Study On Wound Healing Of Mice By Self-assembled Short Peptide Hydrogel Combined With HAMSCs

ObjectiveTo investigate the growth,proliferation,cell cycle,stemness-related gene expression and differentiation of human amniotic mesenchymal stem cells(hAMSCs)in RADA and RGD peptide hydrogels,and to study the synergistic effect of hydrogels and hAMSCs on the healing of wounds in mice with total cortical defects.Methods1.The microstructures,secondary structures and rheological properties of the peptide RADA and RADA-RGD(named as RGD)hydrogels were analysied by transmission electron microscopy,circular dichroism spectroscopy and and rotational rheometer,respectively.The hAMSCs were separated by Collagenase II and trypsase,then the cells were cultured in T75 flasks and purified to P3 to establish hAMSCs,the hAMSCs were encapsulated into the 3D RADA and RGD hydrogels.The morphology of each group was observed.Live/Dead and CCK-8 assay were used to detect the survival and proliferation of hAMSCs in the hydrogel scaffold,respectively;The cell cycle analysis of the encapsulated hAMSCs was performed by a flow cytometry;At last,the mRNA expression levels of the stemness-related,growth factors,inflammatory factors and osteogenic differentiation related genes were analyzed by RT-PCR in both 2D flasks and 3D hydrogel culture over four days.2.C57 mouse(N = 120)were randomly divided into six groups: serum-free medium(control)group,RADA hydrogel group,RGD hydrogel group,hAMSCs transplantation group,RADA+hAMSCs transplantation group and RGD+hAMSCs transplantation group.On the 4th,8th,16 th and 24 th days after modeling,the wounds were photographed and HEstaining was performed to evaluate the the gross morphology and the proportions of the wound area.The expression of keratin 14,CD31 and PCNA were detected by immunohistochemistry and immunofluorescence,respectively.The mRNA expression levels of the growth factors in wound area were detected by RT-PCR.Results1.Results showed that peptides RADA and RGD can form β-sheet nanofibers and scaffold hydrogels with pore sizes of about 5-200 nm.RADA hydrogel has much better gelation performance than RGD hydrogel.Moreover,we found that hAMSCs could maintain long-term growth and proliferation in these peptide hydrogels with a higher survival rate.Under 3D culture conditions,the mRNA expression of stemness-related,growth factors,inflammatory factors and osteogenic and adipogenic differentiation related genes of hAMSCs decreased when compared with that in 2D flasks,while the expression of β-Tubin III increased.2.Combined with hAMSCs,the hydrogles could effectively promote the collagen deposition,expression of pre-traumatic EGF,bFGF and TGF-β,wound re-epithelialization,shorten wound healing time and improved the healing quality.Fluorescent double staining of CD31 and MAB1281 showed that RGD hydrogel combined with hAMSCs group could promote angiogenesis through differentiation in wound area.Conclusion1.The peptide hydrogels could provide better growth environments for the hAMSCs,the mRNA expression of stemness-related,growth factors,inflammatory factors and osteogenic and adipogenic differentiation related genes of hAMSCs decreased when compared with that in 2D petri dish,while the expression of β-Tubin III increased which may indicate the neurogenic differentiation potential of hAMSCs in soft hydrogels.2.Combined with hAMSCs,the hydrogles can could effectively promote the collagen deposition,expression of pre-traumatic EGF,bFGF and TGF-β,wound re-epithelializationand neovascularization,significantly shorten the wound healing time and improved the healing quality.