Improving Osteointegration And Osteogenesis Of Three-Dimensional Porous Ti6Al4V Scaffolds By Polydopamine-Assisted Magnetic Nanoparticles Coating

Background:The excellent biocompatibility and anti-corrosion property has made titanium(Ti)become the promising material for load-bearing and bone-contacting medical substitution in clinical practice.The titanium implants can be placed into a defect site to treat bone defects caused by trauma or tumors.The good interfacial interactions of the bone-implant interface is a critical factor for the successfully biomechanical stability.However,precisely because of the bio-inert,it makes the poor ability of the implant to neighbor bone cells adhere and anchor together.Thus,the appropriate surface-modified titanium implants with high osteoconduction and osteoinduction is increasingly desired for bone regeneration.In recent years,a facile and versatile surface modification technique based on mussel-inspired polymerization of dopamine(PDA)at alkaline pHs on various substrates is attracting considerable attention and has been applied to the surface modification of titanium orthopedic implants.In the previous research group,gelatin magnetic nanoparticles were used to perfuse porous titanium alloy scaffolds,and then hybrid magnetic titanium alloy scaffolds were produced by freeze-drying techniques.The magnetic hybrid scaffolds have been significantly improved by in vitro and in vivo experiments of cell adhesion,proliferation and osteogenic differentiation.However,the magnetic hybrid scaffold prepared by the freeze-drying technique is not stable,and the gelatin will degrade and absorb soon after it enters the body,and the magnetic nanoparticles inside will fall off.In order to further improve the preparation of magnetic hybrid scaffolds,we chose dopamine as a binding agent onto the Ti substrate to build a bridge between the magnetic Fe304 particles and metal matrix.In the past,there was no research on the polyaniline-assisted magnetic nanoparticle-modified titanium metal.Therefore,we first designed a solid titanium plate,and then combined the material and medical theories and methods,using the rapid prototyping technology to precisely control the structural characteristics of the material.To improve the microscopic mechanical properties of materials;to prepare nano magnetic particle coatings by using poly dopamine assisted technology,so that titanium alloy stents have a certain magnetic properties,enhance the osteoinduction of the material,and apply a certain strength of static magnetic field outside the material,making The magnetized titanium alloy stent produces a synergistic effect,promotes the osteogenic effect of the material,and finally improves the biological adaptability of the porous titanium bone repair material.The relationship between the structural design of the porous titanium scaffold,functional modification of the interface,and the biological adaptability of the implant is further explored and elucidated,providing an important basis for the development of new artificial bone repair materials.This is of great significance in improving the level of research and development of bone tissue biomedical materials and achieving more ideal bone defect repair.Objective:To construct a uniform and stable coating with paramagnetic response on the surface and inter of three-dimensional porous Ti6A14V scaffolds by using the poly dopamine-assisted coating technology,and to impart the ability to regulate cell proliferation and differentiation on the surface inter of three-dimensional porous Ti6A14V scaffolds.Methods:Titanium samples were prepared by cutting cylindrical rods(15 mm in diameter and 10cm in length)of pure titanium.The Fe304 nanoparticles(20 mg)and 3,4-Dihydroxyphenethylamine hydrochloride(dopamine hydrochloride)were dispersed in the Tris buffer solution(10 mM,pH 8.5,100 mL)to 2 mg/mL.Porous Ti6A14V scaffolds(pTi)were fabricated by an EBM system.The SEM,EDS,EDS mapping,CAG,AFM were used to surface characterization.The cytotoxicity of the samples was assayed by the LIVE/DEAD kit for mammalian cells.Cell proliferation was measured by cell count kit-8(CCK-8)assay.SEM was employed when cells had attached on different samples at 2 days.The cells were stained by rhodamine-phalloidin of filamentous actin(F-actin),the nuclei were counterstained with 4',6 diamidino-2-phenylindole(DAPI).The ALP activity after 7,10,14 days of incubation were examined to evaluate the bone differentiation activity of surface modified titanium substrates.The mRNA expression levels of ALP,COL-1,OPN,OCN and Runx2,which could reflect the grade of osteogenic differentiation of hBMSCs were quantitatively determined by quantitative RT-PCR tests at 14 days.The status of a fraction of cells grown at the bottom of the culture dish was visualized,by means of ALP and alizarin red S(ARS)staining.18 New Zealand rabbits were randomly divided