Preparation Of 3d Fibroin/chitosan Blend Porous Scaffold For Tissue Engineering Via A Simplified Method

Last century, with the rapid development of tissue engineering, significant progress has been made in the research field of tissue repair and regeneration for injured tissues. Especially in the recent decades, with the applications of biodegradable materials in tissue engineering, a series of scaffolds with excellent biological properties have provided the cells with good templates for their adhesion, proliferation and differentiation. The discovery of such a large number of biocompatible materials have proved modern medicine a new tissue engineering prospect.At the same time, materials which is made of regenerated silk fibroin (RSF) and chitosan (CS) are boardly studied and applicated in biomedical areas. The research not only involves the preparation, improvement and application of a single kind of material, but also tries to blend them together to combinate the advantages of each other. As we all know, naturally-occuring silk fibroin (SF) has good biocompatibility and obtained certain methods to prepare the three-dimensional (3D) scaffold with the RSF solution can provide it with excellent mechanical properties. CS with strong water absorbency is a powerful feature conducive to wound repair, its antibacterial inhibitory effect has been widely used in many fields. If these two kinds of materials can be effectively mixed, the prepared scaffold should have an excellent application. However, for the different characteristics of the two materials (the conformation of the high molecular weight SF molecule is easy to transform in the chitosan solution which is dissolved in acetic acid), it is always a difficult problem to mix them together. Although there have been few research teams successfully blended the CS with serious degradated RSF solution, the relatively low molecular weight will not only affect the mechanical properties of the scaffold, but also affect the degradation rate of the scaffold. So. the blend of with high molecular weight RSF with CS has not been reported yet.To solve this problem and blend high molecular weight RSF and CS together, which can improve the water absorbency and ensure the mechanical properties of the blend scaffold, the degummed silk were dissolved in LiBr-H2O in this paper. So a significant decrease of the molecular weight of SF can be avoided during the silk fiber dissolution process. In this work, we have tried our best to use less acetic acid than many reports to prevent the conformation convert of the SF. At the same time, we developed a simple and flexible method to manufacture the 3D blend porous RSF/CS scaffold, by freezing at-20℃overnight and treating with ethanol and diluted NaHCO3 solution sequentially. No crosslinker or other toxic reagents which may hinder the application of the scaffold in biomedical field were used in this method. In addition, we have improved the freeze-drying methods;-5℃ethanol was used as denaturant. With the ice dissolved out and porous structure was formed. It is not only time saving but also improve the water absorption of the scaffold.It has been proved that the blend RSF/CS scaffolds prepared via this method were effectively mixed and revealed excellent mechanical properties. They were homogeneous porous structured. No macroscopic phase separation was observed. Obviously interactions between the two molecular were proved by XRD and FTIR test results. SME images showed that the pore sizes of blend RSF/CS scaffolds were decreased from about 200μm to 50μm with the increase of the initial concentration of RSF solution. The blend RSF/CS scaffolds took only less than 2 min to reach swelling equilibrium which is greatly improved than RSF scaffolds. We can regulate the internal pore size of scaffolds not only by adjusting concentration of the blend solution, but also by adjusting the freezing temperature. Our experiments demonstrate that the blend RSF/CS scaffolds prepared by low temperature ethanol can absorb water more and faster than pure RSF scaffolds and RSF/CS scaffolds with butanol as denaturant.The results of MTT and RT-PCR tests indicated that the chondrocytes grew very well in these blend RSF/CS porous scaffolds. Those tests results indicated that the RSF/CS scaffold could support the growth of chondrocytes from the beginning of the seeding and the blend RSF/CS scafford's highest expression level of three mRNAs (mRNAs of aggrecan, collagenⅡand Sox-9) suggested that chondrocytes had been proliferated the most and show the best activity in RSF/CS porous scaffolds. SEM and histological evaluation of hemotoxylin-eosin (H&E) (general connective tissue stain) and toluidine blue (TB) (sulfated proteoglycans stain) further confirmed these findings. The blend RSF/CS scaffolds can support the chondrcytes secrete more GAGs and collagen II. These suggested that the blend RSF/CS scaffolds prepared via this new method could be a promising candidate for applications in tissue engineering.In short, we have prepared a 3D blend RSF/CS scaffold with pore size adjustable in this experiment. Such blend RSF/CS scaffold is prepared with high molecular weight RSF solution and has excellent water uptake abbility. It can support the growth of chondrocytes very well and showed excellent biological properties. It is easy to prepare this kind of blend RSF/CS scaffold with required shape and achieved certain biological requirement via our method. With the development of further tissue engineering applications research, the blend RSF/CS scaffold prepared via this new method could be a promising candidate for tissue engineering.