Premature induction of hypertrophy during in vitro chondrogenesis of human mesenchymal stem cells correlates with calcification and vascular invasion after ectopic transplantation in SCID mice

OBJECTIVE: Functional suitability and phenotypic stability of ectopic transplants are crucial factors in the clinical application of mesenchymal stem cells (MSCs) for articular cartilage repair, and might require a stringent control of chondrogenic differentiation. This study evaluated whether human bone marrow-derived MSCs adopt natural differentiation stages during induction of chondrogenesis in vitro, and whether they can form ectopic stable cartilage that is resistant to vascular invasion and calcification in vivo. METHODS: During in vitro chondrogenesis of MSCs, the expression of 44 cartilage-, stem cell-, and bone-related genes and the deposition of aggrecan and types II and X collagen were determined. Similarly treated, expanded articular chondrocytes served as controls. MSC pellets were allowed to differentiate in chondrogenic medium for 3-7 weeks, after which the chondrocytes were implanted subcutaneously into SCID mice; after 4 weeks in vivo, samples were evaluated by histology. RESULTS: The 3-stage chondrogenic differentiation cascade initiated in MSCs was primarily characterized by sequential up-regulation of common cartilage genes. Premature induction of hypertrophy-related molecules (type X collagen and matrix metalloproteinase 13) occurred before production of type II collagen and was followed by up-regulation of alkaline phosphatase activity. In contrast, hypertrophy-associated genes were not induced in chondrocyte controls. Whereas control chondrocyte pellets resisted calcification and vascular invasion in vivo, most MSC pellets mineralized, in spite of persisting proteoglycan and type II collagen content. CONCLUSION: An unnatural pathway of differentiation to chondrocyte-like cells was induced in MSCs by common in vitro protocols. MSC pellets transplanted to ectopic sites in SCID mice underwent alterations related to endochondral ossification rather than adopting a stable chondrogenic phenotype. Further studies are needed to evaluate whether a more stringent control of MSC differentiation to chondrocytes can be achieved during cartilage repair in a natural joint environment.     

Genetic enhancement of matrix synthesis by articular chondrocytes: comparison of different growth factor genes in the presence and absence of interleukin-1

OBJECTIVE: To determine whether articular chondrocytes express growth factor genes delivered by adenoviral vectors and whether expression of these genes influences matrix synthesis in the presence and absence of interleukin-1 (IL-1). METHODS: Monolayer cultures of rabbit articular chondrocytes were infected with recombinant adenovirus carrying genes encoding the following growth factors: insulin-like growth factor 1 (IGF-1), transforming growth factor beta1 (TGFbeta1), and bone morphogenetic protein 2 (BMP-2). As a control, cells were transduced with the lac Z gene. Cultures were also treated with each growth factor supplied as a protein. Levels of gene expression were noted, and the synthesis of proteoglycan, collagen, and noncollagenous proteins was measured by radiolabeling. Collagen was typed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The effects of growth factor gene transfer on proteoglycan synthesis in the presence of IL-1 were also measured. RESULTS: The expression of all transgenes was high following adenoviral transduction. Proteoglycan synthesis was stimulated approximately 8-fold by the BMP-2 gene and 2-3-fold by the IGF-1 gene. The effects of BMP-2 and IGF-1 genes were additive upon cotransduction. Synthesis of collagen and noncollagenous proteins, in contrast, was most strongly stimulated by the IGF-1 gene. In each case, collagen typing confirmed the synthesis of type II collagen. IL-1 suppressed proteoglycan synthesis by 50-60%. IGF-1 and TGFbeta genes restored proteoglycan synthesis to control levels in the presence of IL-1. The BMP-2 gene, in contrast, elevated proteoglycan synthesis beyond control levels in the presence of IL-1. CONCLUSION: Transfer of growth factor genes to articular chondrocytes can greatly increase matrix synthesis in vitro, even in the presence of the inflammatory cytokine IL-1. This result encourages the further development of gene therapy for the repair of damaged cartilage.     