into three groups:pTi,PDA@pTi and Fe304/PDA@pTi group,preparing models of lateral femoral epicondyle bone defects and place implants.Observing the postoperative general condition of the animals,and at postoperative 6 weeks and 12 weeks,using micro-CT in detection of bone ingrowth into the scaffolds and hard tissue slicing with VG staining to observe the ability and efficacy of repair of rat lateral femoral epicondyle bone defects.Results:The EDS data analysis showed that PDA and Fe304 nanoparticles are successfully immobilized on the surfaces of pTi through a one-pot surface immobilization strategy.The images observed by EDS elemental mapping indicate that Ti,C,N,O and particularly Fe are uniformly distributed on the three-dimensional porous Ti6A14V scaffolds.Small particles appeared on the three-dimensional porous Ti6A14V scaffolds coated with PDA/Fe304 nanoparticles observed by SEM.Fe304 nanoparticles coating significantly increased the surface roughness.The Fe304 nanoparticles coating samples exhibited saturation magnetization of 0.398 emu/g,indicating the Fe304 nanoparticles has been incorporated successfully on the three-dimensional porous Ti6A14V scaffolds.The LIVE/DEAD(?)viability/cytotoxicity kit was used to qualitatively assay the cytotoxicity of the different three-dimensional porous Ti6A14V scaffolds.A larger number of live and spindle cells with obvious cell-cell junction and formed network link can be observed from the PDA@pTi and Fe304/PDA@pTi samples.A small number of round cells hardly any intercellular connection can be noticed on the surface of pTi substrates.The group of Fe304/PDA@pTi showed significantly greater cell proliferation than the group of pTi and PDA@pTi according to CCK-8 assay.The fluorescence intensity and quantity of F-actin on the group of Fe304/PDA@pTi was best compared with those on pTi and PDA@pTi groups.The mitosis of hBMSCs cells was activity and the number of cells increased over time.The quantities of lamellipodia extensions between the adjacent cells on the surface of Fe304/PDA@pTi groups is significantly more than the other two groups.The ALP activity in Fe304/PDA@pTi groups was much better than the other two groups.Compared with the unmodified pTi groups,the Fe304/PDA@pTi groups showed significantly higher gene expression of ALP,COL-1,OPN,OCN and Runx2 in hBMSCs after 14days of osteogenic induction culture.However,the difference for ALP,OCN,and Runx2 gene expressions between the PDA@pTi and pTi groups has no statistical significance.The results of ALP and alizarin red S(ARS)staining show that the three-dimensional porous Ti6A14V scaffolds promote osteogenic differentiation of hBMSCs.At 6 weeks postoperatively,there was a small amount of bone ingrowth in the Fe304/PDA@pTi group,and rare bone ingrowth was observed in the pTi group and the PDA@pTi group.After 12 weeks of surgery,the Fe304/PDA@pTi group had a large number of bone ingrowth inside of scaffolds,and new bones cling to the scaffold,the bones are tightly bound to the stent,and more vascular tissue formation can be observed.In the pTi group,there was only a small amount of bone ingrowth,and the bone was not tightly bound to the stent and there was less vascular tissue production.At each detection time point,the Fe304/PDA@pTi+SMF group had better adhesion and proliferation capacity to hHBMSCs than Fe304/PDA@pTi,and cell migration was more pronounced.LIVE-DEAD kit staining showed that there were more cells in the Fe304/PDA@pTi+SMF group,and the connections between the cells were more abundant,and the Fe304/PDA@pTi+SMF group showed more abundant F-actin when stained with phalloidin and DAPI.Comparing with the results of Fe304/PDA@pTi group in experiment 2,it was found that the OCN,OPN and COL-1 in Fe304/PDA@pTi+SMF group were significantly higher than those in Fe304/PDA@pTi group.ALP,RUNX-2 Also increased,but there no statistically significant.The alizarin red staining of the bottom of the dish revealed that there were more red-calcium nodules in the Fe304/PDA@pTi+SMF group.Alkaline phosphatase staining showed consistent results with alizarin red.In animal experiments,the 6-week postoperative results showed that the amount of bone growth in the Fe304/PDA@pTi+SMF group was greater than Fe304/PDA@pTi without magnetic field,and the results were more obvious at 12 weeks.The bone and the stent were tightly bound together.Conclusion:It was demonstrated that the PDA-assisted Fe304 nanoparticles coating surface immobilization strategy could endow the modified three-dimensional porous Ti6A14V scaffolds with significant osteogenesis activity.It can significantly promote the biological activity and obviously promote the bone cells on the adhesion and proliferation.This study demonstrated the combinatory effects of applying an external SMF with the use of magnetic three-dimensional porous Ti6A14V scaffolds on the osteoblast differentiation and bone formation.