The Repair Effect And Mechanism Of PDGF-BB/SA/Dex Hydrogel Loaded With BMSCs On A Mouse Model Of Full-Thickness Skin Excision Wounds

Skin is the largest organ of the body and plays important roles in protecting against mechanical damage,microbial infection and extreme temperature,but it is also highly susceptible to external environmental aggressions.Wound healing is one of the most complex processes in the body and is also a well-orchestrated dynamic and interactive process.Stem cell transplantation offers new hope for the treatment of skin lesions,but the loss and low survival rates of transplanted stem cells in the wound limit their therapeutic efficacy.Tissue engineering techniques have opened a new path to improve the transplantation of stem cells.Injectable hydrogel,with high water content and porous structure,has promising applications in tissue engineering.Sodium alginate（SA）has been widely used in various biomedical fields such as tissue regeneration,drug delivery and cell embedding due to its good biocompatibility and low toxicity.Dextran（Dex）hydrogels could promote the infiltration of vascular cells and endothelial cell migration to the wound surface to increase neovascularization during treatment.So,SA/Dex injectable hydrogels could provide a good microenvironment for stem cells,reduce cell loss and also release growth factors to improve their suitability as a drug delivery system.Platelet derived growth factor-BB（PDGF-BB）is a key glycoprotein factor that can promote the biological behavior of stem cells.Studies have shown that PDGF-BB could regulate the migration,chondrogenic and myogenic differentiation ability of bone marrow mesenchymal stem cells（BMSCs）in hydrogels.However,the short half-life limits its bioavailability.Numerous studies have shown that injectable hydrogels can slowly release PDGF-BB to improve its efficacy in different tissue engineering.However,the therapeutic effects of SA/Dex hydrogels loaded with both PDGF-BB and BMSCs on wound healing have not been reported.ObjectiveThis project proposes to synthesize injectable SA/Dex hydrogels and fabricate PDGF-BB/SA/Dex composite hydrogel with sustained release of PDGF-BB.Then,the biocompatibilities of SA/Dex and PDGF-BB/SA/Dex hydrogels were systematically studied.Finally,the effect and mechanism of BMSCs/PDGF-BB/SA/Dex hydrogel transplantation in a mouse model of full-thickness excision wound were examined by a series of assays.MethodsPart I:Preparation,optimization and characterization of SA/Dex hydrogelsFour SA/Dex injectable hydrogels,denoted as SA,SD,SD₂ and SD₄,were synthesized by ionic cross-linking reaction;the gel time was measured by test tube inversion method;the in vitro weight loss and water content of hydrogels were measured by dry weighing method;the elastic modulus of hydrogels was measured by rotational rheometry;The internal microstructure of the hydrogels was observed by scanning electron microscope（SEM）.CCK-8,acridine orange（AO）-propidium iodide（PI）staining were used to detect the effects of the four hydrogels on the survival and proliferation of BMSCs and to evaluate the cytocompatibility of the hydrogels;hemolysis assay was performed to evaluate the hemocompatibility of the four hydrogels;the hydrogels were injected subcutaneously and the material was taken at specific time points.Hematoxylin&Eosin（H&E）staining was performed to detect the inflammatory reaction of the hydrogel transplantation sites and to evaluate the histocompatibility of the hydrogels.Part II:Effects of PDGF-BB and PDGF-BB/SA/Dex composite hydrogel on the proliferation,migration and endothelial cells（ECs）differentiation of BMSCsCCK-8 was used to detect the effect of 0,10,20,50 and 100 ng/m L PDGF-BB on the proliferation of BMSCs;Transwell was used to detect the effect of 0,10 and 20ng/m L PDGF-BB on the migration of BMSCs;CD31,von Willebrand Factor（v WF）immunofluorescence and q RT-PCR were used to detect the effect of 20 ng/m L PDGF-BB on the ECs-differentiation of BMSCs;CCK-8,AO-PI staining assay were performed to detect the effect of PDGF-BB/SA/Dex composite hydrogel on the survival and proliferation of BMSCs;The effect of PDGF-BB/SA/Dex composite hydrogel on the migration of BMSCs was detected by Transwell;the slow-release ability of PDGF-BB/SA/Dex composite hydrogel was detected by ELISA,and the chemotactic activity of PDGF-BB after slow release was detected by Transwell.Part III:The repair effect and mechanism of BMSCs/PDGF-BB/SA/Dex hydrogel transplantation on a mouse model of full-thickness excision woundA total of 48 C57BL/6 mice mice were used to construct a 0.8 cm diameter full-thickness excision wound models,and randomly divided into Control group（Subcutaneous injection of 100μL saline）,BMSCs group（Subcutaneous injection of10⁶ Di I fluorescently labeled BMSCs）,PDGF-BB/SA/Dex group（Subcutaneous injection of 100μL PDGF-BB/SA/Dex