Adenovirus-mediated gene transfer of insulin-like growth factor 1 stimulates proteoglycan synthesis in rabbit joints

OBJECTIVE: To examine the effect of insulin-like growth factor 1 (IGF-1) on the regulation of cartilage synthesis and other articular events in vivo. METHODS: A first-generation adenoviral vector expressing human IGF-1 (AdIGF-1) from the cytomegalovirus promoter was constructed. Particles of AdIGF-1 (5 x 10(9)) were injected through the patellar tendon into normal rabbit knee joints and rabbit knee joints with antigen-induced arthritis (AIA), with the same dose of a control adenoviral vector injected into the contralateral knees. Lavage fluids were obtained from rabbit knee joints on days 3 and 7 postinjection and used for analysis of IGF-1 expression, white blood cell infiltration, and cartilage breakdown. Cartilage chips from rabbit joints were used for assay of new proteoglycan synthesis, and tissues also were harvested from the dissected knees for histologic study. RESULTS: Intraarticular injection of AdIGF-1 resulted in a mean of 180.6 ng/ml of IGF-1 expression in the lavage fluid from rabbit joints. IGF-1 expression stimulated new proteoglycan synthesis in both naive and AIA rabbit knees, but had no significant chondroprotective or antiinflammatory effects. Histologic analysis showed that elevated levels of IGF-1 expression in both normal and arthritic knees had no adverse pathologic effects on synovium or adjacent muscles. CONCLUSION: Gene transfer of IGF-1 into rabbit knee joints promotes proteoglycan synthesis without significantly affecting inflammation or cartilage breakdown. In addition, no adverse effects following intraarticular IGF-1 gene delivery were observed. Thus, local gene transfer of IGF-1 to joints could serve as a therapeutic strategy to stimulate new matrix synthesis in both rheumatoid arthritis and osteoarthritis.     

Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells in pellet cultural system

OBJECTIVE: Pluripotent mesenchymal stem cells (MSC) have been isolated and well characterized from several tissue sources, including bone marrow stroma. MSC from different animals showed slight differences in morphology and in the potential to differentiate. In the present study, we isolated MSC from bovine bone marrow and induced chondrogenesis in order to establish a new experimental model of stem cell research. METHODS: Bone marrow was harvested from 8 calves. For inducing chondrogenesis, MSC were cultured in pellet culture system in a chemically defined medium supplemented with 0 and 10 ng/mL of transforming growth factor beta1 (TGF-beta1). Chondrogenic differentiation was evaluated by histological, immunohistochemical, and in situ hybridization techniques. The degrees of genes expression were measured by quantitative RT-PCR. RESULTS: Metachromatic alcian blue staining and immunoreactivity for type II collagen were detected in both pellet groups (0 and 10 ng/mL TGF-beta1) after 7 days of culturing. In situ hybridization demonstrated strong expression of type II collagen and aggrecan mRNAs in the round cells located at the center region of pellets and at densely organized areas. On the other hand, type I collagen mRNA was strongly expressed in the superficial layer of the pellets. After 20 days of pellet culture, expression of type II collagen mRNA in the cells which were not treated by TGF-beta1 was 1.7-fold higher compared with that treated by TGF-beta1. CONCLUSION: Independent, spontaneous chondrogenesis of bovine MSC in pellet culture occurred without addition of any external bioactive stimulators, namely factors from TGF-beta family, which were previously considered necessary.     

In vitro stage‐specific chondrogenesis of mesenchymal stem cells committed to chondrocytes

Objective

Osteoarthritis is characterized by an imbalance in cartilage homeostasis, which could potentially be corrected by mesenchymal stem cell (MSC)–based therapies. However, in vivo implantation of undifferentiated MSCs has led to unexpected results. This study was undertaken to establish a model for preconditioning of MSCs toward chondrogenesis as a more effective clinical tool for cartilage regeneration.

Methods

A coculture preconditioning system was used to improve the chondrogenic potential of human MSCs and to study the detailed stages of chondrogenesis of MSCs, using a human MSC line, Kp‐hMSC, in commitment cocultures with a human chondrocyte line, hPi (labeled with green fluorescent protein [GFP]). In addition, committed MSCs were seeded into a collagen scaffold and analyzed for their neocartilage‐forming ability.

