3D Printed Porous 5%CS/?-TCP Scaffolds Synergize With Co-cultured HUVECs And HBMSCs To Enhance Angiogenesis And Bone Formation

The establishment of a functional vascular system is crucial during bone development and regeneration.Synthetic bone scaffolds have potential application in repairing large bone defects,however,low biological activity and inefficient vascularization after implantation often results in graft necrosis and prevents the application.The emerging field of tissue engineering holds promise for the development of a much simpler solution for vascularized bone regeneration.Scaffolds and seed cells are essential for vascularized tissue engineering scaffolds.In this study,porous ?-tricalcium phosphate(?-TCP)scaffolds with calcium silicate(CS)were 3D printed,and pre-seeded with co-cultured human umbilical cord vein endothelial cells(HUVECs)and human bone marrow stromal cells(hBMSCs)to construct tissue engineering scaffolds with accelerated vascularization and induced osteogensis.We found that ?-TCP scaffolds doped with 5 % CS(5%CS/?-TCP)were biocompatible,and stimulated angiogenesis and osteogenesis in vitro.To study the real time affect of 5%CS/?-TCP scaffolds on co-cultured HUVECs and hBMSCs,we carried out transwell and cell labeling experiments.Results showed 5%CS/?-TCP scaffolds could enhance BMP-2/VEGF paracrine pathway between HUVECs and hBMSCs to stimulate angiogenesis.Then,we seeded co-cultured HUVECs and hBMSCs on 5%CS/?-TCP scaffolds.Results showed co-cultured cells spreed well on 5%CS/?-TCP scaffolds,5%CS/?-TCP scaffolds could stimulate co-cultured cells to secrete BMP-2 and VEGF into the surrounding environment,and pre-seeded co-cultured cells could form microcapillary-like structures on 5%CS/?-TCP scaffolds in vitro when prolong culture time.These indicated that porous 5%CS/?-TCP scaffolds only seeded with co-cultured HUVECs and hBMSCs without growth factors could engineer vascularized microenviroment contain scaffolds,cells and growth factors.Finally,animal experiments showed that 5%CS/?-TCP scaffolds enhanced early vascularization of the scaffolds and induce ectopic bone formation,and porous 5%CS/?-TCP scaffolds synergize with co-cultured HUVECs to further enhance early vascularization of the scaffolds and ectopic bone formation in vivo.Collectively,pre-seeded co-cultured HUVECs and hBMSCs on porous 5%CS/?-TCP scaffolds constructed a tissueengineering scaffold with accelerated vascularization and induced osteogensis successfully.Thus,our findings suggest that porous 5%CS/?-TCP scaffolds seeded with co-cultured cells show potential for vascularized tissue engineering scaffolds and the treatment of large bone defects.