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.     

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.

     

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 β1 (TGFβ1), 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 TGFβ1, TGFβ3, 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 ∼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.

     

Articular cartilage repair using dedifferentiated articular chondrocytes and bone morphogenetic protein 4 in a rabbit model of articular cartilage defects

OBJECTIVE: To observe redifferentiation of dedifferentiated chondrocytes after transplantation into the joint, and to evaluate the ability of dedifferentiated chondrocytes transduced with adenovirus containing bone morphogenetic protein 4 (BMP-4) to redifferentiate in vitro and in vivo in a rabbit model of articular cartilage defects. METHODS: Monolayer and pellet culture systems were used to evaluate the redifferentiation of dedifferentiated chondrocytes transduced with BMP-4. A rabbit model of partial-thickness articular cartilage defects was used to evaluate cartilage repair macroscopically and histologically, 6 and 12 weeks after transplantation with first-passage, fifth-passage, or transduced fifth-passage chondrocytes. Histologic grading of the repaired tissue was performed. Expression of BMP-4 and the ability of transplanted cells to recover a chondrocytic phenotype were also assessed. RESULTS: BMP-4--expressing dedifferentiated chondrocytes recovered a chondrocytic phenotype in vitro. After transplantation into the joint, some of the dedifferentiated chondrocytes in the defect sites could undergo redifferentiation and formed matrix that displayed positive toluidine blue staining for glycosaminoglycans. Histologic scores of the regenerative tissue revealed significantly better cartilage repair in rabbits transplanted with BMP-4--expressing cells than in the other treatment groups. Staining with toluidine blue revealed expression of BMP-4 in the cells and in the matrix surrounding the cells. CONCLUSION: Some dedifferentiated chondrocytes can redifferentiate after transplantation into the load-bearing joint. BMP-4 can be used to induce redifferentiation of dedifferentiated chondrocytes in vitro and in vivo, which could help enhance articular cartilage repair.     

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.

     

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.     

Bone regeneration using bone morphogenetic protein (BMP)

Bone morphogenetic protein (BMP) is one of the most promising osteoinductive substances and is expected to be applied clinically for bone reconstruction. BMP has restored critical-size bone defects in numerous animal experiments, but the evaluation of bone formation by BMP in primates is a prerequisite for its clinical application. We attempted to restore the mandible bone defects in primates using BMP. The implantation of BMP with the carrier completely regenerated the mandible bone defects in the young primates, and the occlusal function was restored by the dental implants inserted into the regenerated bone. Although the use of BMP alone to regenerate mandible bone defects in old monkeys produced inconclusive results, the combination grafts of BMP and bone marrow, which contained osteoprogenitor cells, were successful. Furthermore, the combination of BMP and the culture-expanded cells derived form bone marrow grafts regenerate the segmental bone defects in the mandibles of old monkeys. Thus, the implantation of BMP with the BMP-responding cells could restore large bone defects even in elderly patients.     

Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells

The development of regenerative therapies for cartilage injury has been greatly aided by recent advances in stem cell biology. Induced pluripotent stem cells (iPSCs) have the potential to provide an abundant cell source for tissue engineering, as well as generating patient-matched in vitro models to study genetic and environmental factors in cartilage repair and osteoarthritis. However, both cell therapy and modeling approaches require a purified and uniformly differentiated cell population to predictably recapitulate the physiological characteristics of cartilage. Here, iPSCs derived from adult mouse fibroblasts were chondrogenically differentiated and purified by type II collagen (Col2)-driven green fluorescent protein (GFP) expression. Col2 and aggrecan gene expression levels were significantly up-regulated in GFP+ cells compared with GFP− cells and decreased with monolayer expansion. An in vitro cartilage defect model was used to demonstrate integrative repair by GFP+ cells seeded in agarose, supporting their potential use in cartilage therapies. In chondrogenic pellet culture, cells synthesized cartilage-specific matrix as indicated by high levels of glycosaminoglycans and type II collagen and low levels of type I and type X collagen. The feasibility of cell expansion after initial differentiation was illustrated by homogenous matrix deposition in pellets from twice-passaged GFP+ cells. Finally, atomic force microscopy analysis showed increased microscale elastic moduli associated with collagen alignment at the periphery of pellets, mimicking zonal variation in native cartilage. This study demonstrates the potential use of iPSCs for cartilage defect repair and for creating tissue models of cartilage that can be matched to specific genetic backgrounds.     

Endothelial precursor cells stimulate pericyte‐like coverage of bone marrow‐derived mesenchymal stem cells through platelet‐derived growth factor‐BB induction,which is enhanced by substance P

Objective

The aim of this study was to evaluate the angiogenicity of a combination of BM‐EPCs and BM‐MSCs in vitro in the presence of SP and its working mechanism.

Methods

BM‐MSCs and BM‐EPCs were cocultured with or without SP. ELISA and RT‐PCR were performed to detect angiogenic factors such as VEGF and PDGF‐BB. N‐cadherin was detected by Western blot analysis. The tubular network‐forming ability was evaluated by a Matrigel tube‐forming assay.

Results

BM‐EPCs coculture with BM‐MSCs strongly stimulated the recruitment of BM‐MSCs onto the BM‐EPC‐generated endothelial tubular network. Upon SP treatment, endothelial branching point, tubule length, and tubular recruitment of BM‐MSCs were further increased and stabilized. The coculture of BM‐EPCs and BM‐MSCs synergistically stimulated expression of VEGF, VEGF receptor, N‐cadherin, and PDGF‐BB, all of which were further enhanced by SP treatment. Blockade of PDGF‐BB by its functional blocking antibodies markedly reduced the BM‐MSC incorporation into the endothelial tubules. SP‐pretreated BM‐MSCs were preferentially incorporated into the preformed BM‐EPC tubular network.

Conclusions

BM‐EPCs along with SP promote the pericyte‐like coverage of BM‐MSCs on endothelial tubules possibly through the induction of PDGF‐BB.     

Adipose‐derived stem cells: A candidate for liver regeneration

The scarcity of donor livers and the impracticality of hepatocyte transplantation represent the biggest obstacles for the treatment of liver failure. Adipose‐derived stem cells, with their ability to differentiate into the hepatic lineage, provide a reliable alternative cell source with clear ethical and practical advantages. Moreover, adipose‐derived stem cells can effectively repair liver damage by the dominant indirect pattern and increase the number of hepatocytes by the secondary direct pattern. In recent years, the development of the indirect pattern, which mainly includes immunomodulatory and trophic effects, has become a hot topic in the field of cell engineering. Therefore, adipose‐derived stem cells are considered to be ideal therapeutic stem cells for human liver regeneration. In this article, we reviewed the advantages of adipose‐derived stem cells in liver regeneration, and explore their underlying mechanisms.