The Mechanism Of Insulin-like Growth Factor1Enhancing The Proliferation And Osteogenic Differentiation Of Human Periodontal Ligament Stem Cells

Insulin-like growth factor1(IGF-1) is a potent mitogenic protein which can enhance the osteogenic differentiation of periodontal ligament (PDL) fibroblasts. However, it remains unclear whether IGF-1can stimulate the osteogenic differentiation and osteogenesis of human periodontal ligament stem cells (PDLSCs). In this study, STRO-1+periodontal ligament stem cells (PDLSCs) were isolated from human PDL tissues, treated with IGF-1, and their osteogenic capacity was investigated in vitro and in vivo.1. culture and identification of human periodontal ligament stem cells in vivoObjective To obtain the purified human periodontal ligamern stem cells.Methods Premolars were collected. hPDLSCs were prepared by enzyme digestion method. Primary culture cells were purified by limited dilution and magnetic cell sorting. The morphology, growth and clones forming were observed under inverted microscope. The proliferation ability, cell cycle were analyzed.Results hPDLSCs were the typical appearance of fibroblast, positive for vimentin and negative for cytokeratin. MTT assay showed that the population double time was45.74±0.91h. Flow cytometry showed thath the proliferation index was25.58%.Conclusions Purified hPDLSCs were prepared by enzyme digestion, limited dilution and magnetic cell sorting methods and the separated cells were mesenchymal stem cells with the high proliferation ability. 2. effects of IGF-1on the proliferation and osteogenic differentiation of human periodontal ligament stem cellsObjective To investigate the effects of IGF-1on the proliferation and osteogenic differentiation of hPDLSCs.Methods MTT assay, cell cycle analysis, morphological appearance, ALP activity, Alizarin red stain, genes/proteins expression were evaluated in vitro.1×106hPDLSCs in IGF-1untreated and treated groups were respectively harvested which were carefully seeded onto the absorbable gelatin sponges immersed with100ng/mL IGF-1and transplantation procedures were performed under renal capsules. All retrieved tissues at day21post-transplantation were processed for HE staining and immunohistochemical staining.Results100ng/mL is the optimal concentration. Exogenous IGF-1can modify the ultrastructure, enchance the alkaline phosphatase activity, the mineralization ability, the proliferation ability of hPDLSCs and increase the expression of osteogenic markers (runt-related transcription factor2, osterix, and osteocalcin) at mRNA and protein levels. In vivo transplantation illustrated that IGF-1treated implants generated more mineralized tissues, and presented stronger expression of RUNX2, OSX and OCN than Control group.Conclusions IGF-1can enhance the proliferation and osteogenic differentiation of hPDLSCs.3. IGF-1enhances the osteogenic differentiation of human periodontal ligament stem cells via ERK and JNK MAPK pathwaysObjective To elucidate why IGF-1can promote the function of hPDLSCs, we investigated whether MAPK pathway in PDLSCs is activated by IGF-1treatment.Results The levels of phosphor-ERK1/2and phosphor-JNKin IGF-1treated cells were upregulated. However, the levels of phosphor-P38were not affected and realtime RT-PCR results showed that IGF-1can not upregulated the expression of RUNX2, OSXmRNA when the ERK and JNK MAPK were inhibited.Conclusion IGF-1may enhance the osteogenic differentiation of hPDLSCs via ERK and JNK MAPK pathways.