However, there is no known treatment that enables full restoration of injured articular cartilage to its original phenotype.
Tissue engineering based on cell and gene therapy is one of the most promising new approaches by which to repair various tissues, including articular cartilage. This process involves the use of various cell types that can repair articular cartilage by acting as chondroprogenitor cells, as gene delivery vehicles that produce a therapeutic protein at the site of interest, or as both. The variety of cells available for use in cartilage tissue engineering ranges from undifferentiated pluripotent stem cells to well-differentiated chondrocytes (6–9, 14, 15). Chondrocytes are a natural and logical choice for articular cartilage repair applications. However, a limited donor-site capacity to provide a large quantity of chondrocytes is a major impediment to autologous chondrocyte transplantation. Therefore, there are ongoing efforts to identify other cell populations that contain chondroprogenitor cells and are easily isolated and expandable.
In light of its availability and the relative ease of muscle cell isolation, skeletal muscle is an attractive source of cells for use in cartilage tissue engineering applications. Adachi et al recently reported comparable healing of cartilage defects treated with collagen gel containing either muscle-derived cells (MDCs) or chondrocytes (16), which suggests that skeletal muscle may contain cells that can aid in cartilage repair. Several studies have provided evidence of the existence of pluripotent stem cells in skeletal muscle that can differentiate into various lineages, including myogenic, hematopoietic, osteogenic, endothelial, and neuronal (17–20). However, the differentiation of muscle-derived stem cells (MDSCs) into the chondrogenic lineage has not yet been demonstrated.
The administration of growth factors that can enhance cartilage healing is another important facet of cartilage repair. Several growth factors, including transforming growth factor β (TGFβ), bone morphogenetic proteins (BMPs), insulin-like growth factor 1, and basic fibroblast growth factor, can improve chondrocyte proliferation and extracellular matrix (ECM) synthesis in vitro and in vivo (21–28). Studies performed in vitro and in the developing skeleton have led to the identification of BMP-4 as a promising candidate for the promotion of chondrogenesis (29, 30). Steinert et al observed particularly high expression of chondrogenic markers in populations of BMP-4–transfected mesenchymal progenitor cells (31).
Although the results of some studies suggest that direct injection of therapeutic proteins can promote cartilage healing, the relatively short half-lives of these proteins in vivo often necessitate high dosages or repeated injections. It also can be difficult to deliver these growth factors to the injury site. Moreover, the possibility exists that unregulated high dosages or repeated injections of growth factors could negatively influence both injured and normal structures and lead to adverse effects, such as hypertrophy or osteophyte formation (32). Gene therapy, a process whereby donor cells are genetically engineered to deliver a therapeutic protein to the site of injury and thereby promote tissue repair, provides a promising alternative to the direct injection of therapeutic proteins.
We designed this study to determine if MDSCs could serve as a source of progenitor cells for cartilage repair and as gene delivery vehicles for BMP-4. We posited that the impressive self-renewal and long-term proliferation ability of MDSCs (17) would enable enhanced and prolonged gene expression within cartilage defects, which would promote cartilage repair. Our primary aim was to evaluate the ability of BMP-4–expressing MDSCs to differentiate into chondrocytes in vitro and repair articular cartilage defects in vivo.
We first cultured cells in a monolayer or in a 3-dimensional pellet culture system either in normal differentiation medium or chondrogenic medium (CM) with or without TGFβ1 supplementation. We then transplanted the transduced MDSCs into cartilage defects created in athymic rats and investigated the distribution of the LacZ transgene in the repaired tissues and the expression of chondrogenic differentiation markers by the donor cells. Finally, we evaluated the regenerative ability of the donor cells and determined the role of ex vivo BMP-4 gene therapy in the healing of articular cartilage.
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- MATERIALS AND METHODS
BMP4-expressing MDSCs used in ex vivo gene therapy produce bone by endochondral ossification, a process by which cartilage and bone form within the injected tissue (34, 37). We know of no other published studies designed to investigate the potential of these cells in promoting articular cartilage repair. The secretion of chondrogenic, antiarthritic, or chondroprotective proteins by locally transplanted cells could result in faster repair and restoration of normal hyaline cartilage within lesions. Here we evaluated the ability of BMP-4–expressing MDSCs to undergo chondrogenic differentiation in vitro and in vivo. We also assessed the feasibility of using MDSC-based ex vivo gene therapy to enhance the healing of full-thickness articular cartilage defects in vivo.
As evidenced by the results of this study, environmental stimuli, such as culture medium conditions and circulating growth factors, are essential to the differentiation of stem cells toward a specific lineage. MDSC and MDSC-B4 cultured in monolayer in normal differentiation medium did not undergo chondrogenic differentiation. When cells were cultured in CM they began to express CII, an indicator of chondrogenesis. We detected significantly more CII-positive colonies in the MDSC-B4 cultures than in the MDSC cultures maintained in CM for 4 days. This finding demonstrates the importance of BMP-4 secretion by the transduced cells and indicates that BMP-4 influences the differentiation of multipotent MDSCs toward the chondrogenic lineage.
