Biosafety And Osseointegration Ability Of New Medical Ultrafine Grainβ-Type Titanium Alloys

On the basis of d-electronic design theory,South China University of Technology combined two new ultrafine-grained β-type titanium alloys prepared by mechanical ball milling and discharge plasma sintering.These alloys have the advantages of low elastic modulus,high mechanical strength,and wear resistance.Therefore,they can be used as a candidate material in the field of biomedicine.As a new implantable material,the alloys’ biological safety must be verified before clinical application.In addition,the difference in osteogenesis between the two titanium alloys and commercial pure titanium should be analyzed.The purpose of this study was to evaluate the biosafety of Ti-Nb-Zr-Ta-Si and Ti-Nb-Zr-Sn alloys and compare the differences in osseointegration and osteogenesis between the two new titanium alloys and pure titanium implants.Clinical applications and options provide experimental support.Method:Biosafety assessment:1.Cytotoxic response test: MG-63 cells were co-cultured with pure titanium and titanium alloy disks.The cell proliferation rate of the different materials was calculated by the CCK-8 method to evaluate the cytotoxicity of the two new titanium alloy materials.2.Acute toxicity test: The extracts of pure titanium and two new titanium alloys were prepared and injected intravenously into mice.The general state and toxicity of the mice were observed and recorded.The evaluation criteria of the reaction experiments were used to evaluate the acute toxicity of the materials.3.Hemolysis experiment: Blood was collected from rabbit ear artery.Anticoagulated rabbit blood was prepared,mixed with titanium alloy extract,centrifuged,and measured with a spectrophotometer to calculate the hemolysis rate of the two new titanium alloys.4.Oral mucosal stimulation experiment: The material sample was sutured and fixed to the mucosa of the cheek pouch on both sides of the golden hamster.The body condition of the golden hamster and the changes in the mucosa in the mouth were closely observed after the operation.The samples were observed by HE staining.Evaluation of osseointegration ability:Pure titanium implants,Ti-Nb-Zr-Sn implants,and Ti-Nb-Zr-Ta-Si implants were implanted into the femur of 27 rabbits,and nine rabbits were sacrificed at 4,8,and 12 weeks after implantation.1.The pull-out test was used to determine the force value of the implants at the moment of detachment,and the osseointegration ability of the three materials at 4,8,and 12 weeks was compared.2.Tetracycline hydrochloride and alizarin red fluorescent stains were injected at 3 weeks and 1 week before the animals were sacrificed,respectively.After the animals were sacrificed,hard tissue sections were obtained,and the fluorescent marker bands were observed by laser scanning confocal microscopy.The distance between the marker bands was measured using graphics software to calculate the bone mineralization deposition rate of titanium and the two alloys.3.Non-decalcified hard tissue sections were stained with methylene blue-acid fuchsin staining solution,observed and recorded under an optical microscope,and measured using graphics processing software to calculate the bone contact rate of the three materials.Result:Biosafety assessment1.Cytotoxic reaction experiment: The effects of the two new alloys on cell proliferation behavior were similar to those of pure titanium,showing no obvious toxicity and no inhibition of cell growth.2.Acute toxicity test: No mice died during the observation period,and no symptoms of poisoning or adverse reactions were observed within 72 h.The weight gain was within the normal range,indicating that the two new alloys resulted in no acute toxicity.3.Hemolysis test: In accordance with national standards,a hemolysis rate of less than 5% is a negative reaction.The hemolysis rates of the two new alloys were 0.8% and 1.3%.Thus,no hemolysis reaction would result from the two new alloys.4.Oral mucosal stimulation experiment: At 2 weeks postoperatively,direct observation of the buccal sac mucosa in contact with the material showed no congestion,erosion,or inflammatory seepage.The HE staining results also showed that the buccal mucosa epithelium was intact,with no epithelial abnormality or excessive proliferation.No inflammatory cell infiltration was observed in connective tissue,and the two new alloys presented no obvious mucosal irritation.Evaluation of osseointegration ability:1.The results of the pull-out experiment showed that the bonding strength of the bone interface of Ti-Nb-Zr-Ta-Si alloy implants at 4 weeks was higher than that of Ti-Nb-Zr-Sn alloy implants and pure titanium implants,with a significant difference(p <0.05).At 8 and 12 weeks,there was no significant difference in the osseointegration strength of the three implant materials.2.At 4 weeks,the bone mineralization deposition rate of Ti-Nb-Zr-Ta-Si alloy implants was significantly higher than that of pure titanium implants(p <0.05)but not significantly different from that of Ti-Nb-Zr-Sn alloy implants.At 8 and 12 weeks,there was no significant difference in the rate of bone mineralization among the three materials.3.Under the light microscope,a small amount of new bone formed around the implants in each group at 4 weeks,and a small amount of fibrous tissue grew into it.At 8 weeks,the bone tissue around the three implants increased,the cavity structure decreased,and the number of trabecular bones increased.At 12 weeks,the implants of the three materials presented more continuous contact with the bone tissue,and the trabecular bone structure was dense.There was no significant difference in the bone contact rates of the three materials at 4,8,and 12 weeks.Conclusion: Both new ultrafine-grained β-type titanium alloys show good biological safety.Compared with pure titanium and Ti-Nb-Zr-Sn alloy,Ti-Nb-Zr-Ta-Si alloy can promote early osteogenesis and obviously increase the early bone mineralization deposition rate and osteointegration strength.