Results

Coculture of hPi‐GFP chondrocytes with Kp‐hMSCs induced chondrogenesis, as indicated by the increased expression of chondrogenic genes and accumulation of chondrogenic matrix, but with no effect on osteogenic markers. The chondrogenic process of committed MSCs was initiated with highly activated chondrogenic adhesion molecules and stimulated cartilage developmental growth factors, including members of the transforming growth factor β superfamily and their downstream regulators, the Smads, as well as endothelial growth factor, fibroblast growth factor, insulin‐like growth factor, and vascular endothelial growth factor. Furthermore, committed Kp‐hMSCs acquired neocartilage‐forming potential within the collagen scaffold.

Conclusion

These findings help define the molecular markers of chondrogenesis and more accurately delineate the stages of chondrogenesis during chondrocytic differentiation of human MSCs. The results indicate that human MSCs committed to the chondroprogenitor stage of chondrocytic differentiation undergo detailed chondrogenic changes. This model of in vitro chondrogenesis of human MSCs represents an advance in cell‐based transplantation for future clinical use.

     

Human articular chondrocytes secrete parathyroid hormone–related protein and inhibit hypertrophy of mesenchymal stem cells in coculture during chondrogenesis

Objective

The use of bone marrow–derived mesenchymal stem cells (MSCs) has shown promise in cell‐based cartilage regeneration. A yet‐unsolved problem, however, is the unwanted up‐regulation of markers of hypertrophy, such as alkaline phosphatase (AP) and type X collagen, during in vitro chondrogenesis and the formation of unstable calcifying cartilage at heterotopic sites. In contrast, articular chondrocytes produce stable, nonmineralizing cartilage. The aim of this study was to address whether coculture of MSCs with human articular chondrocytes (HACs) can suppress the undesired hypertrophy in differentiating MSCs.

Methods

MSCs were differentiated in chondrogenic medium that had or had not been conditioned by parallel culture with HAC pellets, or MSCs were mixed in the same pellet with the HACs (1:1 or 1:2 ratio) and cultured for 6 weeks. Following in vitro differentiation, the pellets were transplanted into SCID mice.

Results

The gene expression ratio of COL10A1 to COL2A1 and of Indian hedgehog (IHH) to COL2A1 was significantly reduced by differentiation in HAC‐conditioned medium, and less type X collagen protein was deposited relative to type II collagen. AP activity was significantly lower (P < 0.05) in the cells that had been differentiated in conditioned medium, and transplants showed significantly reduced calcification in vivo. In mixed HAC/MSC pellets, suppression of AP was dose‐dependent, and in vivo calcification was fully inhibited. Chondrocytes secreted parathyroid hormone–related protein (PTHrP) throughout the culture period, whereas PTHrP was down‐regulated in favor of IHH up‐regulation in control MSCs after 2–3 weeks of chondrogenesis. The main inhibitory effects seen with HAC‐conditioned medium were reproducible by PTHrP supplementation of unconditioned medium.

Conclusion

HAC‐derived soluble factors and direct coculture are potent means of improving chondrogenesis and suppressing the hypertrophic development of MSCs. PTHrP is an important candidate soluble factor involved in this effect.

     

Articular cartilage repair by gene therapy using growth factor-producing mesenchymal cells

OBJECTIVE: To investigate the repair of partial-thickness lesions in rat articular cartilage by combining cell transplantation with transfer of growth factor complementary DNA (cDNA). METHODS: Mesenchymal cells isolated from rib perichondrium were infected ex vivo with adenoviral vectors carrying bone morphogenetic protein 2 (BMP-2) or insulin-like growth factor 1 (IGF-1) cDNA. The cells were suspended in fibrin glue and applied to mechanically induced partial-thickness cartilage lesions in the patellar groove of the rat femur. The filling of the defects was quantified and the quality and integration of the newly formed tissue were assessed by histochemical and immunohistochemical methods. Uninfected cells or cells infected with a LacZ reporter gene vector served as controls. RESULTS: Transplanted cells were able to attach to the wounded articular cartilage and were not displaced from the lesions by joint movement. Cells infected with both adenoviral vectors AdBMP-2 and AdIGF-1 produced repair cartilage of hyaline morphology containing a type II collagen-positive but type I collagen-negative proteoglycan-rich matrix that restored the articular surface in most lesions. Uninfected cells either failed to fill up the defects or formed fibrous tissue mainly composed of type I collagen. Excessive cells were partially dislocated to the joint margins, leading to osteophyte formation there if AdBMP-2-infected cells were used. These adverse effects, however, were not seen with AdIGF-1-infected cells. CONCLUSION: Stimulation of perichondrium-derived mesenchymal cells by transfer of growth factor cDNA in a partial-thickness defect model allows for satisfactory cartilage restoration by a repair tissue comparable with hyaline articular cartilage.     

