Experimental Study On Skin Regeneration Induced By A Physiochemical Cross-linked Double Network Hydrogel

Part?Preparation and physicochemical characterization of QCS-M-PAM hydrogelObjective To explore the preparation of a double network hydrogel with physical and chemical cross-link.The surface morphology of the QCS-M-PAM hydrogel was observed by scanning electron microscope(SEM).The chemical structure of the hydrogel was analyzed by Fourier infrared spectroscopy(FT-IR),and the physical and chemical characteristics of different components of the hydrogel were characterized.Methods 8g of chitosan and 14.4g of GTMAC were added into 120 ml of distilled water,respectively.The mixture of the three was transferred to a three-neck flask and stirred in reflux at 80?for 36 h.After the reaction,the yellow product was dialysed in distilled water with a dialysis membrane with a molecular weight of 1.4×10~3Da for 72h.Finally,the dialysed product was freeze-dried to obtain the quaternized chitosan with a substitution degree of 18%.At 37?,QCS was dissolved in deionized water(DI)to form a 2%QCS solution.Similarly,acrylamide(AM)is dissolved in deionized water(DI)to form a 28%AM solution.The QCS and AM solutions were then mixed at a ratio of 1:10.Matrigel and AM should be mixed 1:1by volume at first and then stirred continuously for 2-5 minutes.After standing for 20 minutes or ultrasonic defoaming,adding crosslinking agent,accelerator and initiator,and finally vacuuming in a vacuum pump for 60 minutes,the QCS-M-PAM hydrogel with the best effect can be obtained.The surface morphologies of different hydrogels were observed by scanning electronic microscope(SEM),and the swelling ratios of different hydrogels were detected.The composition of the hydrogels were analyzed by Coomassie blue staining.The chemical structure of materials was analyzed by Fourier Transform Infrared Spectroscopy(FT-IR).Results The color of the QCS-M-PAM hydrogel was nearly transparent and had good tensile properties.SEM showed that the QCS-M-PAM hydrogel had excellent pore structure with an average pore size of 28.67±3.3?m,which was in line with the optimal pore size range(20-125?m)required for wound healing.FT-IR could confirm the successful combination of QCS,Matrigel and PAM.The equilibrium swelling rate of QCS-M-PAM hydrogel in vitro was almost 1200%,which showed the potential of application as wound dressing.After Coomassie bright blue staining,the microstructures of different groups of hydrogels were observed.The matrigel was successfully combined into the QCS-M-PAM hydrogel,and the staining degree increased with the proportion of Matrigel.Conclusion The QCS-M-PAM hydrogel synthesized by QCS,Matrigel and PAM has good pore structure and is interconnected with each other.The powerful equilibrium swelling ratio of QCS-M-PAM hydrogel could keep the wet environment of the wound and timely absorb the exudate to create a excellent microenvironment for healing.The addition of Matrigel can improve the biocompatibility of hydrogel,and it contains a variety of components.Part ? Biological detection of QCS-M-PAM hydrogelObjective To investigate the effects of QCS,one of the components of hydrogel,on the survival rate and migration of fibroblasts,as well as the migration of vascular endothelial cells,and study the effects of QCS-M-PAM hydrogel on cell viability,hemostatic effect,antibacterial and fungal effects.Methods The compatibility of different concentrations of QCS for L929 fibroblasts was determined.The opitmal cell viability of QCS was selected for cell experiment.The effect of QCS on migration of L929 fibroblasts was observed at predetermined time point.Then QCS was placed in the lower chamber and the upper chamber of the Transwell to culture vascular endothelial cells,and their effects on the transmembrane migration of vascular endothelial cells were analyzed.Hydrogels of different components were co-cultured with L929 cells to study the effects of on cell viability.The hemostatic effect of QCS-M-PAM hydrogel was detected in liver hemorrhage model of Kunming mouse,and compared with the control group.Germ-positive Staphylococcus aureus(ATCC 1445),S.epidermidis(ATCC 12228),and Gram-negative E.coli(DH10B)were typical bacteria commonly found on the wound.They are used to evaluate the antimicrobial efficacy of hydrogels.A.niger(BNCC 186380)and C.albicans(BNCC 186382)were used to detect the antifungal effect of hydrogels.Results In the concentration range of 10-150?g/ m L,50?g/ m L of QCS showed the highest cell viability and promoted the migration of fibroblasts.In addition,50?g/ m L QCS could also promote the transmembrane migration of vascular endothelial cells,which is of great significance to accelerate wound healing and vascularization.The QCS-M-PAM hydrogel added with Matrigel showed the highest cell viability(>80%).Compared with the control group,QCS-M-PAM hydrogel showed stronger hemostatic effect.QCS-M-PAM hydrogels showed different degrees of antibacterial effect against Staphylococcus aureus,Staphylococcus epidermidis,Escherichia coli,Aspergillus nier and Candida albicans.Conclusion QCS with a degree of substitution of 18% not only has good cytocompatibility,but also maintains a strong broad-spectrum antimicrobial effect.The cell viability of 50?g/ml QCS is higher,and it is beneficial to promote the migration of fibroblasts and vascular endothelial cells.QCS-M-PAM hydrogel is not only good cytocompatibility,but also has strong antibacterial activity against bacteria and fungi.Part ? Mechanical properties and adhesion of QCS-M-PAM hydrogel and effect on macrophage polarizationObjective To explore the mechanical strength and adhesion properties of QCS-M-PAM hydrogel,and the effects of PAM and QCS-M-PAM hydrogel on the polarization of macrophages.Methods The mechanical properties of different hydrogels were carried out on a universal