Angiogenesis Of Cranial Defect Can Be Enhanced By Human Amniotic Mesenchymal Stem Cell In Vivo And In Vitro

Objective:To study the angiogenic characteristics of human amniotic mesenchymal stem cell and positive effect of promoting osteogenesis in cranial defect to seek an alternative seed cell for vascularized bone tissue engineering and provide the theoretic basis for bone regeneration.Methods:hAMSC was isolated from discarded placenta,and certificated via its surface markers by flow cytometry(FCM)and its multilineage differentiation.hAMSCs at the 3rd～5th passages were collected for subsequent experiments.In vitro studies,hAMSC conditional media(CM)was collected and investigated by Human XL Cytoking Array Kit to evaluate expression of proangiogenic factors.Semi-polychain reaction and quantitative-polychain reaction were used to detect the changes of gene expression in EC-induced and non-induced hAMSC and those of gene expression in HUVEC and hAMSC that had cultured with each other by transwells for 1d,4d,7d.Matrigel assay of HUVEC was applied to show support effect of EC-induced hAMSC-CM.And then,3D co-culture system was built to explore cell-cell contract between HUVEC and hAMSC.The proangiogenic proteins in co-culture were dectected by Western-blot to display the advantage on angiogenesis when hAMSC were added to co-culture system.In vivo studies,hAMSCs were delivered to cranial defects of male New Zealand rabbits by fibrin gels.And then,the specimens of the defects was fixed and embedded at 2w,4w and 12w after operation,inspected the neovascularization and osteogenesis in the defects via hematoxylin-eosin staining,masson’s thrichrome staining,immunohistochemistry,Van Gieson’s picro fuchsin staining and Sequential fluorescent labeling.Results:hAMSCs in our study displayed fibroblastic morphology,showed mesenchymal cell special markers by FCM and have potential to differentiate into osteoblast,adipocyte and chondrocyte.In vitro studies,many proangiogenic factors were found in hAMSC-CM,but these factors were downregulated after EC-induction while chemokines were upregulated.The profile of gene expression in hAMSC was complex after co-culturing with HUVEC via tranwell and indicated that cross-talk must be existed between them.There are many proangiogenic proteins were increased in 3D co-culture system.This case seemed to be related to Erk1/2 signaling pathway.In vivo studies,new bone area and vessel area of groups contained hAMSC in rabbit cranial defects were more superior to control group by hematoxylin-eosin staining,masson’s thrichrome staining,immunohistochemistry,Van Gieson’s picro fuchsin staining respectively.The rate of new bone mineralization accelerated by grafting hAMSC was shown by Sequential fluorescent labeling.Conclusion:hAMSC could be served as a promising seed cell for vascularized bone tissue engineering due to its significant advantage in angiogenesis and osteogenesis.