新型骨植入Mg-Zn-Y-Nd-Zr合金的体外降解性能与生物相容性研究
发布时间：2018-09-02 07:15
【摘要】：Mg-Zn-Y-Nd-Zr合金是课题组前期开发的一种新型可降解骨植入用镁合金,通过在合金中添加微量多元化Zn、Y、Nd、Zr元素,并经过挤压加工,镁合金的力学性能大大提高。作为生物医用材料,其生物安全性与生物功能性至关重要,需对其进行全面评价和分析。本论文对Mg-Zn-Y-Nd-Zr合金的体外降解性能及生物相容性进行了系统研究。采用电化学工作站、pH测量、离子浓度检测、失重实验、扫描电子显微镜及能谱分析(SEMEDS)等分析了Mg-Zn-Y-Nd-Zr合金在模拟体液(SBF)、模拟体液+10%胎牛血清(SBF+10%FBS)、DMEM/F12细胞培养基、?-MEM细胞培养基、无酚红MEM细胞培养基五种介质中的降解行为;通过体外细胞培养,采用MTT法、倒置相差显微镜观察等分析了合金对小鼠成纤维细胞(L929)、小鼠颅顶前骨细胞(MC3T3-E1)的细胞毒性、细胞黏附、细胞增殖影响,并通过ALP活性检测、钙化结节染色等研究了合金对骨髓间充质干细胞(BMSCs)向成骨细胞(OBs)诱导分化过程的影响。体外降解实验结果表明,Mg-Zn-Y-Nd-Zr合金在介质中随着时间的延长逐渐发生降解,在SBF中浸泡10 d的平均降解速率为0.0932 mg/(cm2·h),在?-MEM、无酚红MEM、DMEM/F12细胞培养基中的降解非常缓慢,浸泡10d的平均降解速率分别约为0.013、0.013、0.016 mg/(cm2·h)。腐蚀介质成分的不同对合金的降解性能影响很大,在含有蛋白质、氨基酸的环境中合金的降解速率降低;由于不同的细胞培养基中含有不同的营养成分,这种微弱的差异也明显影响了合金的降解:合金在DMEM/F12(用于培养BMSCs细胞)中降解速率大于其在?-MEM(用于培养L929细胞)与无酚红MEM(用于细胞毒性试验)中的降解速率。细胞毒性结果显示,Mg-Zn-Y-Nd-Zr合金浸提液对L929与MC3T3-E1两种细胞均未表现出毒性作用。在最初的浸泡过程中,pH值的升高是造成细胞死亡的根本原因。合金在培养液中短期(10 d)浸泡,释出的Mg2+及合金离子未对两种细胞产生毒性;在长期的浸泡后(60 d),浸泡液中金属离子累积到一定浓度,此时分析致细胞毒性的镁及合金离子浓度分别为,导致L929细胞存活率低于75%时的累积离子浓度为Mg2+:948-1580μg/ml,Zn2+:0.2304-0.384μg/ml,Y3+:0.0395-0.0658μg/ml,Nd3+:0.0448-0.0747μg/ml,Zr4+:0.0098-0.0164μg/ml,导致MC3T3-E1细胞存活率低于75%时的累积离子浓度为Mg2+:1580-2212μg/ml,Zn2+:0.384-0.538μg/ml,Y3+:0.0658-0.0921μg/ml,Nd3+:0.0747-0.105μg/ml,Zr4+:0.0164-0.0230μg/ml。细胞黏附实验结果显示,MC3T3-E1与L929均可在合金表面有一定的黏附;细胞增殖实验结果表明,在培养初期(1至3 d)及浓度较低(20~80%)时,合金浸提液明显刺激了L929的生长;在高浓度(100%,5 d)长期培养时,L929的生长受到了轻微抑制。ALP活性检测与钙化结节染色结果发现,Mg-Zn-Y-NdZr合金明显促进了干细胞向成骨细胞的分化过程,具有一定的成骨诱导性。综上所述,Mg-Zn-Y-Nd-Zr降解速率缓慢且其细胞相容性良好,可进行下一步的动物实验及临床实验。
[Abstract]:Mg-Zn-Y-Nd-Zr alloy is a new type of degradable bone implant magnesium alloy developed by our research group. By adding a small amount of multiple elements of Zn,Y,Nd,Zr to the alloy and extruding, the mechanical properties of magnesium alloy are greatly improved. As a biomedical material, its biological safety and biological function are very important, which need to be comprehensively evaluated and analyzed. In this paper, the biodegradability and biocompatibility of Mg-Zn-Y-Nd-Zr alloys in vitro were studied. Using electrochemical workstation pH measurement, ion concentration measurement, weightlessness test, scanning electron microscope (SEM) and energy dispersive analysis (SEMEDS), the Mg-Zn-Y-Nd-Zr alloy was used to analyze the DMEM / F12 cell culture medium of simulated body fluid (SBF), 10% fetal bovine serum (SBF 10S). The cytotoxicity of the alloy to mouse fibroblasts (L929) and mouse precranial bone cells (MC3T3-E1) was analyzed by in vitro cell culture, MTT assay and inverted phase contrast microscopy. The effects of the alloy on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblast (OBs) (OBs) were studied by ALP activity assay and calcified nodule staining. The results of in vitro degradation showed that the degradation of Mg-Zn-Y-Nd-Zr alloy gradually occurred in the medium with the prolongation of time, and the average degradation rate of the alloy immersed in SBF for 10 days was 0.0932 mg/ (cm2 h), in MEM,DMEM/F12 medium without phenols red) was very slow, and the degradation rate of Mg-Zn-Y-Nd-Zr alloy was very slow. The average degradation rate of soaking for 10 days was about 0.013 ~ 0.013 ~ 0.016 mg/ (cm2 h).), respectively. The degradation rate of the alloy was decreased in the environment containing protein and amino acid because of the different nutrient components in different cell culture medium, the degradation rate of the alloy was greatly affected by the different composition of corrosion medium, and the degradation rate of the alloy was decreased in the environment containing protein and amino acid. This slight difference also