新型低弹性模量钛合金TiNbZrTaSi生物相容性及骨整合能力实验研究

发布时间：2018-05-03 22:11

本文选题：d电子合金设计 + 放电等离子烧结　；参考：《吉林大学》2016年博士论文

【摘要】：目的:钛合金的生物相容性优良,是目前比较通用的生物医用金属材料。但在应用过程中依然存在弹性模量相对较高和植入体与骨组织结合力的问题。植入体与骨组织弹性模量的差异会造成界面应力传导不良,影响周围骨组织的再生、改建,降低骨密度和骨强度,引发骨植入体松动甚至失效。华南理工大学开发了一种名义成分为(Ti-35Nb-7Zr-5Ta)98Si2的新型β型钛合金,该合金采用d电子合金设计理论设计,放电等离子烧结法(SPS)制造,具备低弹性模量(37GPa)、高强度、高塑性以及超细晶结构。其中,Ti-35Nb-7Zr-5Ta合金体系已经被证实具有良好的生物相容性和较低的弹性模量,而硅(Si)元素具有增加非晶形成和晶粒细化的功能,同时也具有良好的相容性。(Ti-35Nb-7Zr-5Ta)98Si2合金的弹性模量远低于Ti-6Al-4V且更接近人骨。本研究旨在通过评价该合金的耐蚀性、生物相容性、生物活性以及骨整合能力,探讨其生物安全性及骨科应用价值,为其应用到临床提供一定的理论依据。方法:(1)耐腐蚀性评价:将合金试样在生理盐水中浸泡1d、7d、14d、30d、60d,分别比较浸泡过程中生理盐水的PH值变化、浸泡前后合金试样的重量变化和扫描电镜下表面形貌改变,以探究其耐腐蚀性能。(2)生物相容性评价:制备(Ti-35Nb-7Zr-5Ta)98Si2合金浸提培养液,用浸提液培养L-929小鼠成纤维细胞,在培养1d、2d、3d、5d、7d时通过光镜观察细胞生长状态并行MTT法细胞毒实验,结合医用材料细胞毒性评价标准对合金细胞毒性进行分析;受试小鼠腹腔注射受试材料生理盐水浸提液,观察小鼠注射后24h、48h、72h的状态,参照全身毒性反应评价标准分析合金的急性全身毒性;将合金试样直接敷贴于受试实验动物皮肤或在皮内注射合金浸提液,观察局部反应,以评价试样皮肤/皮内刺激性;合金试样植入实验动物骨内,3个月后取出试样及其周围骨组织,硬组织切片、HE染色,显微镜观察,评价材料植入骨组织后所引起的局部反应。(3)生物活性评价:小鼠前成骨细胞MC3T3-E1与(Ti-35Nb-7Zr-5Ta)98Si2合金试样共同培养,分别于1h、3h、6h进行吖啶橙染色,显微镜下计数表面粘附细胞并与Ti-6Al-4V合金对比,评价新合金对成骨细胞粘附的影响;对与成骨细胞共培养24h的(Ti-35Nb-7Zr-5Ta)98Si2合金表面喷金,扫描电镜下观察粘附细胞的形态;Annexin V-FITC法染色成骨细胞,流式细胞仪检测细胞的凋亡率;ELISA法检测成骨细胞白细胞介素-6/碱性磷酸酶的分泌量,评价细胞炎性反应及(Ti-35Nb-7Zr-5Ta)98Si2合金对成骨细胞早期分化的影响;MC3T3-E1细胞与(Ti-35Nb-7Zr-5Ta)98Si2合金共培养30d,茜素红染色细胞外基质矿化结节,评价新合金对成骨细胞晚期分化及细胞外基质矿化的影响。(4)骨整合能力评价:选择6只健康成年新西兰大白兔作为受试动物,体重2.5~3kg,每只受试动物双侧股骨分别植入Ti-6Al-4V和(Ti-35Nb-7Zr-5Ta)98Si2合金,每侧股骨植入3个。术后3个月处理受试动物。肉眼及X线检查材料-骨界面及周围组织的情况;取出带有植入材料的动物股骨行硬组织切片,并进行Masson染色及甲苯胺蓝染色,计算骨结合率(BCR);进行力学推出实验,以最大推出力评价植入材料与骨组织的结合强度。结果:(1)材料在生理盐水中浸泡1d、7d、14d、30d、60d,各时间点检测溶液PH值,未见明显变化;浸泡后合金试样的重量没有明显减小;通过扫描电镜比较合金表面浸泡前和浸泡后的形貌,表面光洁度相似,未发现腐蚀痕迹,结果表明(Ti-35Nb-7Zr-5Ta)98Si2合金的耐腐蚀性能良好。(2)小鼠成纤维细胞L-929在合金浸提液中生长状态良好,没有发现明显细胞毒性,细胞形态与普通培养基培养的细胞无差异;MTT检测结果证实(Ti-35Nb-7Zr-5Ta)98Si2合金的细胞毒性在0~Ⅰ级,符合生物安全标准;腹腔注射受试材料浸提液的小鼠未出现腹膜刺激症状、呼吸抑制、运动减少、发绀、腹泻、震颤及死亡等反应,证明新合金不会引起全身毒性;表面敷贴合金试样和皮内注射浸提液的实验动物未出现局部不良反应,证实新合金对皮肤及皮内无刺激性;骨植入后切片观察,合金植入体周围未发生骨组织的坏死、炎症等副反应。(3)吖啶橙染色结果表明(Ti-35Nb-7Zr-5Ta)98Si2合金表面粘附细胞未出现坏死或凋亡,表面粘附细胞计数与Ti-6Al-4V合金相近,二者无显著差异;扫描电镜观察材料表面显示成骨细胞在新合金表面生长状态良好;细胞凋亡实验表明(Ti-35Nb-7Zr-5Ta)98Si2合金组的细胞凋亡率与Ti-6Al-4V及阴性组无明显差别;成骨细胞白细胞介素-6和碱性磷酸酶的分泌量未见明显增高;成骨细胞与受试材料共培养30d后,(Ti-35Nb-7Zr-5Ta)98Si2组细胞外基质的矿化程度与Ti-6Al-4V组相同,二者均明显高于阴性对照组。(4)合金试样植入骨中3个月后肉眼观察:植入物周围无明显红肿、化脓及组织坏死;X线摄片示材料周围未见明显透光区,材料与骨组织结合较好,无明显密度减低和炎性改变;硬组织切片染色结果显示植入体与周围骨组织结合良好,(Ti-35Nb-7Zr-5Ta)98Si2组的骨接触率(77.45%)大于Ti-6Al-4V组(73.31%);力学推出实验结果显示(Ti-35Nb-7Zr-5Ta)98Si2的骨结合力(377N)大于Ti-6Al-4V(284N)。结论:(1)新型超细晶低弹钛合金(Ti-35Nb-7Zr-5Ta)98Si2的生物相容性良好,没有细胞毒性和组织排斥反应,是一种安全性良好的生物医学合金。(2)该合金具耐腐蚀性良好、生物活性较高,骨整合能力优秀,是有很好应用价值的生物医学植入材料。
[Abstract]:Objective: titanium alloy has good biocompatibility and is a common biomedical metal material at present. However, there is still a relatively high elastic modulus and the binding force of the implant and bone in the application process. The difference in the elastic modulus of the implant and bone tissue will cause the poor interfacial stress conduction and influence the regeneration of the surrounding bone tissue. Remodeling, reducing bone density and bone strength, causing bone implants loosening and even failure. A new type of beta titanium alloy with nominal component (Ti-35Nb-7Zr-5Ta) 98Si2 was developed by South China University of Technology. The alloy was designed by D Electronic alloy design theory and produced by discharge plasma sintering (SPS) with low modulus of elasticity (37GPa), high strength and high plasticity And the superfine crystal structure. Among them, the Ti-35Nb-7Zr-5Ta alloy system has been proved to have good biocompatibility and low modulus of elasticity, while silicon (Si) elements have the function of increasing amorphous formation and grain refinement, and also have good compatibility. (Ti-35Nb-7Zr-5Ta) the