We also investigated the effect of TGFβ1 on MDSC and MDSC-B4 cultures in vitro. Research has shown that TGFβ1 has a significant effect on chondrogenesis by mesenchymal stem cells derived from bone marrow (35, 38–43) and periosteum (44, 45). The addition of TGFβ1 to the MDSC cultures in CM increased the number of CII-positive colonies. However, the addition of TGFβ1 to the MDSC-B4 cultures in CM slightly decreased the number of CII-positive colonies. This finding suggests that exposure to TGFβ1 (10 ng/ml) for 4 days did not enhance the chondrogenic differentiation exhibited by MDSC-B4.
Studies involving embryonic stem cells and mesenchymal progenitor cells also have demonstrated the beneficial effect of BMP-4 on chondrogenic differentiation in vitro (30, 31, 46). Other research groups have used the culturing of cells in a micromass pellet culture system as an assay for chondrogenesis, and this approach appears to be optimal for assessing the chondrogenic differentiation of mesenchymal stem cells (35, 41–43). Here we used the pellet culture system and found that BMP-4 plays an important role in promoting the chondrogenic differentiation of MDSCs. This finding confirms the results obtained from the monolayer cell culture. BMP-4 promoted aggregation of the cells, as evidenced by the size and structure of the pellets, and led to their adoption of a chondrocyte-like morphology. The MDSC formed smaller, less condensed pellets that did not contain chondrocyte-like cells, whereas the MDSC-B4 formed bigger, more condensed pellets that contained cells that displayed hypertrophic chondrocyte morphology. This study shows that, under appropriate culture conditions, BMP-4–expressing MDSCs can acquire a chondrocytic phenotype in vitro.
To validate these in vitro results, we attempted to use genetically engineered MDSCs to repair articular cartilage in vivo. We began by demonstrating that transduction of the MDSCs with the LacZ marker gene enabled detection of the transplanted cells within the treated defects for up to 12 weeks after transplantation. Adachi et al (16) previously reported that MDCs and chondrocytes used for osteochondral transplantation expressed LacZ in the repaired tissues for only up to 4 weeks after transplantation. During the cartilage healing process, 4 weeks of protein expression may be insufficient to generate mature repaired tissue. Our results, compared with those generated by MDC or chondrocyte transplantation (16, 47, 48), indicate that the better survival of transplanted MDSCs led to significantly enhanced and prolonged LacZ transgene expression within the cartilage lesions.
LacZ-positive and Alcian blue–positive hypertrophic chondrocytes were present within the repaired tissues 4 weeks after transplantation of MDSC-B4. Furthermore, we were able to colocalize β-gal with CII, a chondrogenic marker, by immunohistochemistry. These findings indicate that the transplanted cells acquired a chondrocyte-like phenotype and participated in the repair of articular cartilage.
Macroscopic examination of the knee joints before preparation for histologic examination was predictive of the histologic results. Regenerated cartilage harvested from the MDSC- and MDSC-B4–treated defects received significantly higher histologic grading scores than did repaired tissue harvested from the control defects 8 and 12 weeks after transplantation. Furthermore, the repaired cartilage obtained from the MDSC-B4 treatment group received significantly higher scores than that obtained from the control or MDSC treatment groups 24 weeks after transplantation. This finding indicates that continuous endogenous BMP-4 supplied by MDSCs genetically modified to express BMP-4 over an extended period of time can enhance articular cartilage healing. Although we observed no ossification of the reparative tissue during the 24-week healing period in this study, we cannot exclude the possible occurrence of ossification at a later time.
Scaffold optimization is an essential step to ensure successful cartilage tissue engineering. Many researchers have chosen to focus on the study of cell-seeded matrices in an effort to engineer synthetic cartilage in vitro for implantation in vivo. The use of cells suspended in fibrin glue instead of solid grafts enables the suspended cells to settle even in narrow clefts, and fast-forming clots are able to prevent any initial displacement (31). Research has demonstrated that fibrin glue has no adverse effects on cell viability and is a suitable matrix for applications designed to promote chondrogenesis (49). Furthermore, some findings suggest that fibrin and fibronectin, the primary components of fibrin glue, could play a role in cell–matrix and matrix–matrix interactions (50). Our study confirmed that a fibrin glue matrix allows MDSCs to proliferate and acquire a chondrocyte-like phenotype after osteochondral transplantation.
This study demonstrates the feasibility of using ex vivo BMP-4 gene therapy involving retroviral transduction of MDSCs to repair articular cartilage. Skeletal muscle is a promising source of progenitor cells that can undergo chondrogenic differentiation in vitro and in vivo under proper stimulation. The MDSCs used here served as good carriers of a therapeutic gene and enabled the delivery of appropriate amounts of BMP-4 protein to the injury site. This technique resulted in improved repair of articular cartilage that persisted for 24 weeks after transplantation. Using other populations of MDSCs isolated from normal mice and retrovirally transduced to express BMP-4, we have been able to produce similar results in vitro and in vivo (data not shown). These findings suggest that BMP-4 gene therapy based on retrovirally transduced MDSCs is a potential strategy by which to improve articular cartilage healing.