| Background Recently,porous titanium scaffolds with distinguished mechanical properties,less stress shielding,and better new bone ingrowth has received considerable attention.Porous titanium scaffolds with controllable geometry,pore size,and porosity are the materials of choice for use as substitutes for bone repair.Supply of nutrients and oxygen from blood vessels is of vital importance in osteogenesis and survival of porous titanium scaffolds.New blood vessel formation or angiogenesis is required for the scaffold to be functional.Although there has been considerable development of surface modifications of porous titanium scaffolds,insufficient bone growth and blood vessel ingrowth into the porous titanium scaffold have limited their further application.Therefore,a biofunctional coating with both osteogenesis and angiogenesis properties is required for porous titanium scaffold long term fixation and survival which are crucial for their clinical applications.Magnesium metal is a degradable material,and magnesium ions play an important role in human bone metabolism.In recent years,a large number of studies have reported that magnesium-based alloys have better biological activity compared with titanium alloys,and can promote new bone growth in fracture repair and bone defect reconstruction.However,due to the disadvantages such as rapid degradation of magnesium-based alloys,poor corrosion resistance,and lack of mechanical properties after degradation,the use of magnesium-based alloys as an implant material in orthopaedics is limited.In recent years,there have been many studies focusing on how to improve the corrosion resistance of magnesium alloy materials.However,the direct preparation of magnesium metal as a biodegradable coating on the surface of Ti6Al4 V scaffold has not been studied yet.The biological activity,osteogenic effect,and vascularization ability of magnesium metal coatings need to be further explored and studied.Methods: 1.The porous Ti6Al4 V scaffold was prepared by electron beam melting technology(EBM),and the magnesium coating was prepared on the inner and outer surfaces of the porous Ti6Al4 V scaffold material by pulsed bias arc ion plating.SEM was applied to observe the morphology of magnesium coating on the surface of the porous Ti6Al4 V scaffold,and the elemental composition was determined by X-ray energy spectrum analysis.2.MC3T3-E1 cells were used to study the in vitro osteogenic activity of the magnesium coated porous Ti6Al4 V scaffold.Human umbilical vein endothelial cells(HUVEC)were used to examine the in vitro pro-angiogenic activity of the magnesium coated porous Ti6Al4 V scaffold.The MC3T3-E1 and HUVEC cells were incubated with the extraction of the magnesium coated porous Ti6Al4 V scaffold to measure the cell biocompatibility.The alamar Blue colorimetric assay was used to detect the proliferation of MC3T3-E1 and HUVEC cells;the Calcein-AM/PI double staining kit was used to detect the viability of MC3T3-E1 and HUVEC cells.CLSM was applied to observe the adhesion and extension of the MC3T3-E1 and HUVEC cells.Alkaline phosphatase staining,alizarin red calcium nodule staining,real-time quantitative PCR were used to detect the osteogenesis of MC3T3-E1 cultured with the extraction of magnesium coated porous Ti6Al4 V scaffold.The vascularization-promoting effect of bioactive magnesium coated porous Ti6Al4 V scaffoldextraction on HUVEC was detected by wound healing test,Transwell test,tube formation test and real-time quantitative PCR.3.In vivo experiments: 42 adult New Zealand white rabbit were used to evaluate the long term osteogenic performance and early vascularization performance of bioactive magnesium coated porous Ti6Al4 V scaffold using rabbit femur acetabular bone defect model.The 6th,9th,and 12 th months were selected as time points for Micro-CT and histological staining to analyze the long-term osteogenic effect of bioactive magnesium coated porous Ti6Al4 V scaffold.12 weeks post-operation was seleceted to do the fluorescent labeling experiment.Since calcein,alizarin red and tetracycline could not remain in the experimental animals for a long period of time.For the study of early vascularization,microangiography and Micro-CT were performed at the 2 4,and 8 week post-operations.Results: 1.Scanning electron microscopy results showed that regular,uniform and dense magnesium grains were distributed on the inside and outside surface of the porous Ti6Al4 V scaffold,which was well combined with the surface of the material.The results of energy spectrum analysis showed that single-phase magnesium coating existed on the surface of the porous Ti6Al4 V scaffold.2.The results of Alamar Blue colorimetric assay showed that the bioactive magnesium coated porous Ti6Al4 V scaffold could promote the proliferation of MC3T3-E1 cells and HUVEC cells on the 4,7,and 14 day of cell culture compared with bare porous Ti6Al4 V scaffold(p <0.05);CLSM results showed that the expression of vinculin and the CN ratio in MC3T3-E1 cells and HUVEC cells co-cultured with the bioactive magnesium coated porous Ti6Al4 V scaffold extraction was significantly higher(p < 0.05),and the CN ratio were significantly higher than bare porous Ti6Al4 V scaffold(p < 0.05);bioactive magnesium coated porous Ti6Al4 V scaffold could promote the alkaline phosphatase activity and the formation of extracellular matrix mineralization and calcium nodules of MC3T3-E1 cells;bioactive magnesium coated porous Ti6Al4 V scaffold could promote the migration,invasion and tubule formation of HUVEC cells.Real-time quantitative PCR results show that magnesium ions could promote the expression of osteogenic genes ALP,Col-1,OPN,OCN and angiogenesis-related genes VEGF,HIF-1?;3.Micro-CT results showed that BV/TV of the bioactive magnesium coated porous Ti6Al4 V scaffold was significantly higher at the 6,9,and 12 month compared with bare porous Ti6Al4 V scaffold(p < 0.05);The result showed that the fluorescence intensity of the bioactive magnesium coated porous Ti6Al4 V scaffold was much higher at 12 weeks than bare porous Ti6Al4 V scaffold(p < 0.05).Similar results to Micro-CT tests in 6,9 and 12 month were observed in histological examination of VG staining.The BV/TV of bioactive magnesium coated porous Ti6Al4 V scaffold group was significantly higher(p < 0.05),and had better bone tissue and osseointegration.Microvascular perfusion imaging revealed that BVV/TV value in the bioactive magnesium coated porous Ti6Al4 V scaffold(21.2±3.2%)was significantly higher than that in the bare porous Ti6Al4 V scaffold(10.9±1.8)at 2 weeks after implantation(p <0.05).At 4 weeks,the BVV / TV value of the bioactive magnesium coated porous Ti6Al4 V scaffold was 35.3 ± 4.5%,which was significantly higher than that of the bare porous Ti6Al4 V scaffold group(15.5 ± 3.4%,p <0.05).Similar results were observed after 8 weeks of implantation.Conclusions: 1.A porous Ti6Al4 V scaffold with excellent porosity,pore size and other parameters was prepared successfully,and the bioactive magnesium coating on porous Ti6Al4 V scaffold surface was fabricated successfully using multi-arc ion plating method which were verified using scanning electron microscopy(SEM)and X-ray energy spectrum analysis.The method produces a tightly bound high purity magnesium metal coating.2.In vitro studies confirmed that the bioactive magnesium coated porous Ti6Al4 V scaffold can promote the proliferation,adhesion and extension of MC3T3-E1 cells and HUVEC cells.It could promote the migration,invasion,and tubule formation of HUVEC cells,and promoted MC3T3-E1 cells osteogenic differentiation and vascularization of HUVEC cells.3.Through in vivo studies,it was confirmed that the bioactive magnesium coated porous Ti6Al4V scaffold had a good ability to promote new bone formation,and osseointegration.At the same time,the bioactive magnesium coated porous Ti6Al4 V scaffold promoted vascularization of the surrounding tissue and provided sufficient nutrition for new bone formation inside the scaffold material. |
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