[Fabrication of pre-vascularized cell sheets based on rabbit bone marrow mesenchymal stem cells]

To explore a new method of developing a pre-vascularized cell sheets.

Bone marrow mesenchymal stem cells (BMSCs) from 3-week-old Japanese white rabbits were harvested and cultured. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were added into the culture medium to differentiate into endothelial like cells (ECs) from BMSCs (experimental group), and non-induced cells served as the control group. The cell morphology was observed; and the von Willebrand factor (vWF) and CD31 immunofluorescent staining was used to identify the induced BMSCs. The 2nd generation BMSCs were seeded on a cell culture dish at a cell density of 9 x 10(4) cells/cm2 and cultured for 14 days to form a thick cell sheet, and ECs from BMSCs were then seeded on the BMSCs sheet at a cell density of 5 x 10(4) cells/cm2 to develop pre-vascularized cell sheets and cultured for 3, 7, and 14 days (group A); non-induced BMSCs sheet and only ECs from BMSCs were used as group B and group C, respectively. The CD31 immunofluorescent staining and histological analysis were performed to evaluate the pre-vascularized cell sheet.

BMSCs changed from long fusiform to cobblestone-like morphology after induced by VEGF and bFGF. The expressions of CD31 and vWF were positive in experimental group, but were negative in control group, which suggested that BMSCs have the ability to differentiate into ECs under this condition. After the ECs were seeded on the BMSCs sheet, the ECs migrated and rearranged; intracellular vacuoles and networks were observed. Furthermore, immunofluorescent staining for CD31 also revealed a developing process of tube formation after the ECs were seeded on the BMSCs sheet. The histological evaluations indicated the microvessel lumen formed. However, no similar change was observed in groups B and C.

BMSCs have the ability to differentiate into ECs after induced by VEGF and bFGF. ECs from BMSCs can develop into vascular network constructs when seeded on the BMSCs sheet, which provides a new method for engineering pre-vascularized tissue construction.