Study Of Chitosan/silk Microfibers Composite Materials And Functional Silk Microfibers

Chitosan(CS)and silk fibroin(SF)are both natural polymers with outstanding properties,which have been deeply studied in tissue engineering and regenerative medicine.Due to good hemostasis effect and antibacterial property,chitosan become a nice option for wound dressings,but pure chitosan materials often exhibit insufficient mechanical properties and high swelling ratios.As silk fibroin is noted for excellent mechanical properties and good biocompatibility,we can combine chitosan and silk fibroin to fabricate their composites with optimized performance.Compared with regenerated silk solution,silk microfibers(mSF)retain the original nature of silk fibers to a great extent,especially the molecular shtucture that dominates the excellent mechanical properties.In this research,we prepared CS/mSF membrane and CS/mSF scaffold by adding silk microfibers into chitosan solution,and mainly discussed the application of CS/mSF membrane for skin wound healing.We also prepared functional silk microfibers,which meant the surface of silk microfibers could be deposited with bone-like calcium phosphate(CaP)minerals via biomineralization.We discussed the mechanism and process of silk microfibers as biotemplates to regulate the growth of minerals,and verified their biocompatibility as well as promotion effect for bone repair.This research includes the following three parts:(1)The fabrication and application of CS/mSF membraneWe first prepared silk microfibers by alkaline hydrolysis,which was a simple and effective method.The length of silk microfibers could be regulated by adjusting the hydrolysis time periods.The results indicated that after hydrolysis for 6 hours the length of silk microfibers was over 200 ?m with significant variations,which decreased to 50-200?m with relatively homogeneity after 12 hours,and to less than 20 ?m with even samller particles after 24 hours,respectively.Given the length and uniformity,we selected silk microfibers of 50-200 ?m for further studies.Then we prepared Pure CS membrane and CS/mSF membranes by solvent envaporation,and carried out a series of tests and characterization of physical properties.The results indicated that silk microfibers significantly improved the mechanical properties of Pure CS membrane,which were mainly due to interaction among silk microfibers and partially to mSF-CS matrix adhesion.The hydrophobility of silk microfibers reduced affinity between water molecules and the membranes,thus decreasing the swelling ratio of Pure CS membrane.The incorporation of silk microfibers also increased the substrate roughness of Pure CS membrane,which offered attachment and support for L929 cells and promoted their proliferation.Based on the impressive performance of CS/mSF membranes,we further use them as wound dressings for skin repair on rats.The wound healing and histological staining results suggested that chitosan and silk microfibers exerted a synergistic effect as the repair process continued,and they both accelerated the wound healing.After 21 days of wound repair,CS/mSF membrane exhibited the best healing effect,not only at the macroscopic level but also at the microscopic level.(2)The fabrication and characterization of CS/mSF sacffoldsWe prepared Pure CS scaffold and CS/mSF scaffolds by phase separation and freeze drying.We fisrt investigated the influence of pre-freeze temperatures on microstructure of chitosan scaffold,and found they displayed lemellar structure and porous structure when the pre-freeze temperatures were-20 and-80 ?,respectively.Then we chose-20 ? as pre-freeze temperature and compared the porous structure of different scaffolds prepared under such condition.The results indicated that the porosity and pore size decreased with the content of silk microfibers increasing,and they were significantly lower than those of Pure CS scaffold when the content of silk microfibers reached 100%and 200%.Silk microfibers embedded in chitosan matrix increased the density of scaffold and thickness of pore wall,leading to improvement in elastic modulus and compressive stress of composite scaffolds.Both Pure CS scaffold and CS/mSF scaffold exhibited good biocompatibility,but CS/mSF scaffold was better for the proliferation of rBMSCs cells due to higher pore wall roughness.(3)The fabrication and application of mSF/CaP compositesSilk microfibers could work effectively as organic template to regulate the nucleation and growth of inorganic minerals when put into 1.5 × SBF for mineralization.The deposition of minerals on silk microfibers showed a time-dependent manner,and the process included four stages of nucleation,growth,self-assembly and aggregation.After mineralization for 7 days,the surface of silk microfibers was covered with a complete mineral layer with hierarchical structures,which indicated a perfect combination of organic template and inorganic minerals.According to the analysis of elemental composition and crystallographic properties,we deduced the minerals were one type of bone-like calcium phosphates partly doped with carbonate.In order to verify the biocompatibility of mineralized silk microfibers,we co-cultured with MG-63 cells.The results suggested that silk microfibers without mineralization,silk microfibers after mineralization for 3 days and silk microfibers after mineralization for 7 days could all promote the growth and proliferation of MG-63 cells,but silk microfibers after mineralization for 7 days achieved the best result.Furthermore,we introduced mineralized silk microfibers into system of Acrylamide-Hyaluronic acid-Bisphosphonate and successfully fabricated self-healing composite hydrogel via coordination bonds between Bisphosphonate and CaP.Then we implanted this into a rat cranial defect model and found the hydrogel could effectively promote bone regeneration.In this research,we took full advantage of silk microfibers.On one hand,we added them into chitosan membrane and chitosan scaffold as reinforcement fillers,which was simple to manipulate and could significantly improve the comprehensive performance of those composites.On the other hand,we used them as organic template to regulate the nucleation and growth of bone-like minerals,not only achieving a perfect combination of organic template and inorganic minerals,but also endowing silk microfibers more functionality.