Study On Preparation And Application Of A Novel Mg-Sr Co-doped MHAw-based Extruded And Enhanced By SiO₂ Complexation Porous Bone Defect Filling Scaffold

Background:With the advancement of industry,changes in transportation in modern society,and the development of diagnosis and treatment of bone-related diseases,every year around the world,the number of patients with bone defects caused by trauma(mechanical,falling and traffic accidents),infections,tumors and deformities is as high as millions.Although the bone tissue has strong regeneration ability and can achieve complete regeneration,when the bone defect size is large,reaching or exceeding the critical size,it cannot regenerate and repair itself after appropriate and proper treatment,and fractures and other injuries still cannot heal after prolonged treatment time,resulting in bone nonunion,requires bone grafting for repair and reconstruction.Compared with soft tissue,bone tissue regeneration time and treatment period are longer,especially bone defect or bone nonunion,which has always been a common challenge faced by orthopaedic surgeons and patients.The development of bone tissue engineering(BTE)has brought new hope for the treatment of bone defects.The commonly used treat methods are autologous bone transplantation,allograft bone transplantation,xenograft bone transplantation and artificial bone replacement.These methods have one or more of the disadvantages of increased surgical site,limited sources,immune rejection,disease spread,mismatched mechanical properties,high treatment costs,and poor efficacy,resulting in limited application.Therefore,the development of new bone substitute materials and the realization of green,feasible and effective treatment of bone defects are the arduous tasks of the BTE.Objective:Bone substitute materials for bone defects therapy need to have the following characteristics:Appropriate structure simulates the local microenvironment of bone tissue;Appropriate mechanical strength supports the defect area and adapts to bone mechanics;Good biocompatibility without immune rejection;Good osteoconductivity allows osteogenic stem cells to adhere,proliferate and crawling;Good osteoinductivity to induce osteogenic differentiation of osteogenic stem cells;Appropriate porosity and pore sizes provide space for cells and blood vessels to grow in the defect area,and create a foundation for the delivery of nutrients and the output of metabolic wastes.Based on the above requirements,the aims of this study are:1.Develop a raw material that can be used to prepare bone defect filling scaffolds and incorporate osteogenic active elements,and using this raw material,a group of porous bone filling scaffolds with good mechanical properties and similar to bone in composition and structure are prepared through several simple methods such as self-made mold extrusion,freeze drying,and sintering;2.After the characterization and physicochemical properties of the scaffolds were tested,the cytocompatibility,osteoconductivity and osteogenic inductive ability of these scaffolds were evaluated by in vitro cell culture;3.The scaffold was implanted in the defect area through the animal model of calvarial bone defect,and the biocompatibility and bone repair effect of the scaffold were evaluated after routine feeding,so as to evaluate its potential value in the treatment of bone defect.Methods:1.Micron hydroxyapatite whiskers(mHAw)were synthesized by heating in a water bath.The whiskers were doped with Mg2+or Sr2+or Mg2+and Sr2+,which are essential trace active elements of bone during the heating "sintering"process.Pure mHAw,Mg-doped mHAw,Sr-doped mHAw and Mg-Sr co-doped mHAw were prepared.After the four kinds of mHAw were prepared into "paste"slurry,the "lotus root-shaped" bionic porous scaffold was extruded through an extrusion molding die,dried in a freeze dryer,and then cut into a size of about 8mm*8mm.The cubes were soaked with silica gel and then calcined in a muffle furnace at 450℃ to form SiO2 complexes to improve the mechanical properties of the scaffolds.The hydrophilicity,pore size,surface morphology,surface roughness,element distribution,element content,mechanical properties and release kinetics of osteogenic active elements of the prepared scaffolds were detected and analyzed.2.To evaluate the osteogenic ability of the scaffolds by in vitro experiments,culture Sprague-Dawley(SD)rat bone marrow mesenchymal stem cells(rBMSCs)on the scaffolds or using scaffold