Examination of Vascular Capacity of Scaffold Free Human Microtissue During Osteogenesis

Abstract

Vascularization is crucial strategy for bone tissue constructs. To mimic natural bone structure, researchers benefit from co-culture strategy by using stem cell and endothelial cells. The correlation of them supports proliferation of both cells. Moreover, the combination of them contributes vascularization marker expression such as Vascular Endothelial (Ve)-cadherin, Platelet Endothelial Cell Adhesion Molecule (PECAM) and Vascular Endothelial Growth Factor (VEGF). Detection of expression rate of markers may be limited in monolayer. Three dimensional scaffold free microtissues (3D SFMs) are used to understand natural expression rate of vascular markers and to form in vivo mimetic environments. To analyze proliferation and vitality of SFMs, diameter measurements and live and dead assay were performed. For vascular analysis, relative Ve-cadherin, PECAM and VEGF mRNA fold changes were calculated by qPCR. Moreover, VEGF immunofluorescence staining was performed to analyze expression of VEGF. Diameters and vitalities of SFMs coculture increased compared with only Human Umbilical Vein Endothelial Cells (HUVEC) and only Human Bone Mesenchymal Stem Cell (hBMSC) groups. mRNA expression of vascular marker was higher than only HUVEC and only hBMSC groups. The highest VEGF protein expression rate was observed in coculture rather than the other groups. hBMSC:HUVEC coculture showed that association of both cell contributes proliferation of cell viability. Moreover, the coculture showed upregulation of vascular marker and high VEGF protein expression. Co-culture system may be preferred to understand more straight responses in terms of vascularization during osteogenic differentiation. HIGHLIGHTS • Endothelial cells support proliferation and viability of stem cells• The coculture of endothelial and stem cell promoted to vascular capacity• VEGF is one of the key protein for regulation of vascularization in coculture