Construction Of Polysaccharidebased Hydrogel Dressings Based On Skin Wound Microenvironment For Injury Repair

Skin wound healing is a multi-stage process involving multiple cellular and molecular interactions,in which damaged tissues are repaired and regenerated through the regulation of cellular behaviors and dynamic remodeling of the extracellular matrix（ECM）.Minor changes of the wound microenvironment caused by exogenous or endogenous factors may affect cell activation and recruitment,which in turn affect the wound healing process.In recent years,based on the“wet healing”theory,plenty of hydrogel dressings have been developed to create a suitable healing microenvironment for wounds and are therefore widely used in clinical wound care.Benefiting from the biocompatibility,high hydrophilicity,degradability and easy gel formation of natural polysaccharides,most of the hydrogel dressings on the market are made by them.However,these dressings are mainly dedicated to isolation and moisturization to reduce infection and alleviate local symptoms.They also lack bioactivity to improve the microenvironment of wounds at the molecular level,which makes the problem of delayed wound healing still exist and puts a higher demand on the development of hydrogel dressings.By further studying the cellular molecular mechanism of wound healing,polysaccharides with different structural functions and biological functions were screened as raw materials of hydrogel dressings for different types of wounds in this thesis.Through structural design,functional modification,and drug loading,the prepared hydrogels possess enhanced bioactivity,which can regulate the wound microenvironment at multiple stages,induce specific behaviors of key cells,and activate relevant pathways.As a result,the hydrogels can accelerate the wound repair and regeneration,and rebuild the skin barrier.The wound healing process consists of four phases:hemostasis,inflammation,proliferation and remodeling,which occur in overlapping chronological order.However,some intrinsic pathologies such as diabetes will prevent the wound from successfully passing through the inflammatory phase,and finally leads to the chronic wound.The persistent inflammation and excessive oxidative stress are the marked features and important mechanisms of chronic wound,which make it difficult to heal.Based on this,in the Chapter2,the hydrogel dressing（Cur/HH gel）with anti-inflammatory and antioxidant capacity was designed and constructed,and basic studies on the treatment of chronic diabetic wounds was carried out at the cellular and molecular level.The biofunctional glycosaminoglycan hyaluronic acid（HA）and heparin（Hep）were respectively modified byβ-cyclodextrin and adamantane to form hydrogel framework by supramolecular assembly,and the natural small molecule curcumin is loaded in the hydrogel at the same time.Hep captured monocyte chemotactic protein-1（MCP-1）to reduce the influx of immune cells to the wound site.Curcumin scavenged reactive oxygen species（ROS）and block inflammatory pathways to inhibit the polarization of macrophages to the abnormal M1 phenotype.Hep and curcumin synergistically disrupted the vicious cycle between ROS and inflammation at the wound site,allowing it to pass through the inflammatory phase smoothly.At the same time,HA endowed hydrogel with ECM mimicking function,which promoted the proliferation and migration of fibroblasts and keratin-forming cells at the wound site,thus effectively accelerating re-epithelialization and granulation tissue formation.By combining the functions of these components,the constructed Cur/HH gel achieved the purpose of modulating the wound microenvironment and regulating cellular behavior in multiple ways,which thus promoted the efficient healing of diabetic chronic wounds.In addition to chronic inflammation,the lack of blood supply to wound caused by vascular microcirculation disorder complicated with diabetes is also an important reason for delayed healing of diabetic wounds.Macrophages expressing high levels of inducible nitric oxide synthase（i NOS）not only induce inflammation,but also inhibit the availability of endothelial nitric oxide synthase that regulates vascular function.Therefore,regulation of nitric oxide synthase（NOS）is a potential treatment for diabetic wounds.Moreover,considering the easy dissolution of the amorphous hydrogels and the inconvenience caused by multiple dressing changes,the hydrogel patches have more advantages in the long-term treatment for chronic wounds.Based on the material design and study in Chapter 2,a polyvinyl alcohol crystalline network was introduced to enhance the HA-Hep hydrogel system in Chapter 3.A dual network hydrogel patch（HHP）was formed by repeated freeze-thawing,loaded with i NOS-specific inhibitor（S）-Methylisothiourea sulfate（SMT）and classical vascular endothelial growth factor（VEGF）.The dual network structure allowed for enhanced gel structure and mechanical properties which matched skin mechanics.The gel had a suitable mesh size to achieve effective binding between heparin and VEGF for rapid release of SMT and slow release of VEGF.The effectiveness of SMT in down-regulating nitric oxide expression and inhibiting inflammation was verified by cytological experiments.These studies provided the possibility for the follow-up of anti-inflammatory and angiogenesis synergistic therapy in diabetic wounds.In the previous two chapters of this thesis,the designed hydrogels were used to regulate the inflammation of chronic diabetic wounds and restore them to the normal healing process similar to acute wounds.After the inflammatory phase,wound repair enters a lengthy proliferative and remodeling phase,which requires the synergistic regulation of multiple cells and growth factors（GFs）to achieve good healing quality.The platelet-rich plasma gel（PRP gel）,which is used clinically,is rich in GFs required for wound repair,but its poor mechanical properties and burst release of GFs reduce its therapeutic effect.In this regard,from the perspective of clinical safety and easy clinical translation,sodium alginate（SA）,a natural polysaccharide commonly used in clinical dressings,was selected as the raw material in Chapter 4 and combined with PRP for innovation.The SA/PRP dual network hydrogel（DN gel）was constructed by the one-step activation of thrombin/calcium chloride.Fibrinogen in PRP formed the first network,and the second network was formed by chelation between SA and Ca²⁺,while the secreted GFs were trapped homogeneously inside the dual networks.Compared with the PRP gel,the prepared DN gel presented good mechanical properties,stability and injectability,and exhibited pro-longed release behavior of bioactive factors.Cytological experiments showed that DN gel could more effectively promote the proliferation and migration of fibroblasts and endothelial cells.In studies of full-thickness defect wound healing in rats and pigs,compared with the PRP gel,DN gel owned the ability to better regulate cell behavior and GFs secretion at the wound site,thus more effectively promoting re-epithelialization,granulation deposition and angiogenesis,which finally improved the speed and quality of wound healing.This DN gel based“one-step”preparation strategy had good potential for clinical translation benefiting from its low cost and high efficacy.In summary,aimed at different types of skin wounds and their microenvironmental characteristics,polysaccharides（HA,Hep,SA）with different structural and biological functions were selected to fabricate hydrogel through supramolecular assembly,freeze-thawing crystallization,and ion chelation,respectively.These hydrogels were used for therapeutic drug（curcumin,SMT,VEGF,PRP）delivery at the same time.The designed hydrogel dressings can spatiotemporally improve the wound microenvironment in multiple dimensions and promote the wound healing with high efficiency and quality.These works provide values for the future development of wound management.