Preparation Of Keratin Biomimetic Bone Scaffold Based On Conformation Induction And Rapid Mineralization And Its Performance

As a major country in animal husbandry and textiles,China produces a large amount of wastes such as poultry feathers,short wool with no textile value,and cattle and sheep skins every year.These solid wastes are natural resources rich in keratin.The keratin extracted from these poultry feather wastes not only has excellent biocompatibility and natural degradability,but also has unique biological characteristics.It has broad applications prospects in the fields of biomedicine,tissue engineering and new renewable materials.Therefore,recycling keratin wastes and developing green and renewable keratin-based materials have important theoretical research value and potential social and economic benefits for protecting the natural environment,making full use of natural resources,and realizing sustainable social and economic development.Because of its good biocompatibility,keratin materials cause less immune response,and the degradation products are non-toxic,thus become a hot research as bone scaffold materials.However,this kind of scaffold generally has the defects of low mechanical strength and insufficient osteoinductive performance,which makes it difficult to provide a relatively stable microenvironment for cell regeneration and proliferation on it.This has become a constraint on the use of keratin scaffolds in bone repair.Studies have found that the secondary conformation and regularity of protein materials are important factors that affect its mechanical properties.Natural protein materials,such as spider silkor wool,exhibit excellent mechanical strength.These protein materials have similar chemical composition,However,in terms of chemical structure,wool fibers are mainly composed of α-helix conformation,while Spider silk are in regular β-sheet conformation.Tthe regenerated keratin is affected by swelling and reducing agents during the extraction process,thus the regular molecular conformation structure is destroyed.This shows that the secondary conformation and the degree of regular arrangement affect the mechanical properties of protein materials.This research starts with protein conformation and regulates the secondary structure of regenerated keratin on a mesoscopic scale,thereby improving the mechanical strength of the material.On this basis,an electrochemical mineralization method was designed to quickly mineralize the keratin scaffold to prepare a biomimetic bone scaffold.In this study,keratin scaffold was fabricated by mesoscopic molecular conformation and macroscopic structure manipulating.Silk fibroin will used to induced mesoscopic keratin molecular conformation transition,effect of conformation inducer on the regulation of interaction between keratin and silk fibroin will be investigated to illuminating the mechanism of controllable transition of molecular conformation.In addition to revealing the relationship between conformation transition and mechanical strength of keratin scaffold.Further,macroscopic structure of keratin scaffold will be decorated by electrodeposition.Rules of hydroxyapatite structure evolution will be studied to establish rapid mineralization method and achieve effective improvement of osteoinductive and mechanical properties of keratin biomimetic scaffold.This work will provide theoretical basis for mechanical property improvement of protein materials and enrich the construction of biomimetic bone scaffold.The mainly research content is as follows:1.First,keratin was extracted from feathers biomass.The relationship between secondary structure and mechanical property of the material was comprehensively studied from the point of protein microstructure.With silk protein as the conformation transition template,the de-crosslinked molecular chains in keratin were assembled on the template to form a regular arrangement,and then a mechanical-enhanced keratin scaffolds was prepared.The results show that using silk fibroin with high β-sheet content as template could promote the transformation of the secondary structure of regenerated keratin.The content of β-sheet conformation of regenerated keratin can increase from 10% to about 25% with the increase of template dosage and the content of random coil conformation exhibited decrement.And with the increase of β-sheet content in the regenerated keratin structure,the strength of the scaffold showed a satisfying improvement.2.On the basis of preparing the mechanical reinforced scaffold,the scaffold was rapidly mineralized by electrochemical method,and the influence of different electrodes and mineralization factors on the formation of calcium phosphate salt was studied.The results show that the type of metal electrode affect the crystal type and morphology of calcium phosphate salt.The main deposit on the stainless steel electrode is hydroxyapatite,while other electrodes are accompanied by the formation of calcium hydrogen phosphate dihydrate.The mineralization factors mainly affect the thickness of the sedimentary layer and the shape of calcium phosphate crystals.When the deposition voltage is 4 V,the mineralization time is 1.5 h,and the electrode spacing is 6 cm,a uniform and high-quality mineralized layer could be formed.3.In order to explore the influence of keratin scaffold on cell behavior,the biocompatibility and osteoinductive of the scaffold was studied.The blood compatibility and MTT study show that the scaffold has no obvious toxicity and exhibited good cell compatibility.Bone marrow mesenchymal stem cells(BMSCs)seeded on the scaffold exhabited obvious proliferation and diffusion.The expression of osteogenic genes by RT-PCR study found that the expression of related genes can be maintained at a high level,and the expression of osteogenic genes on the mineralized scaffold is higher than that of the unmineralized scaffold.