Preparation Of Keratin/Chitosan Hydrogel Composite And Preliminary Study On Its Biological Characteristics

Objective:The purpose of this research is to synthesize a keratin/chitosan hydrogel composite material,explore ways to improve the performance of the composite material,and illustrate the biological safety,tissue compatibility and degradability of the material by studying the effect of the composite material on cells and animals,To provide support for the follow-up keratin/chitosan hydrogel composite material combined with stem cells to repair peripheral nerve damage.Method:(1)The preparation of keratin/chitosan hydrogel composite materials and the formulation of the composition ratio: cross-link composite materials of different concentrations by ionic cross-linking,using Fourier transform infrared reflectance,scanning electron microscopy,and measuring the porosity,swelling rate and tensile strength of the material.The stretchability determines the method of preparing the hydrogel material.(2)The leaching solution extracted from the keratin/chitosan hydrogel composite material was added to U266(human myeloma cells),mouse spleen fibroblast cells(m SFC),and human skin mesenchymal stem cells(Skin Mesenchymal stem cells(s MSC)culture system,set up a control group at the same time,culture in a 37℃ 5% CO2 incubator for 24 hours,and use Annexin V-FITC/PI cell apoptosis detection kit to detect the apoptosis of gel co-cultured cells Determination.(3)By implanting the keratin/chitosan hydrogel composite material under the skin of the rat,the survival status of the rat was observed,the weight change of the rat was recorded,and the expression of inflammatory factors was detected by ELISA and the effect on the main organs and the surrounding implant The tissues were stained with H&E to evaluate the biocompatibility and degradability of the materials.Result:1、The prepared keratin/chitosan hydrogel composite is a semi-transparent solid film with adjustable thickness and shape.2、Scanning electron microscopy observed irregularities and roughness on the surface of the material.In the chitosan hydrogel composite material with a concentration gradient of 1%-4% keratin,as the concentration of chitosan increases,the concentration of keratin decreases correspondingly.,The pore size of the composite material gradually becomes smaller.3、FTIR is used to confirm the presence of functional groups of the two components,chitosan and keratin,after mixing,and to exclude any possible chemical modification caused by the process of mixing and preparing composite materials.4、The porosity of the keratin/chitosan hydrogel composite measured with absolute ethanol is above 70%,and the porosity of the keratin/chitosan composite at 3% concentration reaches 97%.5、The swelling of the keratin/chitosan composite material in the p H 7.4 PBS buffer reached the maximum within 30 minutes,and the 3% hydrogel composite material with the highest swelling rate was around 800%.6、Although there is no significant difference in maximum tensile length and elongation at break,the 3% keratin/chitosan hydrogel composite is higher than other concentrations in terms of maximum tensile force and tensile strength.7、Cells were cultured with the extract of the composite material,and the results of flow cytometry showed that there was no significant difference in apoptosis between the experimental group and the control group,indicating that the keratin/chitosan hydrogel composite material has no cytotoxic effect.8、Analyzing the survival status and body weight changes of the rats,the expression of inflammatory factors and the H&E staining of the tissues found that the implants were nontoxic to the rats and had good biocompatibility and degradability.Conclusion:1、The keratin/chitosan hydrogel composite was prepared by casting and freeze-drying.2、The keratin/chitosan hydrogel composite material has a rough surface,high porosity and swelling rate after being cross-linked with sodium tripolyphosphate,and the cross-linked material has high mechanical strength.3、The keratin/chitosan hydrogel composite material has no cytotoxic effect and has good biological safety.In vivo tests performed by implanting under the skin of rats prove that the composite material is not toxic to animals,and has good biocompatibility and degradability.4、Keratin/chitosan hydrogel composite material can be used as a good matrix and support material for stem cell culture,and used for peripheral nerve repair.