In Vitro and In Vivo Osteogenesis of Human Orbicularis Oculi Muscle-Derived Stem Cells

ABSTRACT

BACKGROUND: Cell-based therapies for treating bone defects require a source of stem cells with osteogenic potential. There is evidence from pathologic ossification within muscles that human skeletal muscles contain osteogenic progenitor cells. However, muscle samples are usually acquired through a traumatic biopsy procedure which causes pain and morbidity to the donor. Herein, we identified a new alternative source of skeletal muscle stem cells (SMSCs) without conferring morbidity to donors. METHODS: Adherent cells isolated from human orbicularis oculi muscle (OOM) fragments, which are currently discarded during ophthalmic cosmetic surgeries, were obtained using a two-step plating method. The cell growth kinetics, immunophenotype and capabilities of in vitro multilineage differentiation were evaluated respectively. Moreover, the osteogenically-induced cells were transduced with GFP gene, loaded onto the porous β-tricalcium phosphate (β-TCP) bioceramics, and transplanted into the subcutaneous site of athymic mice. Ectopic bone formation was assessed and the cell fate in vivo was detected. RESULTS: OOM-derived cells were fibroblastic in shape, clonogenic in growth, and displayed phenotypic and behavioral characteristics similar to SMSCs. In particular, these cells could be induced into osteoblasts in vitro evidenced by the extracellular matrix calcification and enhanced alkaline phosphatase (ALP) activity and osteocalcin (OCN) production. New bone formation was found in the cell-loaded bioceramics 6 weeks after implantation. By using the GFP-labeling technique, these muscle cells were detected to participate in the process of ectopic osteogenesis in vivo. CONCLUSION: Our data suggest that human OOM tissue is a valuable and noninvasive resource for osteoprogenitor cells to be used in bone repair and regeneration.