significantly affected the degradation rate of the alloy: the degradation rate of the alloy in DMEM/F12 (used for cultured BMSCs cells) was higher than that in L929 cells (used for culture) and MEM without phenol red (for cytotoxicity test). The results of cytotoxicity showed that the extract of Mg-Zn-Y-Nd-Zr alloy had no toxic effect on L929 and MC3T3-E1 cells. The increase of pH during initial immersion is the root cause of cell death. The Mg2 and alloy ions released from the alloy were not toxic to the two kinds of cells after soaking in the medium (10 d) for a short period of time, and the metal ions accumulated to a certain concentration after a long immersion (60 d), immersion solution). 姝ゆ椂鍒嗘瀽鑷寸粏鑳炴瘨鎬х殑闀佸強鍚堥噾绂诲瓙娴撳害鍒嗗埆涓，
本文编号：2218650
[Abstract]:Mg-Zn-Y-Nd-Zr alloy is a new type of degradable bone implant magnesium alloy developed by our research group. By adding a small amount of multiple elements of Zn,Y,Nd,Zr to the alloy and extruding, the mechanical properties of magnesium alloy are greatly improved. As a biomedical material, its biological safety and biological function are very important, which need to be comprehensively evaluated and analyzed. In this paper, the biodegradability and biocompatibility of Mg-Zn-Y-Nd-Zr alloys in vitro were studied. Using electrochemical workstation pH measurement, ion concentration measurement, weightlessness test, scanning electron microscope (SEM) and energy dispersive analysis (SEMEDS), the Mg-Zn-Y-Nd-Zr alloy was used to analyze the DMEM / F12 cell culture medium of simulated body fluid (SBF), 10% fetal bovine serum (SBF 10S). The cytotoxicity of the alloy to mouse fibroblasts (L929) and mouse precranial bone cells (MC3T3-E1) was analyzed by in vitro cell culture, MTT assay and inverted phase contrast microscopy. The effects of the alloy on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblast (OBs) (OBs) were studied by ALP activity assay and calcified nodule staining. The results of in vitro degradation showed that the degradation of Mg-Zn-Y-Nd-Zr alloy gradually occurred in the medium with the prolongation of time, and the average degradation rate of the alloy immersed in SBF for 10 days was 0.0932 mg/ (cm2 h), in MEM,DMEM/F12 medium without phenols red) was very slow, and the degradation rate of Mg-Zn-Y-Nd-Zr alloy was very slow. The average degradation rate of soaking for 10 days was about 0.013 ~ 0.013 ~ 0.016 mg/ (cm2 h).), respectively. The degradation rate of the alloy was decreased in the environment containing protein and amino acid because of the different nutrient components in different cell culture medium, the degradation rate of the alloy was greatly affected by the different composition of corrosion medium, and the degradation rate of the alloy was decreased in the environment containing protein and amino acid. This slight difference also significantly affected the degradation rate of the alloy: the degradation rate of the alloy in DMEM/F12 (used for cultured BMSCs cells) was higher than that in L929 cells (used for culture) and MEM without phenol red (for cytotoxicity test). The results of cytotoxicity showed that the extract of Mg-Zn-Y-Nd-Zr alloy had no toxic effect on L929 and MC3T3-E1 cells. The increase of pH during initial immersion is the root cause of cell death. The Mg2 and alloy ions released from the alloy were not toxic to the two kinds of cells after soaking in the medium (10 d) for a short period of time, and the metal ions accumulated to a certain concentration after a long immersion (60 d), immersion solution). 姝ゆ椂鍒嗘瀽鑷寸粏鑳炴瘨鎬х殑闀佸強鍚堥噾绂诲瓙娴撳害鍒嗗埆涓，
本文编号：2218650
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