Inflammation location, but not type, determines the increase in TGF-beta1 and IGF-1 expression and collagen deposition in IBD intestine

BACKGROUND AND AIMS: Inflammatory bowel disease (IBD) is frequently complicated by extracellular matrix (ECM) changes that may result in fibrosis. Transforming growth factor (TGF)-beta1 and insulin-like growth factor (IGF)-1 mediate numerous ECM changes. Our aim was to determine whether TGF-beta1 and IGF-1 are involved in intestinal ECM collagen regulation and what impact the inflammatory infiltrate has on their expression. METHODS: TGF-beta1 and IGF-1 mRNA and protein were assessed in fibrosed Crohn's disease (CD), inflamed CD, inflamed ulcerative colitis (UC), and control intestine using in situ hybridization and immunohistochemistry. Collagen types I and III were quantified by electron immunohistochemistry. RESULTS: In CD, increased TGF-beta1 and IGF-1 mRNA expression was transmural. In UC, the increase was confined to the lamina propria and submucosa. In both, distribution of TGF-beta1 and IGF-1 protein matched mRNA expression and coincided with the distribution of the inflammatory infiltrate. An increase in the collagen type III:I ratio in both CD and UC also coincided with the inflammatory infiltrate. CONCLUSIONS: These findings suggest that TGF-beta1 and IGF-1 are involved in intestinal ECM remodeling in IBD, and their enhanced expression depends on the presence and location of inflammatory infiltrates rather than the type of IBD.     

Potent induction of chondrocytic differentiation of human adipose-derived adult stem cells by bone morphogenetic protein 6

OBJECTIVE: Recent studies have identified an abundant source of multipotent progenitor cells in subcutaneous human adipose tissue, termed human adipose-derived adult stem cells (ADAS cells). In response to specific media formulations, including transforming growth factor beta1 (TGFbeta1), these cells exhibit significant ability to differentiate into a chondrocyte-like phenotype, expressing cartilage-specific genes and proteins such as aggrecan and type II collagen. However, the influence of other growth factors on the chondrogenic differentiation of ADAS cells is not fully understood. This study was undertaken to investigate the effects of TGFbeta1, TGFbeta3, insulin-like growth factor 1, bone morphogenetic protein 6 (BMP-6), and dexamethasone, in various combinations, on the chondrogenic potential of ADAS cells in alginate beads. METHODS: The chondrogenic response of alginate-encapsulated ADAS cells was measured by quantitative polymerase chain reaction, 3H-proline and 35S-sulfate incorporation, and immunolabeling for specific extracellular matrix components. RESULTS: Significant differences in chondrogenesis were observed under the different culture conditions for all outcomes measured. Most notably, BMP-6 up-regulated AGC1 and COL2A1 expression by an average of 205-fold and 38-fold, respectively, over day-0 controls, while down-regulating COL10A1 expression by approximately 2-fold. CONCLUSION: These findings suggest that BMP-6 is a potent inducer of chondrogenesis in ADAS cells, in contrast to mesenchymal stem cells, which exhibit increased expression of type X collagen and a hypertrophic phenotype in response to BMP-6. Combinations of growth factors containing BMP-6 may provide a novel means of regulating the differentiation of ADAS cells for applications in the tissue-engineered repair or regeneration of articular cartilage.     

Cartilage repair in a rat model of osteoarthritis through intraarticular transplantation of muscle‐derived stem cells expressing bone morphogenetic protein 4 and soluble flt‐1

Objective

The control of angiogenesis during chondrogenic differentiation is an important issue affecting the use of stem cells in cartilage repair, especially with regard to the persistence of regenerated cartilage. This study was undertaken to investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the blocking of VEGF with its antagonist, soluble Flt‐1 (sFlt‐1), on the chondrogenesis of skeletal muscle‐derived stem cells (MDSCs) in a rat model of osteoarthritis (OA).

