Preparation Of Natural Silk Micro-and Nanofiber-based Three-dimensional Scaffolds And Regulation Of Their Structure And Properties

Nanofiber scaffolds have promising applications in the field of tissue engineering due to their extracellular matrix-like(ECM)structural properties.The natural silk micro and nano fibers(SNF)obtained by stripping from silk fibers have a high aspect ratio and retain the good mechanical properties,excellent biocompatibility,and degradable properties of natural silk,which have great potential in the field of biomaterials.However,SNF as a tissue engineering scaffold material has not been studied.How to assemble one-dimensional SNFs into structurally tunable three-dimensional porous scaffolds is a key challenge.Due to the lack of effective bonding between fibers,three-dimensional materials formed from SNF lack water resistance,limiting their application in tissue engineering.In this paper,inspired by the protein fiber-glycosaminoglycan composite structure in the natural ECM structure,a variety of natural polysaccharides were used to regulate the assembly of silk micro-nano-protofibrils to construct three-dimensional porous scaffolds,and the effects of fiber slurry concentration,polysaccharide ratio and cross-linking method on the morphology,structural stability,mechanical properties and on cell growth behavior of porous scaffolds were systematically investigated.First,natural silk micro nano fibers were prepared using chemical solvent pretreatment and physical mechanical action,and the preparation process was simple,efficient,and green.The in vitro degradation study of SNF showed that the degradation loss rate of fibers was 9.4%at 13 weeks in PBS,and the degradation loss rate could reach about 40%at the 13th week in the presence of protease XIV,indicating the good degradability of SNF.Meanwhile,SNF suspensions can be prepared into different shapes of films and porous materials,indicating that the dispersed SNF suspensions have good ability to form biomaterials.Secondly,to improve the mechanical properties and water resistance of SNF porous materials,chitosan(CS)was introduced to construct a physical cross-linked network,thereby regulating the assembly of SNF,and composite porous scaffolds with tunable structure,properties and good biocompatibility were prepared.The water stability results showed that the scaffold incorporating 5.0%CS maintained good stability and structural integrity in an aqueous environment,which may depend on the hydrogen bonding interaction between CS and SNF.The results of porosity and specific surface area showed that the porosity and specific surface area decreased gradually with the increase of CS content and SNF suspension concentration.However,the scaffold with the addition of 5.0%CS content still has 98.5%porosity and 135 m~2/g specific surface area.The mechanical properties showed that the compressive strength increased from 18.3 k Pa to 21.8 k Pa and 30.6 k Pa as the addition amount increased from 1.0%to 2.5%and 5.0%.Experiments on rat bone marrow mesenchymal stem cells showed that CS-regulated porous scaffolds have good biocompatibility and can support rapid cell proliferation.Further,the feasibility of SNF scaffolds as a biomineralized template was investigated.the CS-regulated scaffolds were able to induce hydroxyapatite(HAP)deposition on the fiber surface after mineralization in simulated body fluids,forming a protein micro-nanofiber/hydroxyapatite mimetic structure.Osteoblast culture experiments showed that the mineralized scaffolds were biocompatible and able to support rapid cell proliferation.Finally,composite porous scaffolds were prepared by chemically grafting hyaluronic acid(HA)onto SNF via carbodiimide(EDC)to investigate the effect of chemically cross-linked glycosaminoglycans on the structural properties of the scaffolds.First,the HA-modified SNF was naturally dried into a film to study the grafting behavior of HA.The contact angle test showed that when the mass ratio of HA to SNF in the grafting reaction solution gradually increased to 10.0%,the contact angle reached its lowest value,suggesting that the maximum grafting rate might be achieved.Experiments with rat bone marrow mesenchymal stem cells in culture showed that as the proportion of HA in the reaction solution gradually increased,the HA content on the fibers increased and cell viability gradually improved.Further,porous scaffolds were prepared by adding different levels of HA to SNF suspensions by EDC cross-linking.The water stability results showed that the scaffold with 10.0%HA content maintained good stability and structural integrity in an aqueous environment.The carboxyl group in HA forms a covalent cross-link with the amino group in SNF under the action of EDC,and the grafted HA molecules are linked to each other by intermolecular entanglement,hydrogen bonding and possibly chemical cross-linking,thus conferring stability to the scaffold structure.The porosity and specific surface area show that the porosity and specific surface area decrease gradually with the increase of HA content.However,the added content of 10.0%HA scaffold still has 96%porosity and 117.36 m~2/g specific surface area.The mechanical properties showed that the compressive strength increased from 11.3 k Pa to 14.2 k Pa as the addition amount increased from 2.5%to 10.0%.Osteoblast culture experiments have shown that HA-regulated scaffolds have good biocompatibility and can support rapid cell proliferation.As with CS-regulated scaffolds,after mineralization in simulated body fluids,HA-regulated assembly of SNF scaffolds was able to induce hydroxyapatite deposition on the fiber surface,forming a protein micro-nanofiber/hydroxyapatite mimetic structure capable of supporting rapid cell proliferation.In this paper,we proposed a strategy based on polysaccharide regulation of SNF assembly,prepared SNF 3D porous scaffolds with simulated ECM structure and ultra-high porosity,and elucidated the key role of polysaccharide in regulating scaffold structure and performance.The research in this paper provides a feasible strategy and scientific basis for expanding the biological applications of natural silk and constructing scaffolds based on silk micro-and nanofiber preparations.