The Effect Of Multi-component Hydrogels In Promoting Diabetes Wound Healing

Background: diabetes has become a global public health problem.Long term hyperglycemia caused by poor control of diabetes blood sugar level will lead to damage to the basic structure and function of large blood vessels and microvessels of the body,leading to complications such as diabetes wounds,severely threatening people’s life safety and health,and also causing a heavy economic burden.The treatment of diabetes wounds is a challenge for plastic surgery,which puzzles clinical surgeons around the world.Studies on the effect of diabetes on wound healing have shown that high systemic blood glucose concentration leads to excessive glycosylation of proteins and the formation of advanced glycosylation end products,leading to the expression of proinflammatory cytokines,oxidative damage and changes in extracellular matrix.Hyperglycemia can inhibit the proliferation of fibroblasts,endothelial cells and keratinocytes.So,developing a method that can quickly stop bleeding,inhibit bacterial infection,reduce oxidative damage and tissue inflammation,and promote angiogenesis has become an effective means of wound treatment for diabetes.It is worth noting that the immune dysfunction of diabetes wounds has also become an important target for treatment.The study found that the early stage of diabetes wound showed delayed immune initiation,which was manifested by delayed infiltration of immune cells,while the late stage showed widespread presence of proinflammatory cells.By promoting the early initiation of immune response and continuously inhibiting its inflammatory phenotype,it may be able to achieve immune remodeling of diabetes wounds,and ultimately promote the healing of diabetes wounds.Since hydrogel can fill irregular wounds,maintain a moist environment,and has adjustable mechanical properties and good biocompatibility,it has attracted continuous attention in the treatment of diabetes wounds.Promoting the healing of diabetes wounds by building multi-component hydrogel has become an important research direction.Objective:(1)To explore whether CS-DA/TA/PF/3D-Exos hydrogel,which is constructed for rapid hemostasis,inhibition of bacterial infection,reduction of oxidative damage and tissue inflammation,and promotion of angiogenesis,has good antibacterial,anti-inflammatory,antioxidant,adhesive and hemostatic capabilities;(2)Cell and animal experiments were used to verify whether CS-DA/TA/PF/3D-Exos hydrogel can promote the healing of diabetes wounds;(3)To explore whether OHA/GT/ECM/Cur/CCL2 hydrogel,which aims at the early rapid start of immune function,continues to play an anti-inflammatory role and promotes macrophages to polarize into M2 type,can reshape the immune homeostasis of diabetes wounds;(4)The promotion effect of HA/GT/ECM/Cur/CCL2 hydrogel on diabetes wound healing was verified through in vivo and in vitro experiments.Methods and results:(1)We extracted 3D and 2D exosomes and identified exosomes by means of TEM,NTA and WB.We found that the yield of exosomes extracted from 3D culture was higher than that from 2D culture.Further,by evaluating the regulation of HUVECs,fibroblasts and RAW264.7,we verified in vitro that the advantages of 3D exosomes over 2D exosomes are more suitable for the treatment of diabetes wounds.In the development of hydrogel,we first improved the water solubility of CS by grafting dihydrocaffeic acid(dopamine,DA)onto antibacterial cationic polymer chitosan(CS),and then formed CS-DA/TA by superposing the polyphenol structure tannic acid(TA)with antibacterial,anti-inflammatory and antioxidant effects via the dopamine polyphenol group π-π,Then,the amino group of CS grafted dopamine and the aldehyde group reaction of the crosslinking agent Pluronic F127-CHO(PF)form a dynamic Schiff base bond to form CS-DS/TA/PF,which increases the CS-DS/TA/PF hydrogel with good mechanical properties and self-healing properties.Finally,through the polyphenol structure,it can form a covalent bond with the outer membrane of the exosome to achieve the loading and slow release of the exosome,and construct CS-DA/TA/PF/3D-Exos hydrogel;(2)We