Methods

We investigated the effect of VEGF on cartilage repair in an immunodeficiency rat model of OA after intraarticular injection of murine MDSCs expressing bone morphogenetic protein 4 (BMP‐4) in combination with MDSCs expressing VEGF or sFlt‐1.

Results

In vivo, a combination of sFlt‐1– and BMP‐4–transduced MDSCs demonstrated better repair without osteophyte formation macroscopically and histologically following OA induction, when compared with the other groups. Higher differentiation/proliferation and lower levels of chondrocyte apoptosis were also observed in sFlt‐1– and BMP‐4–transduced MDSCs compared with a combination of VEGF‐ and BMP‐4–transduced MDSCs or with BMP‐4–transduced MDSCs alone. In vitro experiments with mixed pellet coculture of MDSCs and OA chondrocytes revealed that BMP‐4–transduced MDSCs produced the largest pellets, which had the highest gene expression of not only type II collagen and SOX9 but also type X collagen, suggesting formation of hypertrophic chondrocytes.

Conclusion

Our results demonstrate that MDSC‐based therapy involving sFlt‐1 and BMP‐4 repairs articular cartilage in OA mainly by having a beneficial effect on chondrogenesis by the donor and host cells as well as by preventing angiogenesis, which eventually prevents cartilage resorption, resulting in persistent cartilage regeneration and repair.

     

Cartilage repair using bone morphogenetic protein 4 and muscle-derived stem cells

OBJECTIVE: Muscle-derived stem cells (MDSCs) isolated from mouse skeletal muscle exhibit long-time proliferation, high self-renewal, and multipotent differentiation. This study was undertaken to investigate the ability of MDSCs that were retrovirally transduced to express bone morphogenetic protein 4 (BMP-4) to differentiate into chondrocytes in vitro and in vivo and enhance articular cartilage repair. METHODS: Using monolayer and micromass pellet culture systems, we evaluated the in vitro chondrogenic differentiation of LacZ- and BMP-4-transduced MDSCs with or without transforming growth factor beta1 (TGFbeta1) stimulation. We used a nude rat model of a full-thickness articular cartilage defect to assess the duration of LacZ transgene expression and evaluate the ability of transplanted cells to acquire a chondrocytic phenotype. We evaluated cartilage repair macroscopically and histologically 4, 8, 12, and 24 weeks after surgery, and performed histologic grading of the repaired tissues. RESULTS: BMP-4-expressing MDSCs acquired a chondrocytic phenotype in vitro more effectively than did MDSCs expressing only LacZ; the addition of TGFbeta1 did not alter chondrogenic differentiation of the BMP-4-transduced MDSCs. LacZ expression within the repaired tissue continued for up to 12 weeks. Four weeks after surgery, we detected donor cells that coexpressed beta-galactosidase and type II collagen. Histologic scoring of the defect sites 24 weeks after transplantation revealed significantly better cartilage repair in animals that received BMP-4-transduced MDSCs than in those that received MDSCs expressing only LacZ. CONCLUSION: Local delivery of BMP-4 by genetically engineered MDSCs enhanced chondrogenesis and significantly improved articular cartilage repair in rats.     

Inhibition of P-glycoprotein facilitated glycosaminoglycan accumulation during chondrogenesis of human bone marrow mesenchymal stem cells

Aim: P‐glycoprotein (P‐gp) is an adenosine‐5‐triphosphate Binding Cassettes B 1 (ABCB1) transporter that exports various substrates on cellular membrane. Surface expression of P‐gp was decreased during chondrogenesis of human bone marrow mesenchymal stem cells (BM‐MSCs). We examined the role of P‐gp in extracellular matrix deposition during chondrogenesis of human BM‐MSCs. Method: BM‐MSCs were isolated from 16 volunteers after informed consent and incubated for 28 days using three‐dimensional culture methods in chondrogenic medium with and without P‐gp inhibitor (verapamil, 10 μmol/L). Results: Hematoxylin and eosin staining revealed a cartilaginous structure with chondrogenic cells in the lacunae after 2 weeks of culture. Total glycosaminoglycan (GAG) content was increased and rose during pellet culture. Hyaluronan (HA) content of the culture medium decreased with P‐gp inhibitor. Type II collagen deposition decreased with P‐gp inhibitor. Conclusion: Inhibition of P‐gp facilitated GAG accumulation via HA export inhibition during chondrogenic differentiation of human BM‐MSCs. Modulation of P‐gp expression during chondrogenesis would be a possible therapeutic approach for articular cartilage regeneration.     

