The Effect Of Insulin-like Growth Factor â… on Oesteogenesis Of Human Periodontal Ligament Stem Cells Under Simulated Microgravity

The deficiency of periodontal tissue caused by periodontitis is the main cause of tooth missing in adults. Tissue engineering aims at constructing such tissues with physiological structure and functions in vitro, however the effect of gravity influenced the formation of three-dimensional structures in laboratory culture. The emerging of the technique of simulating microgravity in ground based environment sheds lights on this topic.In vivo, the metabolism of the tissues depends on the differentionation of the periodontal stem cells. Thus, to construct periodontal tissue in vitro, it's crucial to culture hPDLSCs and accomplish the regulation of proliferation, differentiation, especially osteogenesis of hPDLSCs. Insulin-like growth factor-â… (IGF-â… ) is one of the main regulator taking part in the above process. It's expressed in the extracellular matrix of periodontal ligament cells, and acts on the target cells through autocrine, and paracrine to sitimulate cell proliferation and the synthesis of matrix proteins. However, no reports on the effect of IGF-I on the proliferation and osteogenesis of hPDLSCs in simulated microgravity were seen.The objective of the study is to culture hPDLSCs in simulated microgravity model, and investigate the effect IGF-â… on the proliferation and osteogenesis of hPDLSCs in simulated microgravity. The study consists of two parts:Part one: establishment of the culture model of hPDLSCs in SMGObjective: to culture and identify hPDLSCs, and set the culture modle of hPDLSCs in SMG.Methods:hPDLSCs were isolated with limited dilution method, and identified with flow cytometry assay, immunohistochemistry stain, osteogenic and adipogenic experiment. Culture of hPDLSCs in simulated microgravity was set with rotary cell culture system. Compare the proliferation of the cells with that cultured in normal gravity (NG).Results:Multipotent of differentionation and cell surface biomarkers, STRO-1 and CD146, were identified in hPDLSCs. Compared with that of NG group, hPDLSCs in SMG group showed much more capability of proliferation.Conclusion:hPDLSCs can be obtained with limited dilution method, proliferation of hPDLSCs was enhanced in simulated microgravity. Part two:the effect of IGF-â… on osteogenesis of hPDLSCs in SMGObjective:to investigate the effect of IGF-â… on osteogenesis of hPDLSCs in SMG.Methods:hPDLSCs were divided into negative control group (NG), positive control group (SMG), and experimental group (SMG+IGF-â… ). Real-time PCR, western-blot, image analysis were used to evaluate the expression of biomarker of osteogenesis, OPG/RANKL and the capability of formation of mineralized nodules.Results:the expression level of biomarkers of osteogenesis in experiment group was higher than that of two control groups, the expression level of OPG/RANKL was lower that that of two control groups. The mineralized nodules were more in experiment group than in two control groups.Conclusion:IGF-â… can promots the osteogenesis of hPDLSCs; mean time, it may take part in the formation of osteoclasts through the regulation of OPG/RANKL, thus exerts dual regulation effect on bone metabolism.In brief, hPDLSCs showed enhance proliferate activity in simulated microgravity, and IGF-â… exerts promotion effects on the osteogenesis and proliferation of hPDLSCs in simulated microgravity. In the mean time, IGF-â… may take part into the process of osteoclasts induction through the regulation of OPG/RANKL. By using the technique of simulated microgravity into the periodontal tissue engineering, we tend to provide laboratory basis for the deep research of delicate regulation of cell growth differention in tissue engineering.