modulus of elasticity of (Ti-35Nb-7Zr-5Ta) 98Si2 alloy is much lower than that of Ti-6Al-4V. Human bone. The purpose of this study was to evaluate the corrosion resistance, biocompatibility, bioactivity and bone integration of the alloy, and to explore its biological safety and the application value of the Department of orthopedics. Methods: (1) corrosion resistance evaluation: the alloy samples were soaked in 1D, 7d, 14d, 30d, 60d in normal saline. The change of pH value of physiological saline during soaking process, the weight change of the alloy sample before and after immersion and the change of surface morphology under scanning electron microscope, in order to explore its corrosion resistance. (2) biocompatibility evaluation: preparation (Ti-35Nb-7Zr-5Ta) 98Si2 alloy extract culture liquid, L-929 mouse fibroblast culture by leaching solution, through the culture of 1D, 2D, 3D, 5D, 7d. The cell growth state of the cells was observed by light microscopy, and the cytotoxicity of the alloying cells was analyzed by MTT method. The toxicity of the alloying cells was analyzed with the standard of cytotoxicity evaluation of medical materials. The experimental mice were injected with saline solution by intraperitoneal injection to observe the state of 24h, 48h, and 72h after injection of the mice. The acute whole body of the alloy was analyzed according to the total body toxicity evaluation criteria. Toxicity; the alloy specimens were directly applied to the experimental animal skin or intradermal injection of alloy extract, and the local reaction was observed to evaluate the irritation of the skin / skin. The alloy specimens were implanted in the experimental animal bone, and the specimens and their surrounding bone tissues were removed after 3 months, and the hard tissue section, HE staining, microscope observation, and the evaluation of the implant bone group were used to evaluate the implant bone group. The local reaction after weaving. (3) Bioactivity Evaluation: mouse osteoblast MC3T3-E1 and (Ti-35Nb-7Zr-5Ta) 98Si2 alloy specimens were co cultured, 1H, 3h, 6h were stained with acridine orange respectively, the surface adhesion cells were counted under microscope and compared with Ti-6Al-4V alloy, and the effect of the new alloy on osteoblast adhesion was evaluated; and the osteoblasts were compared with the osteoblasts. A total of 24h (Ti-35Nb-7Zr-5Ta) 98Si2 alloy was sprayed on the surface of 98Si2 alloy, and the morphology of adhesion cells was observed under scanning electron microscope; Annexin V-FITC staining osteoblasts and flow cytometry were used to detect the apoptosis rate of cells; ELISA method was used to detect the secretion of interleukin -6/ alkaline phosphatase in osteoblasts, and to evaluate the inflammatory response and 98Si2 coincidence (Ti-35Nb-7Zr-5Ta). The effect of gold on the early differentiation of osteoblast; MC3T3-E1 cells and (Ti-35Nb-7Zr-5Ta) 98Si2 alloy co culture 30d, alizarin red staining of extracellular matrix mineralized nodules, evaluation of the effect of new alloy on the late differentiation of osteoblasts and extracellular matrix mineralization. (4) evaluation of bone integration ability: selected 6 healthy adult New Zealand rabbits as subjects Body weight 2.5~3kg, Ti-6Al-4V and (Ti-35Nb-7Zr-5Ta) 98Si2 alloy were implanted in each femur of each animal, and 3 were implanted in each side of the femur. 