Endothelial progenitor cells improve the therapeutic effect of mesenchymal stem cell sheets on irradiated bone defect repair in a rat model.

BACKGROUND: The reconstruction of bone defects is often impaired by radiotherapy since bone quality is compromised by radiation. This study aims to investigate the therapeutic efficacy of the composite cell sheets-bone marrow mesenchymal stem cell (BMSC) sheets cocultured with endothelial progenitor cells (EPCs)-in the healing of irradiated bone defects and the biological effects of EPCs on the osteogenic properties of BMSC sheets.

METHODS: BMSCs and EPCs were isolated from rat bone marrow. BMSCs were used to form cell sheets by the vitamin C inducing method. EPCs were seeded on BMSC sheets to make EPCs-BMSC sheets. Osteogenesis of EPCs-BMSC sheets and BMSC sheets were tested. In vitro osteogenesis tests included ALP, Alizarin Red S, Sirius Red staining, qRT-PCR and Western blot analysis after 3 and 7 days of osteogenic incubation. Subcutaneous osteogenesis was tested by H&E staining and immunohistochemical staining 8 weeks after transplantation. EPCs-BMSC sheets and BMSC sheets were used in the 3 mm defects of non-irradiated and irradiated rat tibias. Micro-CT and histological analysis were used to test the healing of bone defects 4 and 8 weeks after transplantation.

RESULTS: EPCs-BMSC sheets showed enhanced osteogenic differentiation in vitro with increased expression of osteoblastic markers and osteogenesis related staining compared with BMSC sheets. In subcutaneous osteogenesis test, EPCs-BMSC sheets formed larger areas of new bone and blood vessels. The EPCs-BMSC group had the highest volume of newly formed bone in the defect area of irradiated tibias.

CONCLUSIONS: EPCs improved the osteogenic differentiation of BMSC Sheets and enhanced the ectopic bone formation. EPCs-BMSC sheets promoted bone healing in irradiated rat tibias. EPCs-BMSC sheets are potentially useful in the reconstruction of bone defect after radiotherapy.