Construction And Evaluation Of Bletilla Striata Polysaccharide-Based Hydrogels For Wound Repair Application

Trauma is a common surgical problem in clinic.Acute injuries such as burns,lacerations and contusions often cause pain and are associated with a loss of intrinsic protection.Chronic trauma,such as diabetic ulcer,can lead to wound infection,necrosis and scar formation,which can seriously affect the patient’s quality of life.The clinical management requirements of trauma include effective hemostasis,wound closure,prevention of infection and other requirements.At present,common dressings such as gauze and bandage in clinical practice cannot meet the requirements.This highlights the necessity of exploring and developing new multifunctional biomaterial systems.In recent years,hydrogel dressings have received extensive attention in the field of tissue engineering and regenerative medicine.Hydrogels meet the requirements of "wet healing" and can simulate natural soft tissue and extracellular matrix(ECM)components to fill,adhere,stop bleeding and promote wound healing.In this thesis,the effective component of Bletilla striata polysaccharide was modified.Bletilla striata polysaccharide could be partially aldehyde oxidized by sodium periodate and obtained Dialdehyde Bletilla striata polysaccharide(BSP-CHO).The relative molecular weight of BSP-CHO with different contents of aldehyde group decreased,but its antioxidant activity did not lose in vitro.Cationic gelatin(CG)is prepared by grafting gelatin with ethylenediamine,which effectively increases the active amino group.Injectable Bletilla striata polysaccharidegelatin hydrogel(BG-gel)with self-healing activity was obtained from the two materials based on Schiff base reaction.Compared with the synthetic polymer materials,the Bletilla striata polysaccharide and gelatin have good biocompatibility,and no chemical cross-linking agent was introduced into Schiff base reaction.Therefore,the BG-gel hydrogels have good cytocompatibility,blood compatibility and in vivo safety.BG-gel hydrogels can be stably degraded under the skin of rats.With the increase of aldehyde group concentration in BSPCHO,the crosslinking density of hydrogel showed an increasing trend,which was characterized by the decrease of pore diameter,the strengthening of mechanical properties and the backward movement of collapse point.In vitro and in vivo experiments showed that BG-gel hydrogels can promote the migration and fusion of L929 cells,and reduce the bleeding time of rat liver puncture and mouse tail hemorrhage model,with excellent hemostatic performance.The wound healing function of BG-gel hydrogels was verified by rat whole layer skin defect experiment.BG-gel hydrogel can absorb the wound exudate,promote the transport of nutrients and waste on the wound,effectively promote the initial hemostasis of the wound,reduce the inflammation of the wound,accelerate the epithelialization of the wound,and contribute to the wound healing.H&E staining showed that BG-gel hydrogels increased angiogenesis and promoted hair follicle development.Masson staining showed that BG-gel hydrogels could promote the formation of collagen fibers and new epidermis,and increase the expression of collagen in the wound area.Acid-zinc alginate nanoparticles loaded berberine hydrochloride and GHK-Cu were prepared by reverse microemulsion method.Two kinds of drug-loaded nanoparticles were embedded into the hydrogel to prepare the berberine hydrochloride nanoparticles complex hydrogels(BNPs-gel)and GHK-Cu nanoparticles complex hydrogels(GNPs-gel).The two drugs in the composite hydrogel showed "sustained release" behavior,and the addition of drugloaded nanoparticles improved the mechanical properties of the hydrogel.The comprehensive promoting effect of BNPs-gel and GNPs-gel on wound healing was studied in rat whole layer skin defect experiment.BNPs-gel plays a significant antibacterial role in the initial stage of wound healing,and reduces the expression of relevant inflammatory factors,accelerating the transformation of wound from inflammation stage to proliferation stage.GNPs-gel can effectively promote the m RNA expression of growth factors and promote angiogenesis.Immunohistochemical experiments were conducted to investigate the effect of nano-composite hydrogels on angiogenic factor CD31 and cell proliferation index Ki67 during wound healing.The two composite hydrogels effectively promoted the expression of Ki67,and GNPs-gel also promoted the expression of CD31.In this thesis,the effective ingredients of Traditional Chinese medicine Bletilla striata polysaccharide and natural material gelatin were modified to make them into the matrix material for preparing hydrogel.Among them,Bletilla striata polysaccharide not only participated in the construction of hydrogel as a matrix,but also showed a good function of promoting healing,playing the role of " medicine and adjuvant of the same source ".On this basis,drug-loaded nanoparticles were embedded into the hydrogel to construct a multifunctional mixed biomaterial system,so as to achieve sustained controlled release of drugs,improve the local bioavailability of drugs in wound tissues,and promote wound healing.Drugloaded nanoparticles-hydrogel mixed biomaterials are a feasible method to prepare wound dressings.Continuous and controlled release of the drug in the hydrogel effectively increases its bioavailability.To some extent,the drug-loaded nanoparticles also endowed the hydrogel with anti-inflammatory,wound healing and angiogenesis promoting pharmacological properties.It has great application potential in repairing wound and accelerating wound healing.