Research On The Mechanism Of Porous Sr@Zn@SiO₂ Nanocomposites Promoting Bone Defect Repair Through The Regulation Of Smad1/5/9 Target

Background Bone defect is a common problem in clinical practice.The incidence of poor healing occurs in 5-10% during the repair process of bone defect,which seriously affects the quality of life of patients and brings a huge economic burden to the national medical service.There has been no breakthrough in its treatment in nearly 30 years.The main reason is that although relevant mechanisms have been confirmed by studies,there has been no major breakthrough in which methods or drugs can activate these mechanisms and obtain satisfactory repair results,so as to avoid bone transplantation and surgical operations,such as bone transport,autogenous bone,artificial bone and allograft bone.The current research proves that metallic elements are widely distributed in nature and play a great role in osteogenesis,antibacterial and angiogenesis.At the same time,metal elements exist widely in various organs of the human body and play an important role in the normal metabolism of life,so metal elements are very likely to be the key to solve this problem.In recent years,the application of metal elements in medicine is more and more extensive,especially in the repair materials of bone defects.The metal type of bone repair materials accounts for more than 90%,among which titanium is the most widely used.However,pure titanium metal has poor biological activity and can only meet the supporting effect.Therefore,in the field of bone repair,solving the problem of bone non-union through the application of other metals has been the focus of research.Calcium is a more abundant element in bone,while Strontium（Sr）,which is similar to calcium,is a widely distributed element in nature and has been widely concerned.It can not only improve the mechanical properties of bone,but also improve the biological properties of bone.Similar to Zinc（Zn）as an essential trace mineral for human body,in a large number of osteogenic materials synthesis,will be doped with Zn element to promote bone formation.Based on this,Sr@Zn@SiO₂,a nano-composite material,is synthesized for the first time.Zn and Sr are wrapped in porous SiO₂.With the slow-release property of porous SiO₂,the two metallic elements can not only reduce the toxicity effect through slow release,but also play a continuous stimulating role.We creatively apply the material in the field of bone defect treatment,hoping that the application of this material will provide a promising,safe and effective way for the treatment of bone defect.Purpose In this study,Sr@Zn@SiO₂ as a nanomaterial,was creatively synthesized and applied in the field of bone defect repair.The repair effect of this material on bone defect was preliminarily determined through in vivo experiments in rats.At the same time,the effects of this material on the invasion and proliferation ability of rat Bone Mesenchymal Stem Cells（BMSCs）were explored through in vitro experiments,and the related indexes of bone formation were measured and compared,and the role of Sr@Zn@SiO₂ in the field of bone repair was clarified.On this basis,the Smad1/5/9 target was explored,and strive to explain the repair effect of Sr@Zn@SiO₂ on bone defects from the mechanism,providing a new possibility for the treatment of bone defects.Method（1）Preparation of Sr@Zn@SiO₂ and morphological study: The Sr@Zn@SiO₂ nanocomposite was prepared by wrapping Sr and Zn materials with SiO₂ nanomaterials.The microstructure and distribution of elements were observed by Transmission Electron Microscopy（TEM）and element scanning technique.It was confirmed that Sr@Zn@SiO₂ nanocomposites were successfully synthesized.（2）The effect of Sr@Zn@SiO₂ on the repair of bone defects in rats: the model of femoral defects in rats was constructed,and the rats were fed for 8 weeks after the femoral defects were filled with Sr@Zn@SiO₂ and SiO₂,respectively.The sample was scanned with Micro Computed Tomography（micro-CT）to detect Bone Mineral Density（BMD）.The percentage of new Bone Volume to Total Volume（Bone Volume/Total Volume,BV/TV）was statistically analyzed.The femur of rats was collected,sliced and stained,and the repair of bone defect of rats was observed.