Construction Of Micro/Nano Topographical Silk-based Scaffolds For Bone Repair

The human skeleton is a kind of mineralized organ with hierarchical structures from microscopic to macroscopic levels.Bone-related diseases are seriously harmful to tens of millions of people.Preparation of artificial bone scaffolds through bone tissue engineering strategy provides a treatment strategy to overcome the human bone defect disease.Meanwhile,silk proteins are natural macromolecules with many excellent physical and chemical properties,playing an increasingly important role in the field of bone tissue engineering.At present,the frequently used bone tissue engineering strategy based on silk proteins includes silk-based scaffolds,cells and growth factors.The process is as follows:1)Building porous scaffold;2)Seeding mesenchymal stem cells(MSC)in the scaffold in vitro;3)Adding exogenous growth factors to induce MSC differentiation.4)Implanting cells/scaffolds into defected bone to repair the bone defect.On the other hand,silk proteins can be used as a biotemplate by biomimetic methods to control the formation of hydroxyapatite crystals(HAp,the inorganic composition of bone)and then to improve the bone regeneration.However,silk-based tissue engineering is still facing challenges such as insufficient mimetic bone structure,lack of osteoinductivity,need for additional adding of differentiation inducer and poor mechanical property.Therefore,how to effectively simulate the composition and structure of the bone tissue,prepare "smart" silk-based bone repair scaffolds and improve the efficiency of osteogenic differentiation and bone regeneration,are urgent problems need to be solved at present.Aiming at the above key issues,this study used bio-mineralization method and protein processing technology to construct micro/nano-topographical silk-based scaffolds to improve its osteoinductivity and bone repairing ability.The main ideas are as follows:(1)Obtaining the HAp crystals by coprecipitation mineralization to improve the differentiation of MSC:The nanocomposite HAp crystals similar to natural bone were synthesized by a coprecipitation mineralization method.It was found that silk protein mediated the nucleation and growth of nanoneedle-like HAp crystals.The nucleation process of HAp could be turned by changing silk concentration and mineralization time.HAp/silk coatings could promote mesenchymal stem cell growth and improve cell attachment.Most importantly,we found that HAp/silk could significantly promote the differentiation of MSC into osteoblasts.(2)Construction of the topological structures of silk-based films by electrostatic spinning and ice crystal template method to improve bone repairing ability:The silk protein films with bead-like microstructure and nanofiber structure were prepared by electrospinning technology,and the bead-like microstructure could induce the MSC differentiation.On this basis,the ice crystal template method was developed to form a regular ridge structure of silk film.This unique facile approach resulted in the formation of nanoridges on the surface of silk film.The ridged silk protein film promoted cell elongation,which in turn allowed the silk film to stimulate the osteoblastic differentiation of MSC without adding osteogenic inducers.Moreover,the ridged silk protein materials had the capability of inducing the formation of calcified bone-like tissue in a rat subcutaneous model.Hence,nanostructured topographies of silk films are potentially attractive for developing smart biomaterials for bone regeneration.(3)Building bone-like lamella structure of silk porous scaffolds by a freeze casting technology to repair large size bone defect.The bone-like lamella structure(LP)of silk porous scaffolds had a regular and orderly lamella structure.The compressive strength and elastic modulus of the LP scaffolds were higher than those scaffolds obtained from the freeze-drying method.The LP scaffolds could promote the long-term cell viability of MSC(up to 21 days)in a 3D culture environment and accelerate bone tissue regeneration in a rat bone defect model.Hence,the silk-based LP scaffold with excellent bone repair ability provides valuable insight into scaffold construction and bone tissue engineering.In this study,four kinds of silk-based biomaterials with micro/nano-topological structure were prepared by coprecipitation mineralization,electrospinning,ice crystal template method and freezing casting method,which could regulate the differentiation of MSC into osteoblasts,and promote the regeneration of bone tissue.Therefore,this study has comprehensively evaluated the differentiated ability and bone repairing ability of silk-based scaffolds with micro/nano-topological structure.