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.

Keywords: Orbicularis oculi muscle; Osteogenic differentiation; Skeletal muscle stem cells.

Fig. 1

Isolation of SMSCs from OOM myofibers. A Schematic illustration of human OOM anatomic structure. T: the tarsal portion of OOM; S: the septal portion; O: the orbital portion. The rectangular region indicates the harvest site of OMM samples for this study. B By HE staining evaluation, OOM samples comprised striated muscles (pink) with no fat tissue. C By immunohistochemical analysis, OOM positively expressed the myofiber marker of myogenin (brown, cross section of the muscle). D Satellite cells of the OOM myofibers were shown by immunoreaction with an anti-Pax7 antibody (green, arrows, cross section of the muscle). E The isolated adherent cells showed positive fluorescent immunostaining of Myf5 (red) and Pax7 (green) when cultured in vitro for 6 days, indicating their myogenic origin. Cell nuclei were visualized by DAPI. Scale bars for B–E: 100 µm. (Color figure online)

Fig. 2

In vitro proliferation and multilineage differentiation of SMSCs. A Freshly isolated cells formed colonies within 48 h in primary culture. B They proliferated to approximately 85% confluence after 14 days. C The subcultured cells appeared homogenous fibroblast-like morphology after passaging. D After myogenic differentiation for 2 weeks, OOM-derived cells were positive for desmin, a late differentiation marker of the muscle cells, and fused into elongated, multinuleated myotube-like structure. E The chondrogenic differentiation in cell pellet culture was identified by the positive collagen type II staining. F The adipogenic differentiation was confirmed by positive Oil red O staining after adipogenic induction. Cell nuclei were visualized by DAPI. Scale bars: 100 µm. (Color figure online)

Fig. 3

In vitro osteogenic differentiation of SMSCs. A After induced for 15 days, osteogenic differentiation was verified by the positive Alizarin red staining. Inset photograph is the gross view of staining. B The non-induced cells revealed negative staining. C Calcium content within the cells was measured using a colorimetric assay of the solubilized Alizarin red stain. At days of 9, 12 and 15 in culture, more Ca²⁺ was deposited in the osteogenically induced cells compared to the control cells. D Growth rates of osteo-induced and non-induced SMSCs were determined by DNA assay. No significant difference in cell numbers was found between two groups. E Greater ALP activity was expressed when cells were osteo-induced compared to the non-induced cells from day 12. F More OCN was produced in the osteo-induced cells than in the control cells from day 12. Error bars represent SD with n = 6 (*p < 0.05;#p < 0.01). (Color figure online)

Fig. 4

In vivo osteogenic differentiation of SMSCs. A After GFP-transduction, the majority of the cells expressed green fluorescence. B The GFP-labeled SMSCs distributed evenly on the β-TCP ceramics after loading. C SEM observation showed the porous structure of the ceramic scaffold. D 7 days after cell seeding, SMSCs deposited large amounts of extracellular matrix covering the ceramic pores and formed a matrix network through the interconnected pores. E X-ray photograph showed higher density in the cell-TCP composite (top) than in the cell-free scaffold (bottom) at 6 weeks of transplanting in vivo. F Quantitative analysis of X-ray grey values demonstrated significant difference between two groups (n = 6). G HE staining revealed typical woven bone formation (red) after implantation of the GFP-labeled osteo-induced cells. H When observed with a fluorescent microscope, these active osteoblasts lining on the bone surface in the adjacent section of the HE-stained specimen showed the green fluorescence. Scale bars: 100 µm for A–B, 200 µm for C–D and 25 µm for G–H. (Color figure online)