Introduction: In recent years the use of cell sheets for Tissue Engineering and Regenerative Medicine(TERM) purposes presented itself as a valid alternative to the traditional approaches[1]. However, when it comes to the engineering of thick tissues this technology faces some of the barriers of conventional strategies namely the lack of an appropriate vasculature to supply the bulk of the constructs in vivo[2, 3]. We propose the establishment of co-cultures of osteogenic, endothelial and pericyte-like cells to produce a cell sheet construct with the aim of obtaining the in vivo formation of osteogenic tissue with improved vessel formation, maturation and stability. Materials and Methods: Human bone marrow mesenchymal stromal cells (hBMSCs) were isolated and characterized for the expression of pericyte marker CD146 and MSCs markers CD90, CD73 and CD105. hBMSC were either cultured in the presence of TGF-β1 to obtain pericyte-like cells or in osteogenic medium for osteogenic commitment. Human umbilical vein endothelial cells (HUVECs) were isolated and co-cultured with the pericyte-like cells over the hBMSC-derived osteogenic cells in thermo-responsive dishes for 7 days. Cell sheet constructs were transplanted to the dorsal flap of nude mice for 7 and 21 days. The explants were characterized using histology and immunohistochemical techniques. Results and discussion: Flow cytometry results demonstrated that supplementation of standard hBMSCs culture medium with TGF-β1 promoted an increase in the amount of hBMSCs expressing CD146 from approximately 50% to more than 97% of cells. Moreover, increased CD146 expression was associated with a change in cellular morphology from spindle-shape to star-like morphology. Immunocytochemistry performed on the co-cultures showed that induced CD146⁺ hBMSCs and HUVECs migrated and self-organized over an osteogenic cell monolayer, suggesting the existence of cross-talk involving all the co-cultured cell types. Immunohistochemistry analysis of transplanted co-cultured cell sheets revealed the formation of osteogenic tissue expressing osteocalcin and positive for calcium deposition. Moreover, the integration of HUVECs with the host’s vasculature was demonstrated by human CD31 staining. Finally, the analysis of the diameter of human CD146 positive blood vessels showed a higher mean vessel diameter for the co-cultured cell sheet condition, reinforcing the advantage of the proposed model regarding blood vessels maturation and stability in constructs for TERM approaches.      

References

1.       Yang, J., et al., Biomaterials, 2005. 26(33): p. 6415-6422.

2.       Pirraco, R.P., A.P. Marques, and R.L. Reis, J Cell Mol Med, 2009. 14(1-2): p. 93-102.

3.       Sasagawa, T., et al., Biomaterials, 2010. 31(7): p. 1646-1654.