test machine with a weight sensor of 200 N and a cross head speed of 30 mm/min in the guide rail.Hydrogels are made into dumbbell shaped parts in the mold for tensile test and cylindrical parts for compression test.The width and thickness of the hydrogel in the tensile test were 20 mm and 3mm,while the height and diameter of the hydrogel in the compression test were 10 mm and 15 mm.In addition,the adhesion of QCS-M-PAM hydrogel to fresh pig skin tissue,glass and plastic was tested on a universal tester.The skin tissue,glass and plastic surfaces are uniformly made into a rectangle of 50mm× 20 mm.Then the hydrogel is applied to the contact surfaces of the above different materials,and the contact area is 20mm× 20 mm.The shear failure test of hydrogel samples was carried out on a universal testing machine with a 50 N load cell at a speed of 20mm/min under normal temperature and pressure.PAM and QCS-M-PAM hydrogels were co-cultured with RAW 264.7 macrophages,and the effects of hydrogels on the differentiation of macrophages were analyzed by flow cytometry and immunofluorescence staining,and the expression levels of macrophage marker genes were detected by q RT-PCR.Results The elongation at break(1210.1±28.15%)and tensile strength(118.3±8.26 k Pa)of QCS-M-PAM hydrogel were higher than those of the other three groups.The elastic modulus of QCS-M-PAM hydrogel was 24.6±3.4k Pa.The compressive strength of QCSM-PAM hydrogel(398.7±12.53 k Pa)was higher than that of the other three hydrogels.The adhesion strength of QCS-M-PAM hydrogel on pig skin tissue was 1.32±0.14 k Pa and the adhesion force was 0.52±0.03 N.QCS-M-PAM hydrogel can adhere closely to the surface of glass,plastic and pig skin.The flow cytometry results showed that the macrophages in the QCS-M-PAM hydrogel group were more polarized towards M2.Immunofluorescence showed that the macrophages of the QCS-M-PAM hydrogel group had increased fluorescence expression of CD206,and the q RT-PCR results showed TGF-? gene expression level was higher in the QCS-M-PAM hydrogel group.Conclusion QCS-M-PAM hydrogel shows ideal mechanical properties,including ductility,compressibility and resilience.The elastic modulus is similar to that of normal human skin,which is more beneficial to the regeneration of skin defects.In addition,it also form effective adhesion to glass,plastic and pig skin without secondary damage to the skin,and could also promote the polarization of macrophages to M2-type.Part ? Experimental study on repairing full-thickness skin defect of rats with QCS-M-PAM hydrogelObjective To evaluate the effect of QCS-M-PAM hydrogel on repairing full-thickness skin defect in ratsMethods According to the different methods of wound management,32 male SD rats with the body weight of 230-260 g were randomly divided into four groups on average: group A: gauze control group,group B: Tegaderm?,group C: QCS-PAM hydrogel,and group D: QCSM-PAM hydrogel.The materials were sterilized under ultraviolet light and placed in the skin defect of rats.After the operation,the wound healing rate of different treatment groups was evaluated at a predetermined time point,and the regenerative skin tissue of the wound was cut for HE and Masson staining,and the angiogenesis of the wound was analyzed by CD31 staining,and the protein expression levels of inflammatory factors and growth factors at different time points were analyzed.Results In vivo animal experiments showed that the wound healing rate of QCS-M-PAM group was significantly better than Tegaderm? on the 10 th day after surgery(***P < 0.001).The wound healing area of QCS-PAM group was not significantly different from Tegaderm? group,but was greater than that of the control group(*P < 0.05).At this time point,granulation tissue formation was observed in all groups,and obvious re-epithelialization of wound was observed in QCS-PAM and QCS-M-PAM groups.On the 15 th day after surgery,the wound healing degree of QCS-M-PAM hydrogel group was still significantly better than that of the other three groups.Neovascularization,hair follicle and sebaceous gland formation were observed in the QCS-M-PAM group.In contrast,the regeneration of dermal appendages in the other three groups was not as significant as in the QCS-M-PAM hydrogel group.QCSM-PAM hydrogel wound healing scar length was only 780±13.47?m,better than Tegaderm? and QCS-PAM group.EGF expression was higher in the QCS-M-PAM group than in the other three groups,and there was no significant difference between the QCS-M-PAM hydrogel group and the Tegaderm? group.On the 5th day after surgery,the expressions of pro-inflammatory cytokines(TNF-?,IL-1 and IL-6)in the hydrogel groups were lower than those in the other two groups,suggesting that the less inflammatory response in the hydrogel groups.On the 10 th day after operation,the expression level of pro-inflammatory cytokines showed a general downward trend,especially in the QCS-M-PAM hydrogel group.Compared with the QCS-PAM and Tegaderm? groups,the protein expression levels of IL-1 and IL-6 were significantly reduced in the QCS-M-PAM hydrogel group.The expression levels of PDGF and FGF in QCS-M-PAM hydrogel were still significantly higher than those in QCS-PAM and Tegaderm? group 10 days after surgery(**P< 0.01).Conclusion QCS-M-PAM hydrogel showed stronger ability of wound regeneration and repair at skin defects,with more average number of mature blood vessels and hair follicles and shorter scar length,indicating that OCS-M-PAM hydrogel had better effect on wound healing.The expression of inflammatory factors in QCS-M-PAM hydrogel was decreased,and the expression of growth factors was relatively increased.We speculated that QCS-MPAM hydrogel could not only inhibit the polarization of macrophages into pro-inflammatory M1 macrophages,but also may be conducive to the polarization of macrophages into antiinflammatory M2 macrophages.