Preparation Of Silk ? Porous Sponge And Its Application As Tissue Repair Scaffold

Silk fibroin has been widely used in tissue engineering,implantable devices and other biomaterials due to its good biocompatibility and tunable degradability.In the process of tissue repair,we need to regulate the structure and properties of scaffold materials at a large scale toward specific tissue regeneration demands.Silk fibroin protein has different crystal structure,and the difference of crystal type and crystallinity significantly affects the structural properties of the material.Therefore,the regulation of crystallization kinetics can provide an effective method to control the structure and properties of silk fibroin biomaterials.However,the current research on the regulation of silk I structure and its effect on biomaterials is far from enough.In this paper,we proposed to regulate the self-assembly of silk fibroin protein structure through freeze-annealing,construct scaffold material with stable silk I crystal,and systematically study its stability.By regulating the crystallization kinetics of silk I crystal,a silk fibroin three-dimensional porous scaffold with tunable structure and performance was prepared,thus providing a new idea for the design,regulation of structure and performance of silk protein biomaterials.Firstly,the effect of freeze-annealing on silk fibroin crystallization kinetics was studied.After the silk fibroin solution was frozen,the crystallization structure was induced by low temperature annealing.XRD results showed that the crystallinity of the silk fibroin increased with the increase of annealing time.After annealing for 48 h,silk fibroin had typical silk I type crystals.At the same time,the initial freezing temperature and concentration also have a certain effect on the crystallization of silk fibroin.DSC results showed that the thermal stability of the material increased with the increasing of annealing time.The mechanism of freeze annealing-induced silk I crystallization may mainly involve freezing-generated concentration and thermodynamically driven assembly of silk fibroin.During freezing,the silk fibroin solution concentrates and the chain folds.In the process of heating annealing,silk fibroin macromolecules obtain enough energy to move the molecular chain,thus transforming to the silk I structure,which is more stable,but its energy is not enough to induce the formation of silk II structure at low temperature.Further study showed that the crystallinity of Silk I could be controlled in a large range by changing the annealing time.When the annealing time extended from 12 h to 48 h,the crystallinity increased from 17.6% to 38.0%.In spite of the low crystallinity obtained during short annealing time,the scaffolded material can still maintain good water stability.Secondly,the stability of silk I porous scaffolds prepared by freeze-annealing was studied.The crystalline structure,mechanical properties and degradation behavior of silk I porous scaffolds were tested,which was treated by 75% ethanol,dry heat treatment,ultraviolet treatment,high temperature and high pressure(HTHP)treatment and water vapor annealing treatment.XRD results showed that the crystal peak position of silk I did not change significantly after different treatments,indicating that the crystal has good stability.DSC results showed that the thermal stability of the materials did not change significantly after different treatments,but the glass-transition temperature and decomposition temperature increased slightly,which may be caused by the increase of crystallinity.The compression test results showed that the mechanical properties of the materials were enhanced except the ultraviolet treatment,and the mechanical properties of the materials treated with 75% ethanol were the most obvious.The degradation experiment showed that the degradation rate of the material was slowed down after sterilization by 75% ethanol and HTHP.The results showed that after different post-treatment,the material could maintain the stable silk I crystal,but the crystallinity might change in different degrees,thus affecting the mechanical properties and degradation rate of the material.Finally,the porous silk fibroin scaffold with silk I crystal was prepared by freezeannealing,and its pore structure,mechanical properties,degradation behavior and cellular compatibility were systematically studied.SEM observation and pore structure analysis showed that the pore structure of porous scaffolds can be controlled by changing freezing temperature and solution concentration.The lower the initial freezing temperature and the higher the solution concentration,the smaller the pore size and the lower the porosity of the scaffold.The results of the compression test showed that the pore structure and crystallinity of the scaffold can be controlled by changing the freezing concentration and the annealing time of the material,so as to control the mechanical properties of the material.With the increase of solution concentration and annealing time,the mechanical properties of the scaffold were enhanced.In vitro degradation experiments showed that the degradation behavior of materials can be effectively regulated by controlling the annealing time.With the increasing of annealing time,the crystallinity increased and the degradation rate of the scaffold slowed down.Cell experiments showed that the silk I scaffold had good biocompatibility and could support the growth of human umbilical vein endothelial cell.At the same time,the cell activity of the material sterilized by 75% ethanol was lower than that of the material sterilized by HTHP,which may be related to the stiffness of the material caused by different posttreatment.Through this study,a green,efficient,simple and controllable method was established to effectively regulate the aggregation structure of silk fibroin,so as to achieve regulation of mechanical properties and degradation rate of silk fibroin scaffold,to meet different biological applications.At the same time,this study confirmed that the silk I scaffold by freeze-annealing has good stability,thus providing an important theoretical basis for its biological application.