Study Of Osseointegration Effects And Mechanisms Of Novel Metallic Bone Implants

Objectives:Biomedical stainless steel and titanium are the most widely used metallic bone implant material in clinical application,and biodegradable medical magnesium has attracted much attention in recent years for the potential uses in bone internal fixation.However,the traditional medical stainless steel will release harmful nickel ions in vivo,and titanium and titanium alloy have poor osteogenic activity and antimicrobial effect.In addition,pure magnesium is confronted with the problems such as fast degradation rate and unfavourable biosecurity in vivo.These questions are the key factors that influence the clinical application of these materials.Therefore,this paper focus on three kinds of novel metallic bone implant materials:high nitrogen nickel-free stainless steel?HNS?,zinc and magnesium ions co-implanted titanium?Zn/Mg-PIII?and medical high-purity magnesium screw.The effects of the novel HNS in inducing osteogenic differentiation of bone marrow mesenchymal stem cells?BMSCs?and in promoting long-term osseointegration and the related mechanisms were all deeply discussed.Furthermore,the multiple functions of Zn/Mg-PIII with osteoinductivity,angiogeneic activity and antibacterial effect and the involved mechanisms were comprehensively investigated.In addition,the long-term degradation characteristics,effect of bone repair and biosecurity in vivo of high purity magnesium screw were studied systematically.The present study may provide important scientific basis for the development,evaluation and application of the three kinds of novel bone implant materials.Materials and Methods:Characterization of materials such as surface morphology,chemical composition and ions release were examined via SEM?EDS?XPS?XRD and ICP.Cell adhesion,spreading and viability of rBMSCs were measured by fluorescence staining and Cell Counting Kit-8respectively.The effects of the novel HNS and Zn/Mg-PIII in inducing osteogenic differentiation of rBMSCs and the related mechanisms were investigated by real-time PCR analysis,western blot analysis and immunofluorescence detection.To discuss the in vivo osteogenesis and osseointegration effect,the novel HNS implants,Zn/Mg-PIII implants and high-purity magnesium screws were implanted into rabbit tibia and femurs,and then various methods such as sequential fluorescently labeling,Micro-CT assay,histological observation,histomorphometric analysis and biomechanical test were all used.Results:?1?Compared with 316L SS,the novel HNS has a higher nitrogen content?0.93wt%?and manganese content?16.70wt%?,and has lower corrosion current density and water contact angle.?2?The novel HNS could promote cellular adhesion,spreading and proliferation of rBMSCs via the upregulation of the gene expression of integrin?1 and integrin?1,and could enhance the expression levels of osteoblast-related genes?ALP,RUNX-2,OPN and OCN?and bone marker proteins?ALP and OCN?via the activating the phosphorylation of ERK 1/2 protein.?3?More obvious deposited apatite particles and much more newly formed bone tissue were observed on HNS implant surfaces than on 316L SS surfaces.After implantation for 4w,12w and 26w,bone-implant contact?BIC?values and bonding strength at the bone-implant interfaces for HNS group were significantly higher than that of 316 SS group?p<0.05?,increasing with time.?4?The Zn/Mg-PIII surfaces were found to promote initial adhesion activity and proliferation of rBMSCs via the upregulation of the gene expression of integrin?1 and integrin?1;Zn/Mg-PIII increased Zn²⁺and Mg²⁺concentrations in rBMSCs by upregulating the gene expressions of ZIP1 and MagT1 and by inhibiting the expressions of ZnT1,and then enhanced osteogenic differentiation related gene?Runx2,ALP,OPN and OCN?.?5?Mg²⁺ions from Zn/Mg-PIII increased Mg²⁺influx by promoting the expression of MagT1 transporter in human umbilical vein endothelial cells?HUVECs?,and then stimulated the transcription of VEGF and KDR via activation of hypoxia inducing factor?HIF?-1?.In addition,it was discovered that zinc in Zn/Mg-PIII had a certain inhibition effects on oral anaerobic bacteria?Pg,Fn and Sm?.?6?The bone volume around Zn/Mg-PIII implant was significantly more than that around other three kinds of implants.After implantation for 12 weeks,both the percentage of BIC and bonding strength for Zn/Mg-PIII implants are statistically higher than those of the other three kind of implants?p<0.05?,increasing with time;?7?After the novel medical high-purity magnesium screws were implanted in vivo,their volumes were slowly reduced over time,and the percent of remaining material at week 26was 73.57%±8.02%.After implantation for 26w,there were obvious hydroxyapatite deposition and new bone formed on high-purity magnesium screws surface,and bone-implant interfaces showed good osseointegration.?8?During the process of long-term?26w?degradation in vivo,the high-purity magnesium screws had not significantly resulted in the increase of magnesium concentration in serum and urine.The levels of alanine aminotransferase,aspartate aminotransferase,creatinine and uric acid in serum were also not significantly increased,and no pathological tissue reaction were observed in kidney and liver tissues.Conclusions:?1?High nitrogen content in newly-developed HNS?Fe-21Cr-16Mn-2Mo-0.93N?enhanced its surface hydrophilicity compared to 316L SS,and facilitated initial adhesion and spreading of rBMSCs via the upregulation of gene expression of integrin?1 and integrin?1.More importantly,Mn²⁺ions released from HNS could induce osteoblast differentiation of rBMSCs via activating ERK 1/2-dependant signaling pathway;?2?In vivo,HNS promoted new bone formation,bone-implant contact and bonding strength at the bone-implant interfaces,especially with significant advantages in early stability and long-term osseointegration of implants;?3?The newly-developed Zn/Mg-PIII can facilitate the initial adhesion,spreading and proliferation of rBMSCs,and also can increase Zn²⁺and Mg²⁺concentrations in rBMSCs by regulating the expression of zinc transporters?ZIP1 and ZnT1?and magnesium transporters?MagT1?.As a result,the osteogenic differentiation of rBMSCs is induced by the synergistic effects of the increased Zn²⁺and Mg²⁺;?4?Mg²⁺ions released from Zn/Mg-PIII increase Mg²⁺influx by promoting the expression of MagT1 in HUVECs,and then activate HIF-1?which is an essential regulatory factor of VEGF,thus enhancing angiogenesis.In addition,thanks to the antibacterial ability of zinc,Zn/Mg-PIII has a certain inhibiting effects on the growth of oral anaerobes?Pg,Fn and Sm?;?5?In vivo,the osteogenesis,angiogenesis,osseointegration and the bonding strength at bone-Zn/Mg-PIII implant interfaces are all significantly enhanced;?6?New medical high-purity magnesium screws shows slowly long-term degradation behavior and good osteogenesis and osseointegration abilities in vivo,and its degradation products do not cause adverse systemic reactions,showing excellent in vivo biological safety.