evaluated the basic properties,safety and biological functions of the CS-DA/TA/PF/3D-Exos hydrogel.We found that the hydrogel has a continuous porous structure,which can grow,penetrate and nourish cells,rapidly expand and absorb wound exudate,and promote wound healing.We evaluated the antibacterial activity by counting bacterial clone formation,and found that CS-DA/TA/PF/3D-Exos hydrogel has good antibacterial ability.In order to evaluate the biocompatibility of CS-DA/TA/PF/3D-Exos hydrogel,we carried out direct contact hemolysis rate and live/dead cells staining in vitro.The results showed that CS-DA/TA/PF/3D-Exos hydrogel had good biological safety and clinical application potential;(3)To evaluate the efficacy of CS-DA/TA/PF/3D-Exos hydrogel in promoting wound healing in diabetes,we established a full-thickness skin defect model in mice.The study found that the wound healing speed was significantly accelerated after CS-DA/TA/PF/3D-Exos hydrogel treatment.H&E staining showed that the CS-DA/TA/PF/3D-Exos hydrogel group had the best skin maturity,dense epidermis,good dermal tissue,and hair follicles were appendages of mature skin.Further,Masson staining was used to evaluate the collagen deposition in different treatment groups.It was found that the collagen fibers in CS-DA/TA/PF/3D-Exos hydrogel group were thick and dense,showing a more mature phenotype.Immunofluorescence staining results showed that the expression of CD31 in CS-DA/TA/PF/3D-Exos hydrogel group was higher than that in other groups on the 7th day of wound healing,and CD31 and α-The co-expression level of SMA was higher than 7 days,indicating that most of the wounds were in the remodeling stage at 14 days.The above results indicate that CS-DA/TA/PF/3D-Exos hydrogel can well promote the healing of diabetes wounds.Because the hemostatic ability of hydrogel is very important,the hemostatic ability of CS-DA/TA/PF/3D-Exos hydrogel in vivo was evaluated by measuring the amount of bleeding and the hemostatic time in the rat liver injury model and the rat amputation model.The results showed that CS-DA/TA/PF/3D-Exos hydrogel group had good hemostatic ability;(4)We successfully prepared fat acellular matrix(ECM)from liposuction adipose tissue by cleaning,freeze-thawing,homogenizing,trypsin,nuclease,isopropanol and other degreasing and nucleic acid removal steps,and then obtained uniform ECM particles through low-temperature freezing and grinding.Oxidized hyaluronic acid(OHA)was prepared by standard method and further cross-linked with GT,and mixed with ECM particles uniformly.Infrared spectrum detection showed that OHA was successfully prepared and cross-linked with gelatin,and also produced a uniform load with ECM particles.We loaded CCL2 with OHA/GT,loaded Curcumin with ECM particles,and then fully mixed the two components to prepare OHA/GT/ECM/Cur/CCL2 hydrogel.The detection results of CCL2 and Curcumin release from OHA/GT/ECM/Cur/CCL2 hydrogel showed that CCL2 was released rapidly at an early stage,while Curcumin was released slowly and persistently;(5)SEM analysis of the hydrogel shows that OHA/GT/ECM/Cur/CCL2 hydrogel has a continuous porous structure.The rheological results showed that the mechanical properties and stability of the hydrogel were enhanced after the completion of crosslinking and the addition of different components.The hydrogel degradation experiment showed that OHA/GT/ECM/Cur/CCL2 hydrogel would achieve rapid degradation at an early stage,with the degradation rate reaching 50% on the fourth day and 90% on the seventh day.The above results suggested that OHA/GT/ECM/Cur/CCL2 hydrogel could well meet the needs of wound healing.Through direct contact hemolysis test and live/dead cell staining,OHA/GT/ECM/Cur/CCL2 hydrogel has good biocompatibility.In vitro immunofluorescence staining confirmed that OHA/GT/ECM/Cur/CCL2 hydrogel treatment can promote M2 macrophage polarization.These results showed that OHA/GT/ECM/Cur/CCL2 hydrogel can promote M2 macrophage polarization and activate the regeneration process.