extracts solution to detect and evaluate cytotoxicity,cell proliferation,crawling,cytoskeleton,osteogenic-specific gene expression levels and osteogenic differentiation-inducing abilities.At the same time,human umbilical vein endothelial cells(HUVECs)were cultured with the extracts solution of the four scaffolds,and the tube formation of HUVECs induced by the extracts solution of the four scaffolds was observed to evaluate the angiogenesis potential of the four scaffolds.3.The effect of bone repair was evaluated by implanting four kinds of scaffolds in the SD rat calvarial bone defect model.At the same time,a blank control group for bone defect was established.At 1 month and 2 months after the operation,blood was drawn from the right atrial appendage of the rats,test the blood,liver function,kidney function and Mg2+ ion content in serum of rats;After blood collection,the left ventricle was perfused with normal saline,and the heart,liver,spleen,kidney,lung,skull including the bone defect area,and brain were collected,and they were all fixed by immersion in 10%neutral formalin for 1 week.Pathological sections were used to observe the toxic effects of scaffolds on various organs.Micro-CT scanning was used to observe the integration of the scaffolds and the edge of the bone defect in the skull.After decalcification with EDTA,the pathological sections were stained with HE and Masson to observe regenerated new bone.Results:1.Four kinds of whiskers,pure mHAw,Mg-doped mHAw,Sr-doped mHAw,and Mg-Sr co-doped mHAw were successfully synthesized,and four types of target porous biomimetic scaffolds were fabricated:mHAw-based SiO2 complex enhanced porous scaffold(SHA),Mg-doped mHAw-based SiO2 complex enhanced porous scaffold(SMHA),Sr-doped mHAw-based SiO2 complex enhanced porous scaffold(SSHA),and Mg-Sr co-doped mHAw-based SiO2 complex enhanced porous scaffold(SMSHA),by extrusion molding,then soaked with silica gel and sintered in a muffle furnace.The hydrophilicity of SHA is the best(WCA 5°),while the hydrophilicity of SMHA is poor(WCA 8°);SMHA has the smallest pore size(247.40± 23.66 μm),while SSHA has the largest pore size(286.20± 19.04 μm);The Young’s modulus of SHA is the smallest(122.43 ± 28.79 MPa),the Young’s modulus of SSHA is the largest(188.44± 47.89 MPa);the compressive strength of SHA is the smallest(1.72 ± 0.29 MPa),and the compressive strength of SMHA is the largest(2.47± 0.25 MPa).The surface micromorphology is similar to cancellous bone and rough,mapping showed that the osteogenic active elements Si,Mg and Sr were all uniformly distributed and could be continuously released from the scaffold.2.The four scaffolds of SHA,SMHA,SSHA and SMSHA have good cell compatibility.SMHA,SSHA and SMSHA were better than SHA in terms of cell survival,cell proliferation,ALP expression,effects on cell morphology,and osteogenic and angiogenesis gene expression.In contrast,SMSHA had the highest in vitro osteoconductivity,the best comprehensive osteoinductive properties,and the strongest ability to promote the osteogenic differentiation of rBMSCs.However,the results of HUVECs tube formation assay showed that SMHA had the best angiogenesis potential.3.The in vivo evaluation results showed that the four scaffolds had good blood,tissue and organ safety,and blood cells,liver functions,and renal functions were all within the normal range compared with the control group.The pathological staining results of the brain,heart,liver,spleen,lung and kidney were similar to those in the control group,and there were no obvious abnormal pathological changes.The SD rat calvarial defects area was well "integrated" with the scaffold,and each scaffold was well integrated with the periphery of the bone defect.Pathological sections showed that each scaffold induced new bone formation to varying degrees.Conclusion:These composite porous scaffolds not only possess acceptable physical and chemical properties suitable for BTE applications,showed good biocompatibility and biosafety both in vivo and in vitro experiments,but also demonstrate the high osteogenic bioactivity of SMSHA in vitro experiments,in vivo experiments confirmed that each scaffold has no major organ targeting toxicity,and the scaffold is closely integrated with bone tissue,which can be used as a potential bone defect filling material.However,the degradation rates of SiO2 and mHAw still need to be further understand,and the degradation of bone repair materials should match the rate of bone regeneration.