新型骨植入Mg-Zn-Y-Nd-Zr合金的體外降解性能與生物相容性研究
發(fā)布時間：2018-09-02 07:15
【摘要】：Mg-Zn-Y-Nd-Zr合金是課題組前期開發(fā)的一種新型可降解骨植入用鎂合金,通過在合金中添加微量多元化Zn、Y、Nd、Zr元素,并經(jīng)過擠壓加工,鎂合金的力學(xué)性能大大提高。作為生物醫(yī)用材料,其生物安全性與生物功能性至關(guān)重要,需對其進行全面評價和分析。本論文對Mg-Zn-Y-Nd-Zr合金的體外降解性能及生物相容性進行了系統(tǒng)研究。采用電化學(xué)工作站、pH測量、離子濃度檢測、失重實驗、掃描電子顯微鏡及能譜分析(SEMEDS)等分析了Mg-Zn-Y-Nd-Zr合金在模擬體液(SBF)、模擬體液+10%胎牛血清(SBF+10%FBS)、DMEM/F12細(xì)胞培養(yǎng)基、?-MEM細(xì)胞培養(yǎng)基、無酚紅MEM細(xì)胞培養(yǎng)基五種介質(zhì)中的降解行為;通過體外細(xì)胞培養(yǎng),采用MTT法、倒置相差顯微鏡觀察等分析了合金對小鼠成纖維細(xì)胞(L929)、小鼠顱頂前骨細(xì)胞(MC3T3-E1)的細(xì)胞毒性、細(xì)胞黏附、細(xì)胞增殖影響,并通過ALP活性檢測、鈣化結(jié)節(jié)染色等研究了合金對骨髓間充質(zhì)干細(xì)胞(BMSCs)向成骨細(xì)胞(OBs)誘導(dǎo)分化過程的影響。體外降解實驗結(jié)果表明,Mg-Zn-Y-Nd-Zr合金在介質(zhì)中隨著時間的延長逐漸發(fā)生降解,在SBF中浸泡10 d的平均降解速率為0.0932 mg/(cm2·h),在?-MEM、無酚紅MEM、DMEM/F12細(xì)胞培養(yǎng)基中的降解非常緩慢,浸泡10d的平均降解速率分別約為0.013、0.013、0.016 mg/(cm2·h)。腐蝕介質(zhì)成分的不同對合金的降解性能影響很大,在含有蛋白質(zhì)、氨基酸的環(huán)境中合金的降解速率降低;由于不同的細(xì)胞培養(yǎng)基中含有不同的營養(yǎng)成分,這種微弱的差異也明顯影響了合金的降解:合金在DMEM/F12(用于培養(yǎng)BMSCs細(xì)胞)中降解速率大于其在?-MEM(用于培養(yǎng)L929細(xì)胞)與無酚紅MEM(用于細(xì)胞毒性試驗)中的降解速率。細(xì)胞毒性結(jié)果顯示,Mg-Zn-Y-Nd-Zr合金浸提液對L929與MC3T3-E1兩種細(xì)胞均未表現(xiàn)出毒性作用。在最初的浸泡過程中,pH值的升高是造成細(xì)胞死亡的根本原因。合金在培養(yǎng)液中短期(10 d)浸泡,釋出的Mg2+及合金離子未對兩種細(xì)胞產(chǎn)生毒性;在長期的浸泡后(60 d),浸泡液中金屬離子累積到一定濃度,此時分析致細(xì)胞毒性的鎂及合金離子濃度分別為,導(dǎo)致L929細(xì)胞存活率低于75%時的累積離子濃度為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,導(dǎo)致MC3T3-E1細(xì)胞存活率低于75%時的累積離子濃度為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。細(xì)胞黏附實驗結(jié)果顯示,MC3T3-E1與L929均可在合金表面有一定的黏附;細(xì)胞增殖實驗結(jié)果表明,在培養(yǎng)初期(1至3 d)及濃度較低(20~80%)時,合金浸提液明顯刺激了L929的生長;在高濃度(100%,5 d)長期培養(yǎng)時,L929的生長受到了輕微抑制。ALP活性檢測與鈣化結(jié)節(jié)染色結(jié)果發(fā)現(xiàn),Mg-Zn-Y-NdZr合金明顯促進了干細(xì)胞向成骨細(xì)胞的分化過程,具有一定的成骨誘導(dǎo)性。綜上所述,Mg-Zn-Y-Nd-Zr降解速率緩慢且其細(xì)胞相容性良好,可進行下一步的動物實驗及臨床實驗。
[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?yōu)姣掓�х殑闀佸強鍚堥噾紱誨瓙嫻撳害鍒嗗埆涓，
本文編號：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?yōu)姣掓�х殑闀佸強鍚堥噾紱誨瓙嫻撳害鍒嗗埆涓，
本文編號：2218650
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