Research Of Methylacrylylated Gelatin Loaded With Concentrated Growth Factors To Promote Wound Healing

Purpose and significance of the study:Growth factors promote the injured tissue repair mainly by activating related signaling pathways,as well as promoting cell proliferation,differentiation and migration.Exogenous growth factors have been widely used in the repair of clinical refractory wounds.However,exogenous growth factors not only have the risk of local rejection,but also are expensive,complex to prepare,single composition,unstable physical and chemical properties,and easy to be contaminated by microorganisms,which are still challenge problems.Concentrated growth factor(CGF)is the third generation of plasma extract after the development of first generation of platelet-rich plasma(PRP)and the second generation of platelet-rich fibrin(PRF).It uses continuous variable speed centrifugation technology to make platelets in the blood collide and break continuously,releasing more growth factors.It is rich in transforming growth factor-β(TGF-β),epidermal growth factor(EGF),and platelet-derived growth factor(PDGF),vascular endothelial growth factor(VEGF),fibroblast growth factor(FGF)etc.It can promote cell proliferation and differentiation,and enhance the ability of tissue regeneration and healing,which has promising clinical application prospects.However,the jelly-like CGF fibrin gel does not easily adhere to the wound site upon local application alone since its poor plasticity and adhesivity.Furthermore,the growth factor released from CGF fibrin gel shows rapid release,a short half-life,and rapid degradation,making it difficult to match the wound healing cycle.Therefore,a special form of CGF and an adaptive carrier needs to be explored for addressing the above-mentioned challenges and achieving effective delivery and sustained release of growth factors.Gelatin Methacryloyl(GM)is an modified alkene double bond gelatin.It is a biomimetic extracellular matrix material of tissue engineering and often used in the field of tissue regeneration and repair.Here,we prepared GM@CGF hydrogel by physical blend of CGF and GM,which can be used as a wet dressing to cover the wound,and can simulate extracellular matrix and growth factor delivery platform,providing a new treatment method for wound repair.Methods:CGF was extracted by continuous variable speed centrifugation and then mechanically compressed to prepare CGF extract.The latter was physically blended with GM,they can rapidly solidify into GM@CGF hydrogel under UV light.(1)Physical and chemical properties of GM@CGF hydrogel: Scanning electron microscope(SEM)was used to observe the surface morphology characteristics.The ability about sustained release of growth factors from GM@CGF hydrogel was detected by release efficiency test.The feasibility of the hydrogel as a wet dressing was evaluated by the tests of swelling,degradation,self-healing,antioxidant,injectability,and wet tissue adhesion.(2)Biocompatibility of GM@CGF hydrogel: cell compatibility,blood compatibility,histocompatibility and visceral toxicity were used to evaluate the safety of hydrogel as a wound dressing.(3)Biological function of GM@CGF hydrogel: migration test,Transwell test,tube formation test were used to evaluate the ability of hydrogel to promote cell proliferation,migration and differentiation in vitro.(4)Wound healing ability of GM@CGF hydrogel: wound healing test in rabbit back skin was used to evaluate the wound repair efficacy of hydrogel,and tissue PCR was used to explore the potential mechanisms of wound repair promoted by hydrogel.Results:1 Physical and chemical properties of GM@CGF hydrogel: SEM showed that the lyophilized GM@CGF hydrogel had a loose three-dimensional pore structure.The GM@CGF hydrogel reached the swelling equilibrium earlier and degraded more efficiently than the GM hydrogel.The GM@CGF hydrogel can sustained release various growth factors,reach the release equilibrium in 6 hours,and then maintain a high release efficiency for a long time.GM@CGF hydrogel also has good antioxidant properties.In addition,GM@CGF hydrogel has excellent gelation performance,superior injectability and self-healing,it can fill the irregular shape wounds.When the skin is bent,twisted and moisten,the GM@CGF hydrogel still keep adherent to the skin.(2)Biocompatibility of GM@CGF hydrogel: cell staining showed that HUVECs had good vitality and morphology in GM@CGF hydrogel with high survival rate.The CCK-8assay indicated that HUVECs in the GM@CGF hydrogel had higher proliferation activity.In vitro hemolysis test showed that the hemolysis rate of GM@CGF hydrogel was much lower than the international standard.The hydrogel was implanted subcutaneously in nude mice for 14 days and observed by H&E staining,we found that the cells gathered at the edge of GM@CGF hydrogel and infiltrated into it.A large number of neovascularization,densely arranged collagen fibers,and evenly distributed fibroblasts and smooth muscle cells were observed around the GM@CGF hydrogel.The shape and structure of svisceral cells in nude mice were normal,and no obvious deformation and necrosis was observed by H&E staining.The blood biochemical test of nude mice also showed that the hepatorenal function were not obvious abnormal.2 Biological function of GM@CGF hydrogel: The scratch test showed that GM@CGF hydrogel could rapidly reduce the scratch area of HUVECs.Transwell test observed that HUVECs clustered in GM@CGF hydrogel with strong invasiveness.In vitro tube formation test showed that GM@CG hydrogel significantly promoted the HUVECs to form vascular mimicry,with more circle and node structures.(4)GM@CGF hydrogel wound healing ability: After applying GM@CGF hydrogel to back wounds in rabbits,no obvious wounds were observed at day 14,showing a faster healing efficiency than the other groups.MASSON’s and H&E staining showed that at day7,GM@CGF hydrogel group had more granulation tissue filling and epithelial regeneration,more fibroblasts migrated to the wound site,and more vascular beds and collagen fibers wrapped around the wound.At day 14,the new epithelium in GM@CGF hydrogel group was moderately thick with more hair follicles and gland.At day 7,tissue PCR showed that GM@CGF hydrogel significantly increased the expression of wound repair related genes(TGF-β,EGF,Col1 and VEGF).Conclusions:(1)GM@CGF hydrogel has stable gelation performance,3D pore structure,good sustained release performance as well as excellent swelling and degradation properties.It can adhere to the wound and prolong the service time.Besides,The good biocompatibility of GM@CGF hydrogel ensures its safety in wound application.(2)GM@CGF hydrogel can significantly promote cell proliferation,migration and differentiation,and has good biological functions in vitro,which lays a cytological support for the application of GM@CGF hydrogel wound.(3)In the process of wound repair,the autologous GM@CGF hydrogel can sustainably release VEGF,PDGF,TGF-β and other growth factors to form a comprehensive growth factor library in the wound,initiate wound repair,stimulate the expression of cell repair genes,promote granulation tissue formation,collagen deposition,as well as vascular regeneration and reconstruction.Meanwhile,it can provide a moist environment and an extracellular matrix-like structure to guide cells to migrate to the defect,achieving the goal of accelerating wound healing.