（3）Effects of Sr@Zn@SiO₂ on invasion ability and osteogenic markers of bone marrow mesenchymal stem cells: Rat mesenchymal stem cells（BMSCs）were isolated,and the surface markers CD90,CD44,CD11 b and CD45 were identified.Nano-composites of different concentrations were added into the medium,and the spread morphology of the mesenchymal stem cells was observed after co-culture.Cell Counting Kit 8（CCK-8）was used to detect the activity of BMSCs and evaluate the in vitro safety of porous Sr@Zn@SiO₂ nanocomposites.Cell migration and invasion ability was measured using Transwell technique,and Quantitative reverse transcription polymerase chain reaction（quantitative Real-time PCR,q RT-PCR）,Western blot and Enzyme Linked Immunosorbent Assay（ELISA）were used to detect the expression of migration and invasion-related genes.Alizarin red staining was used to determine the amount of calcium deposition in all groups.The activity of Alkaline phosphatase（ALP）was detected by biochemical methods,and the expressions of Runt-related transcription factor-2（Runx2）,Bone Morphogenetic Protein-2（BMP-2）,Osteocalcin（OCN）and Smad1 genes related to osteogenic differentiation were detected by PCR.（4）Sr@Zn@SiO₂ regulates osteogenesis via Smad1/5/9 target: After the BMSCs were co-cultured with the blank control,SiO₂ materials and Sr@Zn@SiO₂ nanocomposites,the protein expressions of Smad1/5/9 and p-Smad1/5/9 were detected by Western blot,and the entry of p-Smad1/5/9 into the nucleus was analyzed by immunofluorescence staining.To explore the mechanism of osteogenesis of this material.Results（1）Preparation of Sr@Zn@SiO₂ and morphological study: Sr@Zn@SiO₂ nanocomposites were successfully prepared by wrapping Sr and Zn materials with silica nanomaterials.The fine structure of the materials was observed by transmission electron microscopy（TEM）,and the distribution of elements was obtained by element scanning,which verified the successful synthesis of Sr@Zn@SiO₂ nanocomposites.（2）The effect of Sr@Zn@SiO₂ on the repair of bone defects in rats: On the basis of establishing the model of supracondylar bone defects in rats,the material filling of the bone defects was carried out.Micro-CT scanning results showed that 8 weeks after implantation,bone mineral density（BMD）and percentage of new bone volume to total volume（BV/TV）in Sr@Zn@SiO₂ group were significantly higher than those in SiO₂ control group.Pathological sections showed that the amount of new bone in the Sr@Zn@SiO₂ group was significantly higher than that in the SiO₂ group,especially around the material.（3）Effects of Sr@Zn@SiO₂ on invasion ability of bone marrow mesenchymal stem cells and osteogenic markers: Rat BMSCs were successfully extracted,isolated and cultured,and CD90 and CD44 surface markers of bone marrow mesenchymal stem cells were identified as positive,while CD11 b and CD45 were negative.The safety of Sr@Zn@SiO₂ nanocomposites was detected by CCK8,and it was found that Sr@Zn@SiO₂ nanocomposites with a concentration below 400μg/ml had no obvious toxicity to BMSCs in rats.After the BMSCs were co-cultured with Sr@Zn@SiO₂ nanocomposites,Transwell detected the cell invasion ability.The results indicated that the invasion ability of BMSCs in the Sr@Zn@SiO₂ group was stronger than that in the control group.Similarly,the expression of Chemokine Receptor 4（CXCR4）and Chemokine Stromal Cell Derived Factor-1（CXCL12）related to cell proliferation was higher than that of the control group.At the same time,the amount of calcium deposition in the experimental group determined by alizarin red staining,the ALP activity detected by biochemical method.The expressions of Runt-related transcription factor-2（Runx2）,Bone Morphogenetic Protein-2（BMP-2）,Osteocalcin（OCN）and Smad1 were higher than those of blank control group.（4）Sr@Zn@SiO₂ regulates osteogenesis via Smad1/5/9 target: Rat BMSCs were cocultured with Sr@Zn@SiO₂ experimental group and blank control group.As the target activator of Smad1/5/9 signal,porous Sr@Zn@SiO₂ nanocomposite promoted the phosphorylation of Smad1/5/9 and activated the expression of downstream osteogenic genes,thus promoting bone repair.ConclusionTo sum up,we synthesized porous Sr@Zn@SiO₂ nanocomposite for the first time,and verified its element distribution and microstructure.Through animal experiments and cell experiments,we preliminarily explored the important role of porous Sr@Zn@SiO₂ nanocomposite in promoting bone repair in vivo and in vitro.At the same time,it was confirmed that Sr@Zn@SiO₂ could regulate the downstream osteogenic genes through the activation of Smad1/5/9 related signal targets,and achieve the result of promoting bone repair.