Silk Fibroin Film Modified By Dopamine And Its Application In Skin Wound Repair

Skin tissue engineering has developed rapidly in recent years and the excellent wound dressing can accelerate healing and functional recovery of wounds.Silk protein is a kind of natural biological macromolecule material,by having its unique excellent physical and chemical properties,which could be processed into membrane,gel,sponge scaffolds and nanofiber etc.In addition,it can promote the wound tissue regeneration,silk protein as biological materials of repairing skin wound is pay more and more attention.But,the good performance of cell compatibility and cell initial adhesion are required for the wound dressingat present,one of the main difficulties which limit the silk protein clinical application is how to improve the cell compatibility of silk protein.In this study,we firstly prepare the silk protein film,including(Bombyx mori)silk fibroin nanofibers and Antheraea pernyi silk fibroin film,then the films are modified by dopamine to improve the performance of cell adhesion,finally the wound model of full-thickness skin is used to verify the good effect of wound healing.(1)Preparation of dopamine-modified silk nanofiber and its application in skin repairSilk fibroin nanofibers were firstly prepared by electrospinning technology.In order to obtain the silk nanofibers with diameter about 400 nm,the concentration of spinning solution,voltage,receiving distance and flow velocity of spinning solution were in detail investigated.According to the various factors,the optimal experimental condition is that the reaction concentration is 7.5%,voltage is 24 KV,receiving distance is 12 cm and spinning fluid flow rate is 0.02 ml/min.Next,we successively synthesized dopamine-modified silk nanofibers,which were confirmed by SEM and XPS,there are obvious dopamine particles coating on the surface of the silk nanofibers and N/C mole ratio close to theoretical value of poly-dopamine.Through the persistence test,we found that dopamine-modified coating could last for 7 days.And the contact Angle and protein adsorption test were also verified that because of adding dopamine,the hydrophilicity and protein adsorption of silk fibroin membrane were significantly increased which were beneficial for cell adhesion.Then we carried out the relevant validation by culturing L929 cells on dopamine-modified silk nanofiber,the results showed that the initial adhesion,spreading and proliferation performance of cells could be significantly increased by these nanofibers.At last,a full thickness skin wound model of SD rats is employed and demonstrated rapid reepithelialization and fast wound healing rate in the group of silk nanofibers modified by dopamine.(2)Preparation of aligned silk nanofibers and its induction stem cells into vascular differentiationIn order to improve the mechanical properties of silk nanofibers,we prepared the aligned silk nanofibers through the magnetic field collecting device which provided effect of magnetic field superimposed electric field by adding nano-Fe3O4 particles to spinning solution;We can observe excellent aligned silk nanofibers with addition of 0.3%nano-Fe3O4 particles and spinning time of 30 min;The result of FTIR analysis indicated that adding 0.3%of nano-Fe3O4 particles did not affect the secondary structure of silk nanofibers;Also,the mechanical test was performed and showed that the aligned silk nanofibers possess high tensile strength and elasticity modulus in its orientation direction.which providing information for mechanical properties of biomaterials to guide the cell differentiation;Moreover,the aligned silk nanofibers have a certain magnetic responsiveness with an extra small quantity of nano-Fe3O4 particles through the vibration sample magnetometer test;The results of cultured mesenchymal stem cells confirmed that the aligned silk nanofibers could induce cell orientation growth and differentiate into blood vessels in basal medium;Finally,we cultured the myoblast C2C12 on the aligned silk nanofiber and found that it could also induce cell to grow along with the direction of orientation,establishing the basis for its application in deep skin trauma muscle repair.(3)Preparation of dopamine-modified tussah silk film and its application in skin repairTo further improve the cellular compatibility of wound dressing,we innovatively use Antheraea pernyi silk fibroin which contains RGD peptide sequence as research material.Firstly,we prepared the Antheraea pernyi silk film and then modified it by dopamine for 8 h and 16 h respectively,the result of AFM showed that the size of dopamine particles and the surface roughness of the Antheraea pernyi silk film both increase gradually with modification time,in addition,the thickness of dopamine coatinghas linear relationshipwith reaction time;The XPS test showed that the N/C mole rate of the dopamine-modified Antheraea pernyi silk film was similar to theoretical value of poly-dopamine,indicating the successful modification of dopamine;The contact angle test showed that the hydrophilicity of the Antheraea pernyi silk film surface was increased after the modification of dopamine;According to the Zisman method,the free energy of the Antheraea pernyi silk film was increased from 20.61 mJ/m2 to 39.95 mJ/m2 and the protein adsorption capacity was significantly enhanced after the modification of dopamine.Then the result of culturing the mesenchymal stem cells on the Antheraea pernyi silk film indicated that the cell adhesion and proliferation increased after modification of dopamine;At the same time,the cell wound scratch assay proved that the Antheraea pernyi silk film modified by dopamine could promote stem cell migration rate to the score point,which is key factorsof promoting wound healing for it can induce the cells migrate to the wound setting;Finally,the results of wound healing in rats indicated that the Antheraea pernyi silk film wound dressing modified by PDA for 16h increased wound healing rate,reepithelialization,dermis proliferation,collagen synthesis and promoted epidermal differentiation into hair follicles and sebaceous glands comparing with original AF films and gauze dressings,and this is beneficial to skin functional recovery.In this study,silk fibroin film,including the silk nanofibers film and Antheraea pernyi silk film,were modified by dopamine could significantly improve the ability of cell adhesion,and the application of silk protein in the field of skin wound repair was expanded.In brief,our study broaded the application range of silk proein biomaterials.