hydrogel）,and BMSCs/PDGF-BB/SA/Dex group（Subcutaneous injection of 100μL of PDGF-BB/SA/Dex hydrogel loaded with10⁶ Di I fluorescently labeled BMSCs）.Wound healing in each group was detected on days 3,7 and 12 after transplantation;H&E and Masson staining experiments were used to detect the quality of wound healing in each transplantation group;Keratin 6（K6）and Keratin 1（K1）immunofluorescence staining was used to detect the epithelial regeneration ability of skin wounds in each group;Western blot and TUNEL staining were performed to detect the expression of apoptosis in mouse skin tissue cells 7 days after transplantation.;Di I staining was used to detect the retention and survival of BMSCs at the injury site at 7 days after transplantation;v WF and SOX9 immunofluorescence assays were used to detect in vivo endothelial cell differentiation of BMSCs and activation of hair follicle stem cells after transplantation;CD31/α-SMA immunofluorescence,q RT-PCR and Western blot were performed to detect the angiogenesis and the expression of PDGF-BB/PDGFR-βand PI3K/AKT/e NOS-related pathway proteins.ResultsPart I:Preparation,optimization and characterization of SA/Dex hydrogels1.SA/Dex injectable hydrogel could be shaped according to the mold,and exhibit proper gelation time,high water content and biodegradability.The elastic modulus was around 200 Pa and SEM results showed that SA/Dex hydrogels with different concentration ratios were alternately connected inside to form a porous mesh structure.2.The good cytocompatibility of SA/Dex hydrogel was beneficial to the proliferation and survival of BMSCs;subcutaneous injection of SA/Dex hydrogel did not cause any obvious inflammatory reaction,which had excellent histocompatibility and blood compatibility.And,the in vivo degradation rate was consistent with that in vitro.So,SD₂ hydrogel was selected as the ideal delivery vehicle for subsequent experiments.Part II:Effects of PDGF-BB and PDGF-BB/SA/Dex composite hydrogel on the proliferation,migration and endothelial cells（ECs）differentiation of BMSCs1.10 and 20 ng/m L PDGF-BB significantlypromoted the survival and migration of BMSCs,in which the promotion effect of PDGF-BB at 20 ng/m L on the proliferation and migration of BMSCs was more obvious after treatment（P<0.05）.And,PDGF-BB significantly improved the ECs differentiation efficiency of BMSCs（P<0.05）.2.The PDGF-BB/SA/Dex composite hydrogel promoted the proliferation of BMSCs on day 3（P<0.05）and had good biocompatibility.PDGF-BB/SA/Dex composite hydrogel released PDGF-BB continuously and steadily.30%of PDGF-BB was released on Day 1,and then entered the slow release process,and the cumulative release rate reached 80%on Day 25.Transwell results indicated that PDGF-BB released on Days 7 and 14 significantly promoted the migration of BMSCs（P<0.05）with high biological activity.Part III:The repair effect and mechanism of BMSCs/PDGF-BB/SA/Dex hydrogel transplantation on a mouse model of full-thickness excision wound1.BMSCs/PDGF-BB/SA/Dex transplantation accelerated wound healing as accompained by more re-epithelialization and collagen deposition（P<0.05）.In addition,PDGF-BB/SA/Dex hydrogel promoted the ECs-differentiation of transplanted BMSCs and proliferation of hair follicle stem cells in the wound（P<0.05）.2.The retention and survival of BMSCs at the wound were significantly increased in BMSCs/PDGF-BB/SA/Dex group.The number of Ki67-positive cells was significantly increased,while TUNEL-positive cells were significantly decreased in skin tissues with lower expression of pro-apoptosis-related proteins Bax and Caspase3（P<0.05）.3.The expressions of CD31 andα-SMA were obviously increased in BMSCs/PDGF-BB/SA/Dex group（P<0.05）,and Western blot results showed that PDGF-BB,PDGFR-β,p-PI3K/PI3K,p-AKT/AKT and p-e NOS/e NOS expression were significantly increased by the transplantation of BMSCs/PDGF-BB/SA/Dex hydrogel（P<0.05）.Conclusion1.SA/Dex hydrogel had high water content,biodegradability and good biocompatibility for the simultaneous delivery of stem cells and PDGF-BB.2.PDGF-BB significantly promoted the survival,migration and ECs differentiation of BMSCs;PDGF-BB/SA/Dex hydrogel sustainably released PDGF-BB and promoted the proliferation and migration of BMSCs.3.BMSCs/PDGF-BB/SA/Dex transplantation significantly promoted wound healing by improving epithelialization,collagen deposition and tissue remodeling.In addition,PDGF-BB/SA/Dex hydrogel promoted the retention of BMSCs,regeneration of keratinocytes,proliferation of hair follicle stem cells and the ECs-differentiation of transplanted BMSCs in the wound.Finally,PDGF-BB/SA/Dex injectable hydrogel accelerated BMSCs-mediated skin wound healing by promoting angiogenesis via the activation of PDGF-BB/PDGFR-β-mediated PI3K/AKT/e NOS pathway,which may provide a new therapeutic strategy for stem cell therapy in wound healing.