Cell-of-origin for heterotopic ossification induced by bone morphogenetic protein 4 in skeletal muscle / 中国组织工程研究

ABSTRACT

BACKGROUND:Heterotopic ossification of skeletal muscle is a clinically serious complication.For heterotopic ossification of skeletal muscles,the cells involved in the process of heterotopic ossification remain unclear. OBJECTIVE:To investigate the involvement of myocytes,fascia cells,and endothelial cells in the process of heterotopic ossification in skeletal muscle and to observe the cell origin of heterotopic ossification in skeletal muscle induced by bone morphogenetic protein 4. METHODS:Both C2C12 cells and the myotubes formed by the C2C12 cells in the induction medium were cultured,and 500 ng/mL bone morphogenetic protein 4 was added to the medium respectively,and whether the C2C12 cells and myotubes continued to proliferate within 10 days under the treatment were observed under a microscope.Myogenic cells(L6,derived from rats)and fibroblast-derived cells(derived from human)were co-cultured.After treatment with 500 ng/mL bone morphogenetic protein 4 and 10 ng/mL transforming growth factor-β,osteogenic and chondrogenic differentiation potential within 21 days were observed using Safranine O staining and Alcian blue staining.Using transgenic animal FVB/N-TgN(TIE2-LacZ)182Sato mice,15 μL of adeno-associated virus-bone morphogenetic protein 4(5×1010 PFU/mL)were implanted in the thigh muscle space of genetic mice for 10 and 14 days.X-gal staining was used to observe the formation of new blood vessel endothelium in the differentiated bone. RESULTS AND CONCLUSION:(1)Bone morphogenetic protein 4 caused myotube breakdown and increased C2C12 cell proliferation.Compared with other groups,the pure fibroblast-derived cell group had a higher area of positive alcian blue and safarin O staining(P<0.05)and a lower area of alkaline phosphatase staining(P<0.05),while the pure L6 group had a bigger area of alkaline phosphatase staining(P<0.05)but a smaller area of positive alcian blue and safarin O staining(P<0.05).(2)Transplantation of adeno-associated virus-bone morphogenetic protein 4-adsorbed gelatin sponge into FVB/N-TgN(TIE2-LacZ)182Sato mice resulted in heterotopic ossification.(3)X-gal staining results demonstrated that there was no obvious staining in chondrocytes and differentiated bones and Tie2+ endothelial cells did not participate in the formation of the alienated bone.(4)These findings verify that fibroblasts are the primary source of osteoblasts during the adeno-associated virus-bone morphogenetic protein 4-induced ectopic endochondral ossification in skeletal muscle,but myogenic cells are the main source of osteoblasts.Tie2+ endothelial cells might not be the cell source for cartilage and bone.