Construction of tissue engineering bone with the co-culture system of ADSCs and VECs on partially deproteinized biologic bone in vitro: A preliminary study

Scaffold-based bone tissue engineering has therapeutic potential in the regeneration of osseous defects. The present study aimed to explore the adhesion and cell viability of a co-culture system composed of vascular endothelial cells PI(−)/Annexin V(+) represents early apoptotic cells, and PI(+)/Annexin V(+) represents late apoptotic cells (VECs) and adipose-derived stem cells (ADSCs) on partially deproteinized biologic bone (PDPBB) in vitro, and determine the optimum time period for maximum cell viability that could possibly be used for standardizing the scaffold transplant into the in vivo system. VECs and ADSCs were isolated from pregnant Sprague-Dawley rats and confirmed by immunostaining with von Willebrand factor and CD90, respectively. PDPBB was prepared using standardized protocols involving coating partially deproteinized bone with fibronectin. PDPBB was incubated in a mono-culture with VECs or ADSCs, or in a co-culture with both of these cells at a ratio of 1:1. An MTT assay was used to assess the adhesion and cell viability of VECs and ADSCs on PDPBB in the three different cultures. Scanning electron microscopy was used to observe the adhesion, cell viability and morphology of the different types of cells on PDPBB. It was observed that the absorbance of each group increased gradually and peaked on the 10th day; the highest absorbance was found for the co-cultured cells group. The difference of cell viability between each cell group was statistically significant. On the 10th day, in the co-cultured cells group, several cells adhered on the PDPBB material and a nest-like distribution morphology was observed. Therefore, the adhesion and cell viability of the co-cultured cells was higher compared with the mono-cultures of VECs or ADSCs. As cell viability was highest on the 10th day, this could be the optimal length of time for incubation and therefore could be used for in vivo experiments.