Effects Of Methanol And Glutaraldehyde On Properties Of Freeze-drying Silk Fibroin/Gelatin Porous Scaffolds

Background:Seed cells, scaffold materials and growth factors are three basic aspects for tissue engineering.The choice of scaffold materials is very important in tissue engineering. Ideal materials of cells scaffold should have good biocompatibility, biodegradability, porosity and certain mechanical properties. Silk Fibroin as a natural macromolecule material with excellent biocompatibility, mechanical properties and degradability is widely used in the field of tissue engineering. Gelatin is one of collagen modified derivatives, it has good biocompatibility and low antigenicity. Academics have prepared tissue engineering stents of silk fibroin/gelatin with excellent properties, and methanol or glutaraldehyde is usually used to be the cross-linking agents, but comparative studies on properties of stents which treated by these two cross-linking agents are rarely reported, so it is necessary to explore and study the different cross-linking impacts on properties of silk fibroin/gelatin composite scaffolds. Our research prepared silk fibroin/gelatin composite porous scaffolds by freeze-drying technology, treated stents with methanol and glutaraldehyde respectively, then we compared structures and properties of stents so as to provide further theory for the preparation of silk fibroin/gelatin composite porous scaffold with more excellent properties.Aim:To investigate and contrast structures and properties of silk fibroin/gelatin porous scaffolds cross-linked by methanol and glutaraldehyde in different temperatures.Methods:Silk fibroin/gelatin composite porous scaffolds with equal proportion were prepared by freeze-drying method in-20℃and-80℃respectively, then each was divided into two groups cross-linked respectively by methanol and glutaraldehyde, finally compared the structures and properties of these four groups.(1) The Microstructures of SF/Gt scaffolds were observed by scanning electron microscope (SEM).(2) The surface phase structures of scaffolds were tested by XRD.(3) The porosities of SF/Gt scaffolds were measured by ethanol displacement method.(4) The water absorption ratios and swelling ratios of scaffolds were measured.(5) The thermal stabilities of SF/Gt scaffolds were tested by thermogravimetric apparatus.(6) The compression mechanical properties of SF/Gt scaffolds were tested by electronic universal testing machine controlled by microcomputer. Results:(1) SEM showed the apertures of composite scaffolds cross-linked by glutaraldehyde were more regular, the connection between holes was more compact, and the aperture sizes were more uniform, while the structure connection of scaffolds cross-linked by methanol was incomplete even included fragments. This implied the pore structures of scaffolds cross-linked by glutaraldehyde were better than the scaffolds cross-linked by methanol.(2) XRD showed the composite scaffolds cross-linked by whether methanol or glutaraldehyde all included β-sheet stable conformations of silk fibroin, there was no statistical significance between them.(3) The porosities of scaffolds cross-linked by glutaraldehyde were higher than the scaffolds cross-linked by methanol(P<0.05), meanwhile we also observed porosities of scaffolds increased with the decreasing of pre-freezing temperatures(P<0.05).(4) The water absorption ratios and swelling ratios of scaffolds cross-linked by glutaraldehyde were all higher than the scaffolds cross-linked by methanol(P<0.05), but the pre-freezing temperatures had a small effect on water absorption ratios and swelling ratios of the scaffolds.(5) The initial thermal decomposition temperatures of four groups were all about280℃, the thermal decomposition temperatures of scaffolds cross-linked by glutaraldehyde were slightly higher than the scaffolds cross-linked by methanol, but there was no statistical significance between the results.(6) The elastic moduli and yield strengths of scaffolds cross-linked by glutaraldehyde were all higher than the scaffolds cross-linked by methanol(P＜0.05), at the meantime we also observed the decreasing of pre-freezing temperatures can improve mechanical properties of the scaffolds(P＜0.05).Conclusion:The SF/Gt scaffolds cross-linked by glutaraldehyde has a more uniform pore distribution, a better holes connection, higher porosities,water absorption ratios, swelling ratios and superior mechanical properties. It provided a theoretical basis for the selection of cross-linking agents of the SF/Gt freeze-drying tissue engineering scaffolds, the results also established the good experimental foundation for our next research on cells-scaffold composite culture in vitro and animal test in vivo. However, glutaraldehyde has a certain toxicity, it may remain in the scaffolds and affect the properties of scaffolds, therefore the trend of future study is researching and applying the scaffold cross-linking agent without toxicity and vestigital.