Abstract 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.
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