Ice-Templated Silk Fibroin/Tricalcium Phosphate/Two-Dimensional Black Phosphorus Composite Scaffold For Osteogenesis And Angiogenesis

Background and objective:Bone defect is a common clinical problem in oral implant surgery,and effective bone repair materials are needed to promote bone regeneration.At present,the commonly used bone repair materials are autogenous bone,allogeneic bone,heterogeneous bone and so on,but they all have some limitations,such as biocompatibility,mechanical properties,degradation rate and osteogenic ability.Therefore,finding a kind of bone repair material with excellent biological and mechanical properties is an important goal of bone tissue engineering.Silk fibroin is a natural protein with good biocompatibility,degradability,wettability and three-dimensional pore structure,and can be used as an organic component of bone repair materials.Tricalcium phosphate is a commonly used inorganic ceramic,which has good osteogenic ability,biological activity and degradability,and can be used as an inorganic component of bone repair materials.Two-dimensional black phosphorus is a new type of two-dimensional material,which has excellent optoelectronic properties,biocompatibility and degradability,and can be used as a functional component of bone repair materials.The composite of these three materials can form a multi-functional bone repair material,which can not only improve the mechanical properties,but also promote bone regeneration and angiogenesis.Ice-templating technique is a method to prepare porous materials.Porous materials with ordered morphology and controllable pore size can be formed by the growth and melting of ice crystals.Silk fibroin /tricalcium phosphate / two-dimensional black phosphorus composite scaffold can be prepared by ice-templating method,which has good pore structure and biological properties and can be used as an effective bone repair material.The purpose of this study is to construct silk fibroin / tricalcium phosphate / twodimensional black phosphorus composite scaffold by ice-templating method,and to study its osteogenesis and angiogenesis,so as to provide a new material choice for bone defect repair.Research methods:Firstly,a few layers of two-dimensional black phosphorus nanosheets were stripped from black phosphorus crystals by liquid phase exfoliation method.Scanning electron microscope,energy spectrum analysis,transmission electron microscope,atomic force microscope,nano-particle size and Zeta potentiometer were used to detect the size,distribution,morphology and thickness of BPNS,the degradation rate of BPNS in vitro and the photothermal properties of BPNS.Then silk fibroin was extracted from silkworm cocoon,SF/TCP scaffold and SF/TCP/BPNS scaffold with radial gradient pore structure were prepared by radial freezing of icetemplating technique,SF/TCP scaffold with axially aligned pore structure was prepared by unidirectional freezing,and SF/TCP scaffold with random pore structure was prepared by traditional freezing method.Zeta potential and Fourier transform infrared spectroscopy were used to detect the stents in each group.Optical scanner,X-ray,scanning electron microscope and micro-CT were used to detect the micro-morphology of the stents,and the compressive strength of the stents.The effects of scaffold materials on the growth of BMSCs and HUVEC were detected by CCK-8 and cell death staining,the blood compatibility of scaffolds was detected by hemolysis test,the osteogenic potential of scaffolds in vitro was detected by ALP staining,and the effect of scaffold materials on angiogenesis in vitro was detected by lumen formation test in vitro.Finally,a rat model of critical skull defect was established to study the osteogenic ability of scaffold materials in vivo.Three rats in each group were killed at 4 weeks and 8 weeks respectively.The specimens of heart,liver,spleen,lung,kidney and other organs were taken for embedded section staining to detect the toxicity of the materials in vivo,and the skull specimens were scanned by micro-CT to detect the guided bone regeneration of the scaffold materials.After decalcification,sections were stained with HE and Masson,and the osteogenic ability of the scaffold materials in vivo was systematically evaluated.Results:In this experiment,the SF/TCP/BPNS composite scaffold material based on ice-templating technique was successfully prepared.The scaffold material is a three-dimensional porous structure with high porosity,interconnected pores and directional pores,which can highly simulate the natural bone structure.The compressive strength of scaffolds with radial micropores is significantly higher than that of axially aligned pores and traditional frozen scaffolds.The synthesized materials have good biocompatibility,and the scaffolds loaded with appropriate concentration of BPNS can significantly promote cell growth,osteogenic differentiation and angiogenesis in vitro.In vivo experiments,SF/TCP/BPNS composite scaffolds based on icetemplating technique showed the best ability to promote bone regeneration in each group.The bone regeneration scaffold material based on ice-templating technique developed in this study accords with the concept of efficient,minimally invasive and accurate medical treatment,and has certain value for improving the success rate of bone grafting and ensuring the aesthetic effect of the planting area.it has good application prospects,economic benefits and positive social benefits.