3 months after the operation, the animals were treated with the naked eye and X-ray material - the bone interface and the surrounding tissue; the animal femur with the implanted material was taken out of the hard tissue section and Masson staining was performed. The bone binding rate (BCR) was calculated by toluidine blue (1). Results: (1) the material was soaked in 1D, 7d, 14d, 30d, 60d in normal saline, and the pH value of the solution was not significantly changed at all time points; the weight of the alloy samples after soaking was not significantly reduced; through sweep, the weight of the alloy was not significantly reduced. The morphology of the alloy before and after immersion was compared with the surface of the alloy, and the surface finish was similar, and the corrosion resistance was not found. The results showed that the corrosion resistance of (Ti-35Nb-7Zr-5Ta) 98Si2 alloy was good. (2) the L-929 of mouse fibroblasts grew well in the alloy extract, and no obvious cytotoxicity was found, and the cell morphology and ordinary medium were found. There was no difference in cultured cells; MTT test results confirmed that the cytotoxicity of (Ti-35Nb-7Zr-5Ta) 98Si2 alloy was at the level of 0~ I, which accorded with the biosafety standard; the mice intraperitoneally injected with the tested material did not have peritoneum stimulation symptoms, respiratory depression, motor decrease, cyanosis, diarrhoea, tremor and death, which proved that the new alloy did not cause systemic toxicity. There was no local adverse reaction in the experimental animals with surface application and intradermal injection, which proved that the new alloy had no irritation to the skin and skin. After the bone implantation, the bone tissue was not necrotic, and the inflammation and other side effects were not occurring around the alloyed implant. (3) the acridine orange staining results showed the surface of (Ti-35Nb-7Zr-5Ta) 98Si2 alloy. The adhesion cells were not necrotic or apoptotic, and the number of surface adhesion cells was similar to that of Ti-6Al-4V alloy, and there was no significant difference between the two ones. The surface of the osteoblasts showed good growth on the surface of the new alloy by scanning electron microscope. The apoptosis experiment showed that the apoptosis rate of the group of (Ti-35Nb-7Zr-5Ta) 98Si2 gold group was not obvious to the Ti-6Al-4V and negative group. The secretion of interleukin -6 and alkaline phosphatase in osteoblasts was not significantly increased; after the osteoblasts were co cultured with the tested material for 30d, the mineralization degree of the extracellular matrix in (Ti-35Nb-7Zr-5Ta) 98Si2 group was the same as that in the Ti-6Al-4V group, and the two were significantly higher than that in the negative control group. (4) the alloy specimens were implanted in the bone for 3 months after implantation. There was no obvious redness, suppurative and tissue necrosis around the material, and no obvious light transmittance was found around the material. The material and bone tissue were well combined with no obvious density reduction and inflammatory change. The results of hard tissue section staining showed that the implant was well combined with the surrounding bone tissue, and the bone contact rate (77.45%) in group 98Si2 (77.45%) was greater than Ti-6Al-4V Group (73.31%); the experimental results showed that the bone resultant force (377N) of (Ti-35Nb-7Zr-5Ta) 98Si2 was greater than Ti-6Al-4V (284N). Conclusion: (1) the biocompatibility of the new ultra-fine grain low elastic titanium alloy (Ti-35Nb-7Zr-5Ta) 98Si2 is good, there is no cytotoxicity and tissue exclusion reaction, and it is a good biomedical alloy with good safety. (2) the alloy has a good resistance. It has good corrosion resistance, high bioactivity and excellent osseointegration ability. It is a biomedical implant material with good application value.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08;R68

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