Fabrication of prevascularized osteogenic differentiated cell sheet based on human bone marrow mesenchymal stem cells and human umbilical vein endothelial cells

doi:10.3969/j.issn.2095-4344.2129

Abstract

Abstract:

BACKGROUND: How to make the engineered bone tissues survive and function after transplantation, the precondition is to have sufficient and effective blood supply, which is one of the main obstacles in the clinical application of the engineered bone tissue.

OBJECTIVE: To explore a new method to acquire vascular networks in vitro to realize vascularized engineered bone tissues.

METHODS: Human bone marrow mesenchymal stem cells were cultured on a cell culture dish at a high density 9×10⁴/cm² for 14 days to form a thick cell sheet. The morphology of the cells was observed under a microscope and cells were stained with alkaline phosphatase and alizarin red. The human umbilical vein endothelial cells at 5×10⁴/cm² were seeded onto the surface of human bone marrow mesenchymal stem cells sheet for 7 days. The formation of vascular network and the characteristics of the sheet were evaluated by microscopic observation, CD31 immunofluorescence staining and type I collagen immunofluorescence staining.

RESULTS AND CONCLUSION: (1) Human bone marrow mesenchymal stem cells proliferated fast and formed an intact sheet, and the sheet cultured in osteogenic-induced medium showed positive staining of alkaline phosphatase and alizarin red. (2) Inverted phase contrast microscope showed that human umbilical vein endothelial cells migrated and aligned arrangement on the osteogenic differentiated cell sheet. The immunofluorescent images for CD31 revealed a progressive process of lumen formation. (3) Positive type I collagen was observed in the pre-vascularized osteogenic differentiated cell sheet. (4) These results indicate that the osteogenic differentiated cell sheet can be formed by continuously culturing. The prevascularizing method using coculture model of culturing endothelial cells onto an osteogenic differentiated cell sheet provides a theoretical guidance for optimizing the construction of prevascularized bone tissue.

Key words: stem cells, bone marrow mescenchyal stem cells, pre-vascularization, cell sheet, vascular endothelial cells, osteogenesis, co-culture, vascularized networks, experiment

CLC Number:

Chen Jia, Yang Yiqiang, Hu Chen, Chen Qi, Zhao Tian, Yong Min, Ma Dongyang, Ren Liling. Fabrication of prevascularized osteogenic differentiated cell sheet based on human bone marrow mesenchymal stem cells and human umbilical vein endothelial cells[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(31): 4934-4940.

Figures/Tables 5

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