| Bone tissue has the ability to self-regenerate,but its regeneration ability is limited,especially for critical size defects.In such cases,medical materials are required to assist in bone tissue regeneration.Bone scaffolds made from autograft or allograft bone can be used to regenerate bone tissue,but these can cause secondary trauma or have reduced biological activity or risk of pathogen transmission.To address these issues,bone tissue engineering has been developed,with the aim of creating functional substitutes for regenerated bone.Biological scaffolds can provide mechanical stability and can either integrate into the bone structure or be degraded and excreted by the body.Natural biological scaffolds are of particular interest due to their outstanding performance in biology and excellent mechanical properties.A difficult research topic in the field of biomimicry is the simulation and construction of the orderly graded combination of organic matter and inorganic minerals to construct bone regeneration and repair materials that can highly restore the structure and function of defective tissue.Silk fibroin and hydroxyapatite have unique mechanical properties,biocompatibility,osteogenic inductivity,and slow degradability,and have become matrix materials for bone tissue engineering.In this study,silk protein was selected as the raw material,and the silk protein/hydroxyapatite composite scaffold was prepared using directional freezing combined with biomimetic mineralization.The scaffold was then co-cultured with cells to evaluate its biocompatibility and osteogenic inductivity.The study’s main research contents are as follows:(1)Using silk fibroin protein as the matrix material,a three-dimensional silk fibroin scaffold with an oriented structure was prepared by directional freezing technology.The effects of the concentration of silk fibroin protein and freezing temperature on the microstructure of the scaffold were systematically studied using scanning electron microscopy(SEM).The results showed that the microstructure of the scaffold gradually became porous with an increase in protein concentration.The aperture size,controlled by adjusting the temperature of the frozen stent,can be within the range of 5-80 μm.The conformational changes of silk fibroin protein were analyzed using infrared spectroscopy(FTIR).The results showed that ethanol and BDDE could cross-link silk fibroin with a physicochemical method.The mechanical properties of the support were tested,and the tensile results showed that the tensile modulus of the support could reach 40 MPa.The modulus of the compression cycle can reach 13 MPa.(2)In order to improve the osteogenic induction ability of the scaffold,a liquid precursor of calcium phosphate(Cap-PILP)was used to mineralize the silk scaffold.The particle size distribution of the mineralized liquid was characterized using dynamic light scattering.The scaffold was moistened by citrate before mineralization.Scanning electron microscopy(SEM)was used to observe the microstructure of the scaffolds before and after mineralization.The nano mineralization of filaments was observed using transmission electron microscopy(TEM).The calcium phosphate crystals mineralized on the scaffold were analyzed using X-ray powder diffraction(XRD).Thermogravimetric analysis(TG)was used to test the content of inorganic substances loaded on the support after a certain time of mineralization.The universal mechanical testing machine was used to carry out a three-point bending experiment on the mineralized bracket.The results showed that the particle size of Cap-PILP mineralized liquid ranges from 100 nm to about 100 nm and can remain stable for 3days.The use of citrate before mineralization has an excellent effect on the wetting of the scaffold,which can make the mineralized calcium phosphate evenly distributed on the scaffold without changing the original morphology.TEM and SEM images showed that the mineralized solution could mineralize the protein scaffold at the nano and micron levels.XRD results showed that after soaking in a weak alkaline environment,the calcium phosphate on the scaffold gradually changed into hydroxyapatite.TG test results showed that the corresponding calcium phosphate content was 26%,30%,and 38.8%,respectively,after 1,12,and 24 hours of mineralization.The results of the three-point bending experiment showed that the maximum bending modulus of the scaffold could reach 2 GPa,which was comparable to that of human cancellous bone tissue.(3)In order to evaluate the biocompatibility and osteogenic induction ability of the scaffold,an in vitro cell co-culture experiment was conducted on the scaffold.The results showed that the scaffolds had no cytotoxicity,and the cells could proliferate well on the scaffolds before and after mineralization.Cell osteoblast differentiation tests showed that calcium phosphate loaded scaffolds had a positive effect on cell osteoblast differentiation without the addition of osteoblast growth factors.Laser confocal microscopy was used to assess the impact of the scaffold on cell morphology,and it was found that the directional stents with an aperture size of 10-20μm had the best ability to guide the direction of cell growth.In conclusion,in this paper,the anisotropic filaments scaffold was prepared by directional freezing technology,and the multi-scale uniform mineralization of the scaffold was carried out with liquid precursor of calcium phosphate,which not only effectively improved the mechanical properties of the scaffold,but also showed the effect of inducing osteogenic differentiation of cells in vitro cell experiments.The scaffolds provided a good extracellular matrix for guiding cell behavior and had potential application value in tissue engineering. |
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