Cell-cell interaction in a co-culture system consisting of CRISPR/Cas9 mediated GFP knock- in HUVECs and MG-63 cells in alginate-GelMA based nanocomposites hydrogel as a 3D scaffold.

The interaction between osteogenic and angiogenic cells through a co-culturing system in biocompatible materials has been considered for successfully engineering vascularized bone tissue equivalents. In this study, we developed a hydrogel-blended scaffold consisted of gelatin methacryloyl (GelMA) and alginate enriched with hydroxyapatite nanoparticles (HAP) to model an in vitro prevascularized bone construct. The hydrogel-based scaffold revealed a higher mechanical stiffness than those of pure (GelMA), alginate, and (GelMA+ HAP) hydrogels. In the present study, we generated a green fluorescent protein (GFP) knock-in umbilical vein endothelial cells (HUVECs) cell line using the CRISPR/Cas9 technology. The GFP was inserted into the human-like ROSA locus of HUVECs genome. HUVECs expressing GFP were co-cultured with OB-like cells (MG-63) within the 3D fabricated hydrogel to investigate the response of co-cultured osteoblasts and endothelial cells in a 3D structure. Cell viability under the 3D co-cultured gel was higher than the 3D mono-cultured. Compared to the 3D mono-cultured condition, the cells were aligned and developed into the vessel-like structures. During 14 days of culture periods, the cells displayed actin protrusions by the formation of spike-like filopodia in the 3D co-cultured model. Angiogenic and osteogenic-related genes such as CD 31, vWF, and osteocalcin (OCN) showed higher expression in the co-cultured versus the mono-cultured. These results have collectively indicated that the 3D co-cultured hydrogel facilitates interaction among cells, thereby having a greater effect on angiogenic and osteogenic properties in the absence of induction media. This article is protected by copyright. All rights reserved.