Fabrication Of Biomimetic Scaffolds With Different Hierarchical Microstructures Of Bone And Its Effect On BMSC

BackgroundThe incidence of bone fractures and musculoskeletal diseases has been increasd by the development of social economy,changes of lifestyle and aging of population.The treatment of orthopedic diseases has drawed national attention.In order to solve the difficulties of repairing and regenerating of bone tissue,the development of new bone tissue engineering materials has become an important subject at present.Improving the physical properties of tissue engineering materials to promote the biological activity are the core task.Studies found that the topographic microstructure which cells live have special biological effect,and the topographic microstructure of bone plays a key role in regulating cell behavior and bone regeneration.Therefore,fabricated bone tissue engineering bionic scaffold with bone microstructure may enhance the interaction with cells.This could give a new insight into bone tiusse engineering research area.Human bone tissue is a complex multi-layered structure that changes from micro to macro.Different levels of bone microstructure can have different effects on cell behavior and differentiation.In this study,we characterized human bone microstructure at different levels which were the extracellular matrix(ECM)and trabecular of bone.Based on the parameters of topographic microstructure,we fabricated biomimetic nanofiber scaffolds and biomimetic metal scaffolds.To understand the impact of structural characteristics on scaffolds,we investigated the Bone Mesenchymal stem cells(BMSC)on biomimetic scaffolds,including BMSC morphology,behavior and differentiation.This study may lay a theoretical foundation for the design,development and safe application of new generation scaffolds.Part Ⅰ:Characterization and Parameter Extraction of Bone Microstructure at Different LevelsObjective:To obtain bone topographic and structural parameters,we establish the foundation for fabricating scaffolds.Methods:In this part of the study,bone samples were collected from femoral head after surgery,and bone samples were partitioned by Micro-CT,pathological HE staining and nanoindentations.SEM and Micro-CT were used to characterize the bone tissue in the relatively normal area,and software processing was used to extract the topographic and structural parameters of different levels of bone tissue.We obtain the parameters of mineralized collagen fiber bundle diameter,angular orientation and other parameters in the bone ECM and the parameters of porosity and pore size in the trabecular structure of the bone.Results:Mechanical properties of bone samples:average elastic modulus=10.01±1.86 GPa,average hardness=357±31 MPa.The topographic parameters of bone ECM:average mineralized collagen bundle fiber diameter=915±22 nm,average mineralized collagen fiber angle trend=37.9±2.1°.The structural parameters of bone trabecula:average porosity=65.4±3.2%,average pore diameter=189±23μm.Conclusion:The bone tissue samples were characterized by SEM and Micro-CT technology,and combined with software processing methods to obtain different levels of bone topographic and structural parameters.Part Ⅱ:Fabrication of Biomimetic PLA/nHA Nanofiber Scaffolds with ECM Topographic Parameters and Its Effect on BMSCsObjective:Based on the obtained bone ECM topographic parameters,we fabricated biomimetic nanofiber scaffolds and investigated its effect on BMSCsMethods:PLA/nHA nanofiber scaffolds were fabricated by electrospinning method.Biomimetic PLA/nHA scaffolds with ECM topographic parameters were constructed by adjusting electrospinning process.The physical and chemical properties of PLA/nHA scaffolds were characterized by SEM analysis,XRD diffraction spectrum,FTIR test and wettability analysis.In vitro cell culture methods were used to compare the cell morphology,cell proliferation,and osteogenic differentiation of BMSCs on PLA/nHA nanofiber scaffolds with different parameters.Results:We successfully fabricated three groups of PLA/nHA scaffolds with different topographic parameters by electrostatic spinning.The diameter of nanofibers of the three groups:S1 group=389±45 nm;S2 group=906±166 nm;S3 group=1204±187 nm;Among them,the topographic parameters of PLA/nHA scaffolds in group S2 were consistent with bone ECM.Cell morphology and proliferation results showed that BMSCs had larger cell spreading areas on the scaffolds in the S1 and S2 groups and cells show more pseudopods embedded on the surface of the S1 and S2 scaffold.Cell proliferation of both S1 and S2 groups at different time points were better than that in the S3 group.Osteogenesis-related marker gene tests showed that the expressions of BMSCs on the S2 group were better than those in the S1 and S3 groups at 3d,7d,and 14d.The results of 21 d calcium deposition experiments showed that the mineralization of BMSCs on S2 group was significantly better than the other two scaffolds.Conclusion:1.The electrospinning method can fabricate biomimetic PLA/nHA scaffolds with ECM topographic parameters of bone.2.PLA/nHA scaffolds have good biocompatibility,different nanofiber diameter could affect the biological activity of scaffolds;3.Scaffolds with nanofiber diameters between 300nm and 900nm could promote the spreading and proliferation of BMSC 4.PLA/nHA scaffolds with ECM topographic parameters of bone(the 900nm interval)could promote BMSC osteogenic differentiation compared to other structure.Part Ⅲ:Fabrication of Biomimetic Ti-40Nb Scaffolds with Structural Parameters of Trabecular Bone and Its Effect on BMSCsObjective:Based on the obtained structural parameters of trabecular bone,we fabricated biomimetic Ti-40Nb scaffolds and investigated its effect on BMSCsMethods:The porous Ti-40Nb alloy scaffolds were fabricated by the SPS method.Ti-40Nb alloy scaffolds with different structural parameters(porosity,pore size)were adjusted by the preparation process.The physical and chemical properties of the porous Ti-40Nb alloy scaffolds were characterized by XRD diffraction spectrum,mechanical test and corrosion resistance test.We investigated in-vitro cell experiments such as the cell morphology,cell proliferation and ALP activity of BMSCs on Ti-40Nb alloy scaffolds with different structural parameters and compared the osteogenic differentiation of BMSCs on Ti-40Nb alloy scaffolds.Results:We successfully fabricated three groups porous Ti-40Nb alloy scaffold by the SPS method,which have trabecular-like porous structure with a pore size between 100 μm and 350 μm.The porosity of the three groups was 13.4%in the S1 group,38.9%in the S2 group,and 61.2%in the S3 group.The structural parameters of the Ti-40Nb scaffold material in the S3 group were consistent with the trabecular bone.The elastic modulus of the three groups of porous Ti-40Nb alloys are S1 group=27.3 GPa,S2 group=22.6 GPa,S3 group=18.4 GPa to match human bone tissue;and the compressive strength is S1 group=452 MPa,S2 group=411 MPa and S3 group=326 MPa,which meets the requirements of the mechanical properties of the stent materials.Blood compatibility and corrosion resistance test results show that porous Ti-40Nb has good biocompatibility and corrosion resistance.Cell morphology and proliferation results showed that BMSCs could grow well on porous Ti-40Nb scaffold material,while the proliferation in the S3 group was better than that in the S1 and S2 groups.The ALP results showed that the ALP activity of BMSC on the S3 group was higher than that of the other two groups at 3d and 7d.Conclusion:1.Biomimetic porous Ti-40Nb with trabecular bone structural parameters can be fabricated by SPS method;2.Porous Ti-40Nb has good biocompatibility,mechanical properties and corrosion resistance.Different structural parameters can affect the biological activity of alloy scaffold materials.3.The porous Ti-40Nb alloy scaffolds with structure parameters of trabeculae bone(61.2%porosity)were beneficial to BMSC proliferation and early osteogenic differentiation compare to other structures.