Adipose-derived Stem Cells(ADSCs)and Platelet-rich Plasma(PRP)loaded Hydrogels For Improving Healing In A Murine Pressure Ulcer Model

Background and ObjectionPressure ulcers are caused by local tissue pressure for a long time,and tissue suffered from continuous ischemia,hypoxia and malnutrition,resulting in tissue ulceration and necrosis,and serious cases can also cause death.It often occurs in patients who are bedridden or wheelchair-bound including elderly patients and patients who suffered from paralysis,coma.Pressure ulcers could cause problems such as unhealing migration,long treatment time,difficult treatment,high cost and repeated attacks,which make patients lose the ability to take care of themselves in normal life and cause heavy burden to individuals,families and society.With the aging of population,the incidence of pressure ulcers is increasing year by year.Effective treatment of pressure ulcers is a difficult problem to be solved.In recent years,hydrogel has been gradually applied to the treatment of pressure ulcer wounds due to its good biocompatibility,degradability,and porous network structure similar to natural extracellular matrix.However,the wound is prone to infection,ischemia,and hypoxia,the single structure and function of the hydrogel cannot meet the needs,so it is necessary to develop a multifunctional hydrogel.A number of studies have shown that stem cells can promote angiogenesis and nerve regeneration in pressure ulcer wounds.However,transplantation of stem cells to pressure ulcer wounds alone has the problem of low survival rate and retention rate of stem cells.Therefore,some researchers have found that hydrogel-loaded stem cells in the treatment of pressure ulcers can significantly improve the survival rate and low retention rate of stem cells,but the survival rate is still very low in the wound area without vascular residue.Platelets,as multifunctional cells,play an important role in angiogenesis,tissue repair and inflammation.Platelet rich plasma(PRP)is a platelet concentrate obtained from whole blood after centrifugation,rich in platelet fibrin and concentrated growth factor.Studies have shown that PRP can promote early wound healing and prevent infection,but the release of growth factors in PRP has a short timeliness and cannot play a long-term effect.Some researchers have found that PRP loaded with hydrogel to treat pressure ulcers can promote the proliferation and migration of stem cells,but the effect is limited.Gelatin and silk fibroin are natural hydrophilic colloid with good adhesion and biocompatibility.Natural polymers of natural hydrogels contain hydroxyl and amine groups,which can be chemically modified to improve mechanical properties and biological stability.In this study,methacrylate anhydride(MA)was used to modify gelatin to synthesize Gel MA.Silk fibroin was modified with glycidyl methacrylate(GMA)to synthesize SFMA.Then,in the presence of ultraviolet light and photoinitiator LAP,GELMA /SFMA composite hydrogel was formed by simple mixing of SFMA solution and GELMA solution.Based on the biological functions and research background of ADSCs,PRP and modified hydrogels,the hypothesis was put forward in this study,that is,using Gelma and SFMA hydrogels as cell and growth factor carriers,to develop ADSCs and PRP hydrogels system.The mechanism and effect of hydrogels GELMA /SFMA,GELMA/SFMA/PRP and GELMA /SFMA/PRP/ADSCs in promoting wound healing were studied and evaluated by material characterization,biocompatibility and mouse pressure ulcer model.Methods(1)Methacrylate silk fibroin(SFMA),methacryloyol gelatin(Gel MA),SFMA/Gel MA composite hydrogel and SFMA/Gel MA/PRP hydrogel were prepared and characterized by 1H NMR,FT-IR,SEM and pore size analysis of hydrogel swelling,rheological properties,compression properties in vitro and PRP release growth factors.(2)The prepared hydrogels were loaded with ADSCs,and cell survival and proliferation of stem cells on the surface and inside of the hydrogels were analyzed by dead or alive cell staining,CCK8,electron microscopy,scratch test and other methods to evaluate the biocompatibility of the hydrogels.(3)The hydrogels prepared in this study(SFMA/ GELMA,SFMA/ GELMA /PRP,SFMA/ GELMA /PRP/ADSCs)were applied to the mouse pressure ulcer model,respectively.The effect of hydrogel loaded with ADSCs and PRP on pressure ulcers was comprehensively evaluated through wound healing evaluation,histological staining,H&E staining,immunofluorescence and Western Blot detection of related indexes(CD31,α-SMA,CD68,CK14,Col-1α)and immunohistochemistry(S100).Results(1)The characterization results of 1H NMR,FT-IR and SEM showed that the prepared hydrogel accords with the typical structure of hydrogel.(2)The results of swelling,rheological,compressibility,in vitro degradation and PRP release of growth factors showed that all hydrogels had good water absorption,good stability and good slow release of growth factors in PRP,and the degradation rate of Gel MA/SFMA hydrogel was slower than that of pure Gel MA hydrogel.(3)The results of biocompatibility evaluation showed that the cells grew on the surface of the hydrogel and the survival rate was high,and with the extension of culture time,the number of ADSCs cells increased,indicating that the prepared Gel MA/SFMA/PRP hydrogel has a three-dimensional structure,which is suitable for cell adhesion and proliferation,and has good biocompatibility.(4)The results of wound healing of mouse pressure ulcers model treated with hydrogel loaded stem cells(SFMA/Gel MA/PRP/ADSCs)showed that with the extension of time,the pressure ulcers wound area of Gel MA/SFMA/PRP/ADSCs group was significantly smaller than that of the control group,and the wound healing time was significantly reduced.The results of HE staining and Masson staining showed that the inflammatory infiltration was the slightest in the Gel MA/SFMA/PRP/ADSCs group,with partial skin accessory structure regeneration,epidermis regeneration and dermis thickening,but necrotic scabs and inflammatory cells could be seen in the dermis of other groups.The number of inflammatory cells in Gel MA/SFMA/PRP group and Gel MA/SFMA group decreased significantly,while there were a large number of inflammatory cells in gauze control group.The results of immunohistochemistry and western blot showed that the expression of CD31,α-SMA,COL-1α and S100 was up-regulated,while the expression of CD68 and CK14 was down-regulated in Gel MA/SFMA/PRP/ADSCs group.Conclusion(1)Gel MA/SFMA/PRP hydrogel can significantly promote the proliferation and migration of ADSCs cells.ADSCs encapsulated in hydrogel can maintain normal cell morphology and high survival time.(2)Gel MA/SFMA/PRP/ADSCs hydrogel can promote wound healing,reduce inflammatory infiltration,increase collagen deposition,promote angiogenesis and nerve regeneration.