ABSTRACT
Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-ß (TGF-ß) super-family, is one of the main chondrogenic growth factors involved in cartilage regeneration. BMP2 is known to induce chondrogenic differentiation in various types of stem cells in vitro. However, BMP2 also induces osteogenic differentiation and endochondral ossification in mesenchymal stem cells (MSCs). Although information regarding BMP2-induced chondrogenic and osteogenic differentiation within the same system might be essential for cartilage tissue engineering, few studies concerning these issues have been conducted. In this study, BMP2 was identified as a regulator of chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. BMP2 was used to regulate chondrogenic and osteogenic differentiation in stem cells within the same culture system in vitro and in vivo. Any changes in the differentiation markers were assessed. BMP2 was found to induce chondrogenesis and osteogenesis in vitro via the expression of Sox9, Runx2 and its downstream markers. According to the results of the subcutaneous stem cell implantation studies, BMP2 not only induced cartilage formation but also promoted endochondral ossification during ectopic bone/cartilage formation. In fetal limb cultures, BMP2 promoted chondrocyte hypertrophy and endochondral ossification. Our data reveal that BMP2 can spontaneously induce chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. Thus, BMP2 can be used in cartilage tissue engineering to regulate cartilage formation but has to be properly regulated for cartilage tissue engineering in order to retain the cartilage phenotype.
Subject(s)
Bone Morphogenetic Protein 2/metabolism , Cell Differentiation , Chondrogenesis , Osteogenesis , Stem Cells/cytology , Stem Cells/metabolism , Adenoviridae/metabolism , Animals , Biomarkers/metabolism , Bone Development , Chondrocytes/pathology , Collagen/metabolism , Extremities/embryology , Fetus/metabolism , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Hypertrophy , Mice , Staining and LabelingABSTRACT
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
BACKGROUND: Bone morphogenetic protein 9 (BMP9) and Wnt/ß-catenin signaling pathways are able to induce osteogenic differentiation of mesenchymal stem cells (MSCs), but the role of Wnt/ß-catenin signaling pathway in BMP9-induced osteogenic differentiation is not well understood. Thus, our experiment was undertaken to investigate the interaction between BMP9 and Wnt/ß-catenin pathway in inducing osteogenic differentiation of MSCs. METHODS: C3H10T1/2 cells were infected with recombinant adenovirus expressing BMP9, Wnt3a, and BMP9+Wnt3a. ALP, the early osteogenic marker, was detected by quantitative and staining assay. Later osteogenic marker, mineral calcium deposition, was determined by Alizarin Red S staining. The expression of osteopotin (OPN), osteocalcin (OC), and Runx2 was analyzed by Real time PCR and Western blotting. In vivo animal experiment was carried out to further confirm the role of Wnt3a in ectopic bone formation induced by BMP9. RESULTS: The results showed that Wnt3a enhanced the ALP activity induced by BMP9 and increased the expressions of OC and OPN, with increase of mineral calcium deposition in vitro and ectopic bone formation in vivo. Furthermore, we also found that Wnt3a increased the level of Runx2, an important nuclear transcription factor of BMP9. CONCLUSION: Canonical Wnt/ß-catenin signal pathway may play an important role in BMP9-induced osteogenic differentiation of MSCs, and Runx2 may be a linkage between the two signal pathways.
