The Scalded Skin Repair Application Of A New Biomaterial Dressing Made From RGD-Recombinant Spider Protein

Spider silks are a diverse family of natural materials with extraordinary mechanical properties such as high tensile strength and extensibility as well as biological compatibility.In recent years,the reported exceptional nature of silk lead to increased interest in silk for biomedical applications.The purpose of this research was to establish optimal technique to produce RGD-recombinant spider silk protein pNSR16 and pNSR32 in engineering E.coli pNSR16/BL₂₁(DE₃)pLysS and pNSR32/BL₂₁(DE₃)pLysS through high cell density fermentation.Additional,the recombinant spider protein pNSR16 and pNSR32 were purified from the output of the high cell density fermentation.Finally the histocompatibility of scaffold biomaterial prepared from purification product were discussed,and used in the deepâ…¡degree scalded skin repair application in SD rats as a new recombinant spider protein dressings.In the beginning,three factors which affect the expression of spider silk protein such as the cell density before induction,the culture temperature and the concentration of Gly/Ala after induction was exploited.The high-cell density fermentation conditions of engineering E.coli pNSR16/BL₂₁(DE₃)pLysS and pNSR32/BL₂₁(DE₃)pLysS was optimized and repeated as follows:The best yield of the spider silk protein expressed in high cell density fermentation in E.coli pNSR16/BL₂₁(DE₃)pLysS is Cell density OD_600nm=30,30℃,and 0.2%(w/v)Gly/Ala while the best yield of the spider silk protein expressed in high cell density fermentation in E.coli pNSR32/BL₂₁(DE₃)pLysS is OD_600nm=30,30℃,and 0.2%(w/v)Gly/Ala.After the optimization of the fermentation process,the recombinant spider silk protein pNSR16 and pNSR32 were purified from the high cell density fermentation in a large scale.Three kinds of scaffolds:recombinant spider silk protein pNS2 which there isn't RGD cell adhesion site on it,pNSR16 containing RGD cell adhesion sites and PVA were prepared by the method in our lab.Their histocompatibility were compared by implanting into SD mouse in vivo and examined at different periods.It was showed that there were well tissue compatibility with the biomaterials of pNS2 and pNSR16,especially for pNSR16,which showed best tissue compatibility. So the pNSR16 and pNSR32 protein which both contain RGD cell adhesion sites but have different Molecular Weight were used as new biological dressings in the SD rats deepâ…¡degree burn wounds skin repair experiment as well as the collagen protein. Scalded rats model was set up through heating a weight(2.0cm and 50g)in boiling water for 15 min,and pressure on the back of mouse 15s.The histology testing of the model is in accord with deepâ…¡degree burn features.Three kinds of biomaterial dressings were applied on the scalded rats model to repair the wound skin.It can be found that recombinant spider silk protein pNSR16 and pNSR32 had shorten the wound healing time,improved wound healing rate and increased the hydroxyproline levels on the wound,all of which were superior to the role of collagen dressings.The best effect of burn wounds skin repair was achieved by pNSR32 protein,the better one is pNSR16 protein,then the collagen protein.In this experiment,collagen membrane material was not so good as the recombinant protein in the affect of burn wounds skin repair.