In Vitro Construction Of Pre-vascularized Osteogenesis Cellular Spheroids Using Low-binding Plates

Objective: The study aimed to construct a kind of pre-vascularized osteogenesis spheroids when rabbit bone marrow mesenchymal stem cells(BMSCs),osteoblast cells differentiated from rabbit BMSCs(OBs)and human umbilical vein endothelial cells(HUVECs)were co-cultured in vitro using low-binding plates,providing a new strategy for the vascularized bone in tissue engineering.Methods:(1)Isolation and culture of rabbit BMSCs: BMSCs were isolated and purified by the method of whole bone marrow adherent.The morphology and proliferation of BMSCs were observed by microscope.(2)Differentiation of BMSCs into osteoblast cells: The second passage BMSCs were treated with an osteoblastinducer reagent(OIR)for 14 days.The morphology changes,characteristics and proliferation of induced cells were observed by inverted microscope.Alizarin red staining was used to identify osteoblast cells.(3)Culture of human umbilical vein endothelial cells(HUVECs): The morphology of HUVECs were observed under inverted microscope.(4)The generation of three-dimensional(3D)BMSCs spheroids: MSC spheroids were generated using round-bottom,low-binding,96-well plates.The effects of different seeding density(1000,5,000,10,000,50,000 per well)on the spheroid formation were investigated.The spheroid diameter was quantified via bright field microscope(n=6 per seeding density)at the time of 1,3,7 day after the BMSC cell suspension was seeded.(5)The generation of co-culture spheroids: BMSCs spheroids,OBs spheroids and co-culture spheroids(BMSCs: OBs: HUVECs=1:1:1)were generated at a density of 50,000 per well.The alkaline phosphatase(ALP)staining and Alizarin red(AR)staining were used to evaluate the osteogenic properties of spheroids.The CD31 immunofluorescence staining and DAPI staining were performed to evaluate the vascularized cellular spheroids.Results:(1)With the whole bone marrow adherent method,we successfully isolated rabbit BMSCs,which were spindle or polygonal in shape and grow in monoclonal way under the inverted microscope.(2)After BMSCs cultured with medium containing an OIR for 14 days,Alizarin red(AR)staining results were positive,mineralized nodules were observed.(3)HUVECs showed good proliferation and arranged like paving stones.(4)BMSCs spheroid were generated using round-bottom,low-binding 96-well plates.Cell aggregates formed compact multicellular spheroids 24 h after suspension culture.The spheroid diameter increased with increasing cell number.After the spheroid cultured for 7 days,the spheroid aggregated more closely and the spheroid diameter decreased.(5)With same seeding density of 50,000 cells per well,the diameter of OBs spheroids is the smallest among the three group.ALP and AR staining at day 7 showed positive result in OBs spheroids and co-culture spheroids.CD31 immunofluorescence staining at day 1,3,7 revealed a progressive process of lumen morphogenesis in co-culture spheroid.Conclusion: Rabbit BMSCs have the ability to differentiate into osteoblast cells.We successfully generated pre-vascularized osteogenesis cellular spheroids by Coculturing of BMSCs,OBs and HUVECs.It provides a new strategy for the construction of vascularized bone tissue engineering.