Abstract

A double-cell sheet (DCS) complex composed of an osteogenic cell sheet and a vascular endothelial cell sheet with osteogenesis and blood vessel formation potential was developed in this study. The osteogenic and vascular endothelial cell sheets were obtained after induced culture of rabbit adipose-derived mesenchymal stem cells. The osteogenic cell sheet showed positive alizarin red, von Kossa, and alkaline phosphatase (ALP) staining. The vascular endothelial cell sheet exhibited visible W-P bodies in the cells, the expression of CD31 was positive, and a vascular mesh structure was spontaneously formed in a Matrigel matrix. The subcutaneous transplantation results for four groups of DCS and DCS-coral hydroxyapatite (CHA) complexes, and the CHA scaffold group in nude mice revealed mineralization of collagen fibers and vascularization in each group at 12 weeks, but the degrees of mineralization and vascularization showed differences among groups. The pattern involving endothelial cell sheets covered with osteogenic cell sheets, group B, exhibited the best results. In addition, the degree of mineralization of the DCS-CHA complexes was more mature than those of the same group of DCS complexes and the CHA scaffold, and the capillary number was greater than those of the same group of DCS complexes and the CHA scaffold. Therefore, the CHA scaffold strengthened the osteogenesis and blood vessel formation potential of the DCS complexes. Meanwhile, the DCS complexes also promoted the osteogenesis and blood vessel formation potential of the CHA scaffold. This study will provide a basis for building vascularized tissue-engineered bone for bone defect therapy. Statement of Significance This study developed a double-cell sheet (DCS) complex composed of an osteogenic cell sheet and a vascular endothelial cell sheet with osteogenesis and blood vessel formation potential. Osteogenic and vascular endothelial cell sheets were obtained after induced culture of rabbit