| Wound healing is an important and urgent problem in surgical field,and it is also a very active research topic.In recent years,as an active substance with excellent physical and chemical properties,Bletilla striata polysaccharides have been widely concerned by scholars at home and abroad,and have broad prospects in wound healing.Purpose In this study,a new type of oxidized Bletilla striata polysaccharide hydrogel was designed with Lactobacillus plantarum as the key bacteria.The hydrogel was not only superior to the traditional Bletilla striata polysaccharide hydrogel,but also could use the lactic acid produced by Lactobacillus plantarum metabolism to affect the physiological microenvironment of wounds,so as to find a new solution to wound healing.The physicochemical properties and structures of polysaccharides before and after oxidation were analyzed.The preparation process and formulation ratio of composite hydrogels were screened,and the basic properties of composite hydrogels were characterized.The pharmacological activities of composite hydrogels were investigated in vivo and in vitro to evaluate the potential of composite hydrogels in wound healing.Methods1.Refined BSP was obtained by extraction and purification process,and oxidized BSP(OBSP)was obtained by periodate oxidation reaction.The physicochemical properties of polysaccharide before and after oxidation was characterized by Fourier-infrared spectroscopy,Nuclear magnetic resonance spectroscopy,total polysaccharide content and molecular weight determination.2.The optimal ratio of OBSP-CS hydrogel was 2%(w/v)OBSP solution and 1.5%(w/v)CS solution according to the mass ratio of 1:1,and the mixture was stirred at48°C and 200 rpm for 4 h.OBSP can be cross-linked with chitosan to prepare hydrogels.Screen the optimal solution concentration,dosage ratio and cross-linking temperature of OBSP to prepare hydrogel,and optimize the preparation process.Lactobacillus plantarum was added at the optimal ratio of OBSP-chitosan to obtain a composite hydrogel.The physical and chemical properties of the hydrogel,such as infrared spectroscopy,scanning electron microscopy,thermal analysis,rheological property,swelling rate and p H value,were measured and analyzed.3.Take S.aureus,P.aeruginosa and E.coli which are common in wound infections as test bacteria.The antibacterial activity of the composite hydrogel in vitro was studied by the method of agar well diffusion and colony counting.4.The CCK-8 method was used to evaluate the toxicity of the composite hydrogel on mouse fibroblasts cells(L929),and the biocompatibility of the composite hydrogel was determined in vitro.5.The composite hydrogel was applied to the wound model of full-thickness skin defect on the back of mice,and the effects of composite hydrogel on wound healing were explored with wound healing rate,pathological tissue staining sections and serum inflammatory factors as observation indexes,combined with liver and kidney toxicity evaluation.Results1.The content of total polysaccharide was 69.23% by phenol-sulfuric acid method.The molecular weight Mw and Mn of polysaccharides were 3.99 × 105 g/mol and 7.09× 104 g/mol,respectively.Compared with BSP,OBSP showed enhanced carbonyl(C=O)stretching vibration absorption peak at 1727.35 cm-1 in FT-IR spectrum.Combined with the new peak at δ9.2 ppm in 1H-NMR spectrum,it indicated that part of hydroxyl group on BSP molecules changed into aldehyde group after oxidation,which indicated the success of oxidation reaction.2.The primary amine group(-NH2)on the chitosan and the carbonyl group(C=O)on the BSP underwent a condensation polymerization reaction to form the imine bond(C=N),that was,a Schiff-base was formed to form hydrogel.The stretching vibration of carbonyl group(C=O)and the characteristic absorption peak of Schiff-base(C=N)in the ATR-FTIR spectrum revealed the successful preparation of hydrogel.SEM results showed that the composite hydrogel had a three-dimensional porous network structure and good physical strength.The rheology showed obvious frequency independence,which was a typical hydrogel behavior.After swelling and equilibrium,the water absorption capacity of the composite hydrogel was 30-40 times of its own weight.The p H value was between 5.86-6.52,which was a slightly acidic wound dressing.3.OBSP-CS-LP hydrogel had better antibacterial activity than OBSP-CS hydrogel through agar well diffusion test and plate counting test,and the antibacterial rate was more than 98%.OBSP-CS-LP hydrogel can effectively prevent bacterial infection by improving the inhibition rate of three kinds of commonly tested bacteria in wound.4.The results of cytotoxicity experiments showed that the survival rate of L929 cells cultured in different concentrations(0.625-10.000mg/m L)of composite hydrogel extract was as high as 75%.CCK-8 method showed good biocompatibility of OBSP-CS-LP hydrogel.5.The experimental results of the full-thickness skin defect on the back of mice showed that after 7 days of treatment,the wound closure rate of the composite hydrogel was high,and the scabs fell off naturally,and almost completely healed after14 days.It was found that the composite hydrogel could accelerate the deposition of collagen and reduce the formation of scar.The results of serum factor determination by ELISA showed that the hydrogel could promote the high expression of VEGF factor,reduce inflammatory,and had no liver and kidney toxicity.Conclusions1.Preparation of hydrogel by oxidative cross-linking method,this method didn’t require harsh conditions,and the dialdehyde BSP prepared by periodate oxidation method is an important modification,which not only improved the mechanical properties of the hydrogel,also avoid the use of toxic cross-linking agents,and is suitable for the preparation of hydrogel probiotic delivery systems.2.The hydrogel wound dressings based on BSP had good biocompatibility,low toxicity and extensive biomedical activity.It had excellent properties in natural polymer hydrogel materials and broad application prospects.3.Faced with the heavy burden of unhealed wounds on society,the transformation of wound microenvironment seems to be a feasible strategy.The BSP hydrogel of compound Lactobacillus plantarum have good antibacterial activity,and the metabolites of Lactobacillus plantarum can also provide a slightly acidic environment for wound healing.A suitable slightly acidic environment in the wound could greatly inhibit the growth of bacteria,which is very important for rapid wound repair.Therefore,the BSP hydrogel dressing of compound Lactobacillus plantarum is more suitable for wound tissue repair. |
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