There is a close correlation between vascularization and bone formation in endochondral ossification as maximum extent of bone formation follows maximum levels of VEGF expression. This suggests that osteogenesis and vascularization are coupled by spatiotemporal regulation of paracrine signaling in which the invading vascular endothelial cells secrete osteogenic morphogens to stimulate cell differentiation and bone formation. The objective of this work was to develop a tissue model to investigate the effect of spatial patterning of mesenchymal stem cells and endothelial progenitor cells and spatiotemporal delivery of osteogenic and vasculogenic morphogens on vascularized osteogenesis in a 3D culture system. To achieve the objective, a 3D co-culture system was developed consisting of a cell-adhesive, degradable polyethylene glycol matrix with gelatin methacrylate-filled microchannels for patterning of human mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC). MSC were encapsulated in the matrix and a combination of MSC+EPC were encapsulated in the microchannels. Self-assembled polyethylene glycol nanogels (PEG NG) were synthesized for timed delivery of BMP-2 and VEGF morphogens. The osteogenic BMP-2 was conjugated to 21-day release NG and added to the MSC-laden matrix. The vasculogenic VEGF was conjugated to 5-day release NG and added to the MSC+EPC-laden microchannels. The 3D tissue model was cultured in osteogenic-vasculogenic medium. At each time point, the tissue model was evaluated for osteogenesis and vasculogenesis by biochemical, mRNA, and protein analysis.
Groups included MSC/EPC patterned tissue model without BMP-2/VEGF (None), with dissolved BMP-2/VEGF, and with BMP2-NG/VEGF-NG. Osteogenic control group was MSC encapsulated in degradable PEG gel with BMP-2 or BMP2-NG. Vasculogenic control group was MSC+EPC encapsulated in gelatin methacrylate with VEGF or VEGF-NG. Based on the results, the extent of vascularized osteogenesis was higher in patterned cellular constructs compared to un-patterned constructs. Further, timed-release of VEGF and BMP-2 in the patterned cellular constructs significantly enhanced the extent of vascularized osteogenesis compared with the direct addition of VEGF and BMP-2. We further discovered that the spatial patterning of MSC and EPC and the spatiotemporal of BMP-2 and VEGF sharply increased the expression of vasculogenic factors bFGF and PDGF and osteogenic factor TGF-β in the tissue constructs. The results suggest that osteogenesis and vasculogenesis are coupled by localized secretion of paracrine signaling factors during bone formation.