ABSTRACT
BACKGROUND/AIMS: Joint cartilage defects are difficult to treat due to the limited self-repair capacities of cartilage. Cartilage tissue engineering based on stem cells and gene enhancement is a potential alternative for cartilage repair. Bone morphogenetic protein 2 (BMP2) has been shown to induce chondrogenic differentiation in mesenchymal stem cells (MSCs); however, maintaining the phenotypes of MSCs during cartilage repair since differentiation occurs along the endochondral ossification pathway. In this study, hypoxia inducible factor, or (HIF)-1α, was determined to be a regulator of BMP2-induced chondrogenic differentiation, osteogenic differentiation, and endochondral bone formation. METHODS: BMP2 was used to induce chondrogenic and osteogenic differentiation in stem cells and fetal limb development. After HIF-1α was added to the inducing system, any changes in the differentiation markers were assessed. RESULTS: HIF-1α was found to potentiate BMP2-induced Sox9 and the expression of chondrogenesis by downstream markers, and inhibit Runx2 and the expression of osteogenesis by downstream markers in vitro. In subcutaneous stem cell implantation studies, HIF-1α was shown to potentiate BMP2-induced cartilage formation and inhibit endochondral ossification during ectopic bone/cartilage formation. In the fetal limb culture, HIF-1α and BMP2 synergistically promoted the expansion of the proliferating chondrocyte zone and inhibited chondrocyte hypertrophy and endochondral ossification. CONCLUSION: The results of this study indicated that, when combined with BMP2, HIF-1α induced MSC differentiation could become a new method of maintaining cartilage phenotypes during cartilage tissue engineering.
Subject(s)
Bone Morphogenetic Protein 2/metabolism , Chondrogenesis , Forelimb/growth & development , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mesenchymal Stem Cells/physiology , Osteogenesis , Animals , Bone Morphogenetic Protein 2/genetics , Cell Differentiation , Cells, Cultured , Forelimb/embryology , Gene Expression Regulation , HEK293 Cells , Humans , Hyaline Cartilage/physiology , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Male , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/metabolism , Mice , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolismABSTRACT
OBJECTIVE: To investigate the effect of co-expression of bone morphogenetic protein 2 (BMP2) and Sox9 on chondrogenic differentiation of mesenchymal stem cells (MSCs) in vitro and provide experimental evidence for tissue engineering of cartilage. METHODS: Mouse embryonic bone marrow MSC C3H10T1/2 cells were infected with recombinant adenovirus expressing BMP2, Sox9 and green fluorescent protein (GFP) for 3-14 days, with cells infected with the adenovirus carrying GFP gene as the control. The mRNA expression of the markers of chondrogenic differentiation, including collagen type II (Col2a1), aggrecan (ACAN), and collagen type X (Col10a1), were determined by real-time PCR. Alcian blue staining was used for quantitative analysis of sulfated glycosaminoglycan in the cellular matrix. The expression of Col2a1 protein was assayed by immunohistochemical staining and Western blot analysis. RESULTS: Adenovirus-mediated BMP2 expression induced chondrogenic differentiation of C3H10T1/2 cells. Overexpression of Sox9 effectively enhanced BMP2-induced expression of the chondrogenic markers Col2a1, aggrecan and Col10a1 mRNAs, and promoted the synthesis of sulfated glycosaminoglycan and Col2a1 protein in C3H10T1/2 cells. CONCLUSION: Co-expression of BMP2 and Sox9 can promote chondrogenic differentiation of MSCs in vitro, which provides a new strategy for tissue engineering of cartilage.
Subject(s)
Bone Morphogenetic Protein 2/genetics , Cell Differentiation , Chondrocytes/cytology , Mesenchymal Stem Cells/cytology , SOX9 Transcription Factor/genetics , Animals , Bone Morphogenetic Protein 2/metabolism , Cartilage/cytology , Cells, Cultured , Humans , Mesenchymal Stem Cells/metabolism , Mice , SOX9 Transcription Factor/metabolism , Tissue EngineeringABSTRACT
Bone morphogenetic protein 2 (BMP2) is one of the key chondrogenic growth factors involved in the cartilage regeneration. However, it also exhibits osteogenic abilities and triggers endochondral ossification. Effective chondrogenesis and inhibition of BMP2-induced osteogenesis and endochondral ossification can be achieved by directing the mesenchymal stem cells (MSCs) towards chondrocyte lineage with chodrogenic factors, such as Sox9. Here we investigated the effects of Sox9 on BMP2-induced chondrogenic and osteogenic differentiation of MSCs. We found exogenous overexpression of Sox9 enhanced the BMP2-induced chondrogenic differentiation of MSCs in vitro. Also, it inhibited early and late osteogenic differentiation of MSCs in vitro. Subcutaneous stem cell implantation demonstrated Sox9 potentiated BMP2-induced cartilage formation and inhibited endochondral ossification. Mouse limb cultures indicated that BMP2 and Sox9 acted synergistically to stimulate chondrocytes proliferation, and Sox9 inhibited BMP2-induced chondrocytes hypertrophy and ossification. This study strongly suggests that Sox9 potentiates BMP2-induced MSCs chondrogenic differentiation and cartilage formation, and inhibits BMP2-induced MSCs osteogenic differentiation and endochondral ossification. Thus, exogenous overexpression of Sox9 in BMP2-induced mesenchymal stem cells differentiation may be a new strategy for cartilage tissue engineering.