(6)To evaluate the efficacy of OHA/GT/ECM/Cur/CCL2 hydrogel in promoting wound healing in diabetes,we established a full-thickness skin defect model in mice.It was found that the wound healing speed of mice was significantly accelerated after treatment with OHA/GT/ECM/Cur/CCL2 hydrogel.H&E staining showed that OHA/GT/ECM/Cur/CCL2 hydrogel group had the best skin maturity.Masson staining was used to evaluate collagen deposition in different treatment groups.It was found that collagen fibers in OHA/GT/ECM/Cur/CCL2 hydrogel group were more mature and arranged more regularly.The results of immunofluorescence staining showed that OHA/GT/ECM/Cur/CCL2 hydrogel had a good ability to promote the polarization of macrophages to M2 type.In addition,OHA/GT/ECM/Cur/CCL2 hydrogel group CD31 and α-The expression level of SMA was the highest.The above results indicate that OHA/GT/ECM/Cur/CCL2 hydrogel can promote the healing of diabetes wounds,which mainly reshapes the immune homeostasis based on macrophages through the multi-component effect,providing a new idea for the treatment of diabetes.Diabetes has become a global public health problem.the global prevalence of diabetes is increasing year by year,which seriously threatens people’s life safety and health,and also creates a heavy economic burden.Long term hyperglycemia caused by poor control of blood sugar level in diabetes will lead to damage to the basic structure and function of large blood vessels and microvessels,and then lead to important organs and peripheral neuropathy.Once the complications of diabetes occur,it is difficult to reverse by drug treatment.Therefore,understanding the pathogenesis of its complications and carrying out early intervention can effectively reduce the decline in quality of life,disability and death caused by diabetes complications.The refractory wound of diabetes is one of the main complications of diabetes.In addition,diabetes patients often face delayed healing or non healing of the surgical wound after undergoing surgery,which can further lead to infection,and may eventually become a chronic disease wound,troubling clinical surgeons around the world.As a graduate student of plastic surgery,we focus on the repair and reconstruction of a variety of tissues,including refractory wounds.Therefore,research on diabetes wounds is also a key issue we need to focus on.Conclusion: We found that CS-DA/TA/PF/3D Exos hydrogel had the properties of3 D Exos sustained release,rapid hemostasis,ROS removal,antibacterial,anti-inflammatory and adhesive properties.Dynamic Schiff base bond and PF micelles make hydrogel have excellent mechanical strength.DA/TA increases the wet tissue adhesion of hydrogel and reduces the risk of infection during treatment.Due to the catechol structure,DA/TA also enhances the rapid hemostasis and exocrine release characteristics of hydrogels by activating platelets and interacting reversibly with 3D Exos.CS-DA/TA/PF and CS-DA/TA/PF/3D-Exos hydrogels showed better hemostasis time and bleeding volume in rat liver injury model and rat tail amputation model.In addition,CS-DA/TA/PF/3D-Exos hydrogel has a good therapeutic effect on wound healing rate,fibroblast migration,epidermal formation,granulation tissue thickness and collagen distribution of full-thickness skin defect model in vivo.CS-DA/TA/PF and CS-DA/TA/PF/3D-Exos hydrogels have shown good biosafety in vitro experiments,and have the ability of antibacterial,antioxidant,immune regulation and promoting angiogenesis.They are ideal strategies for specific treatment of chronic infected wounds.OHA/GT/ECM/Cur/CCL2 hydrogel has good histocompatibility and adhesive properties.Through rapid degradation of OHA/GT and good water solubility,it can achieve rapid release of CCL2 in early stage,promote early recruitment of macrophages,etc.,and thus start the immune response.Through the complex effect of insoluble ECM/Cur,it can continue to play an anti-inflammatory role in diabetes wounds and effectively promote the polarization of macrophages to M2 type.By reshaping immune homeostasis,it effectively promoted the healing rate of diabetes wounds,fibroblast migration,epidermal formation,granulation tissue thickness and collagen distribution,which is a new strategy and new idea for treating diabetes wounds.