Effects Of Silica Nanoparticles/Gelatin Composite Scaffold On Osteogenic Differentiation Of MSCs And Bone Defect Repair In Mice

Research background:There is a growing clinical demand for bone repair materials,and tissue-engineered bone has the advantages of being widely available,avoiding secondary injury to patients from autologous bone,and having a controllable size compared to autologous bone and allogeneic bone;compared to allogeneic bone,tissue-engineered bone has higher osteogenic activity and higher bone repair capacity.The ideal graft material should have similar mechanical characteristics to autologous bone,promote the differentiation of osteoblasts,and support the growth of new bone and blood vessels in the defect.Collagen is the main component of all extracellular matrix of tissues.Collagen is biocompatible,biodegradable and non-toxic.It can be recognized and adhered to by many cell types.However,due to poor mechanical properties of gelatin alone,it degrades too quickly in vivo and cannot meet the standards of bone graft materials.Therefore,it is necessary to add inorganic materials to form composites and crosslinking agents to make it meet the requirements.Silicon-based biomaterials are biocompatible,so they are widely studied and have been used for bone regeneration,angiogenesis,drug delivery,etc.In the bio-glass studied,it has been proved that silicon can promote osteogenic differentiation of osteoblasts,but simple silica has no in vitro biological activity,and too high concentration will inhibit the activity of cells,so it is necessary to add calcium,an indispensable substance for in vitro mineralization.Research purpose:To solve the above problems,this study adopts the sol-gel method.The Si O₂ and Ca（OH）₂ into biological activities of nano-sized Particles（Bioactive Particles BPs）,by covalent bonds to form the stable connection with gelatin.Organic components improve the biocompatibility of the material,increase the toughness of the material,also as the attachment of inorganic nanoparticles,inorganic components improve the hardness of the material,promote osteogenic differentiation.There is a synergy between the two components.Adjusting the ratio of the two can improve the mechanical properties of the material.Biological activity and physical characterization materialsThe research methods:1.1.Physical characterization of the material:the sol-gel method was used to produce 14nm diameter BPs,adjust the ratio of particles and gelatin to produce four ratios of material-BPs/gel-0%,BPs/gel-20%,BPs/gel-50%,BPs/gel-100%.Scanning Electron Microscope SEM（SEM）tested the ability of the material to detect pore size distribution,particle distribution and in vitro mineralization,contact angle test tested the hydrophilicity of the material,mechanical testing machine tested the mechanical properties of the material,Inductively Coupled Plasma Emission Spectroscopy（ICP-OES）detects the release of calcium and silicon from the material.2.In vitro cell experiment:a series of concentration gradient BPs and 4 materials were co-cultured with MSCs of mice respectively.CCK-8 was used to detect the effects of BPs and materials on the growth and adhesion of MSCs.3.In vivo bone defect experiment:four kinds of materials were implanted into C57 mice of bone defect model.The bone formation was detected by Micro-CT and HE staining.4.The preliminary investigation into the osteogenic mechanism of BPs in the material:cells were inoculated into the material,inhibitors were added,and whether BPs affected cell differentiation through Wnt/β-catenin,P38MAK,PI3K was detected.Experimental results:1.The experimental results of the physical characteristics of the material showed that the physical characteristics of the material were closely related to the ratio of gelatin and BPs,the SEM results showed that the pore diameter of the material was more densely distributed as the BPs ratio increased;in the mechanical experiments,BPs could significantly improve the mechanical properties of the material;the experimental results of the contact angle showed that the gelatin could increase the hydrophilicity of the material.2.BPs and composites were co-cultured with cells,and the adsorption and proliferation of BPs and composites were detected by CCK-8 reagent at different time points,and the results showed that the higher the BPs content,the stronger the adsorption capacity of composites on cells,but had no obvious effect on cell proliferation.Osteogenic differentiation of MSCs,and inhibition of differentiation may occur at too high a concentration-more than 4μg/ml.3.In vivo osteogenic experiments showed different results from in vitro cellular experiments,Micro-CT showed that the group with the highest content of BPs-BPs/gel-100%scaffolds could promote the formation of continuous closed neo-cortical bone at the defect,and CTVox data showed that this group of scaffolds formed the most abundant bone tissue at the bone defect,indicating that BPs/gel-100%in vivo repair capacity of bone defects is optimal.4.In preosteogenesis,BPs induced RUNX2 expression through Wnt/β-catenin,P38MAPK and PI3K,and MSCs synthesized secreted ALP through P38MAPK and PI3K,and MSCs synthesized COLⅠthrough P38MAPK.For the expression of late osteogenic genes,all three groups of added inhibitors had the inhibitory effect compared to the BPs group.Genes OCN,OPN,and OSX were least expressed by BPs+LY249002（PI3K inhibitor）;XAV-939（Wnt/β-catenin inhibitor）and SB203580（P38MAPK inhibitor）inhibited the expression of OCN and OPN but the difference between them was not statistically different,although significant;from the inhibition of The degree of analysis,the three inhibitors had significant effect on OPN,indicating that BPs induced the expression of this gene mainly through WNT/β-catenin,P38MAPK and PI3K,while for OCN and OSX,BPs could induce the expression of osteogenic genes through the above three pathways,but not completely,suggesting that there may be other signaling pathways as well.Conclusion:The nanoscale BPs made by sol-gel method can promote osteogenic differentiation in a certain concentration range（≤4μg/ml）,and the composite material formed with gelatin can improve the physical characteristics of the material by controlling the ratio of the two,in vitro and in vivo experiments proved that this composite scaffold can significantly promote osteogenic differentiation and repair the site of bone damage.The in vitro and in vivo experiments demonstrated that this composite scaffold can significantly promote osteogenic differentiation and repair the bone injury.