Blocking vascular endothelial growth factor with soluble Flt‐1 improves the chondrogenic potential of mouse skeletal muscle–derived stem cells

Objective

To investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the effect of blocking VEGF with its antagonist, soluble Flt‐1 (sFlt‐1), on chondrogenesis, using muscle‐derived stem cells (MDSCs) isolated from mouse skeletal muscle.

Methods

The direct effect of VEGF on the in vitro chondrogenic ability of mouse MDSCs was tested using a pellet culture system, followed by real‐time quantitative polymerase chain reaction (PCR) and histologic analyses. Next, the effect of VEGF on chondrogenesis within the synovial joint was tested, using genetically engineered MDSCs implanted into rat osteochondral defects. In this model, MDSCs transduced with a retroviral vector to express bone morphogenetic protein 4 (BMP‐4) were coimplanted with MDSCs transduced to express either VEGF or sFlt‐1 (a VEGF antagonist) to provide a gain‐ and loss‐of‐function experimental design. Histologic scoring was used to compare cartilage formation among the treatment groups.

Results

Hyaline‐like cartilage matrix production was observed in both VEGF‐treated and VEGF‐blocked (sFlt‐1–treated) pellet cultures, but quantitative PCR revealed that sFlt‐1 treatment improved the expression of chondrogenic genes in MDSCs that were stimulated to undergo chondrogenic differentiation with BMP‐4 and transforming growth factor β3 (TGFβ3). In vivo testing of articular cartilage repair showed that VEGF‐transduced MDSCs caused an arthritic change in the knee joint, and sFlt‐1 improved the MDSC‐mediated repair of articular cartilage, compared with BMP‐4 alone.

Conclusion

Soluble Flt‐1 gene therapy improved the BMP‐4– and TGFβ3‐induced chondrogenic gene expression of MDSCs in vitro and improved the persistence of articular cartilage repair by preventing vascularization and bone invasion into the repaired articular cartilage.

     

Chondrogenic differentiation of bovine synovium: bone morphogenetic proteins 2 and 7 and transforming growth factor beta1 induce the formation of different types of cartilaginous tissue

OBJECTIVE: To compare the potential of bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7) and transforming growth factor beta1 (TGFbeta1) to effect the chondrogenic differentiation of synovial explants by analyzing the histologic, biochemical, and gene expression characteristics of the cartilaginous tissues formed. METHODS: Synovial explants derived from the metacarpal joints of calves were cultured in agarose. Initially, BMP-2 was used to evaluate the chondrogenic potential of the synovial explants under different culturing conditions. Under appropriate conditions, the chondrogenic effects of BMP-2, BMP-7, and TGFbeta1 were then compared. The differentiated tissue was characterized histologically, histomorphometrically, immunohistochemically, biochemically, and at the gene expression level. RESULTS: BMP-2 induced the chondrogenic differentiation of synovial explants in a dose- and time-dependent manner under serum- and dexamethasone-free conditions. The expression levels of cartilage-related genes increased in a time-dependent manner. BMP-7 was more potent than BMP-2 in inducing chondrogenesis, but the properties of the differentiated tissue were similar in each case. The type of cartilaginous tissue formed under the influence of TGFbeta1 differed in terms of both cell phenotype and gene expression profiles. CONCLUSION: The 3 tested members of the TGFbeta superfamily have different chondrogenic potentials and induce the formation of different types of cartilaginous tissue. To effect the full differentiation of synovial explants into a typically hyaline type of articular cartilage, further refinement of the stimulation conditions is required. This might be achieved by the simultaneous application of several growth factors.