Fabrication Of Vasularized Modular Bone Tissues Via Coculturing Of HAMSCs And HUVECs

Previously,we developed a novel technique,so-called "microtissue assembly",for fabricating viable centimeter-sized tissue constructs in vitro.However,upon implantation in vivo,new mass transfer limit is anticipated,which will potentially result in tissue necrosis.It was hypothesized that vascularization of in vitro engineered macrotissues would be able to support quick integration with hosts and maximize tissue suvival in vivo.In the present study,we first investigated coculture of human amniotic mesenchymal stem cells(hAMSCs)and human umbilical vein endothelial cells(HUVECs)in two-dimensional(2D)tissue culture plates,focusing on the effects of culture medium(either cell growth medium or osteogenic medium),cell density,and the ratio of cell numbers on the formation of vascular-like structures and the osteogenesis of hAMSCs.We further evaluated the feasibility of creating vascularied bone microtissues with hAMSCs and HUVECs in spinner flasks,with an emphasis on the effect of seeding time point of HUVECs on viability,osteogenesis and angiogenesis of bone microtissues.It was found that vascular-like structures could be formed in both a-MEM growth and osteogenic medium when hAMSCs and HUVECs were cocultured in 2D.The higher the density of HUVECs,the faster the vascular-like structures were formed,and the earlier the structures disintegrated.On one hand,a high density of hAMSCs was beneficial to maintain the vascular-like structures.On the other hand,a high density of HUVECs was disadvantageous for the osteogenic differentiation of hAMSCs,and a low density of HUVECs showed no influence.Collectively,a 1:1 ratio between HUVECs and hAMSCs seemed to be optimal for both vascularization and osteogenesis.In spinner flasks,microtissues were prepared by coculturing hAMSCs and HUVECs,which had high viability and displayed characteristics of both vascularization and osteogenesis.However,in comparison with microtissues made of hAMSCs only,the osteogenesis was slightly less efficient.In addition,the seeding time point of HUVECs did influence the osteogenic differentiation of hAMSCs,and seeding of HUVECs at 7 d post osteogenic induction of hAMSCs was favorable for the osteogenesis.Taken together,it is feasible to fabricate viable,vascularized bone microtissues by coculturing hAMSCs and HUVECs in spinner flasks,which lays a solid foundation of engineering large-sized vascularized bone tissue constructs.