激光增材制造骨科用NiTi形状记忆合金的细胞毒性与生物相容性研究

Cytotoxicity and biocompatibility of additively manufactured nickel-titanium shape memory alloy for orthopaedics

目的:评估激光增材制造的骨科用NiTi形状记忆合金支架的细胞毒性与生物相容性,为开发新型NiTi合金骨科植入物提供基础。方法:利用激光粉末床熔融工艺制备NiTi合金支架,使用扫描电子显微镜观察NiTi合金支架形貌;将样品置于细胞培养基中,在37℃条件下离心搅拌7 d,以制备含100%、50%、10%金属浸出液的稀释液。培养小鼠成骨细胞前体细胞(MC3T3-E1)与人脐静脉内皮细胞(HUEVC),并将其置于不同培养基中处理:对照组(使用正常培养基),10%浸出液组(培养基为10%浸出液,90%正常培养基),50%浸出液组(培养基为50%浸出液,50%正常培养基),100%浸出液组(培养基为100%浸出液)。借助CCK-8试剂盒与Calcein/PI细胞活性与细胞毒性检测试剂盒评估孵育24 h后MC3T3-E1细胞的存活率与凋亡情况。将指数生长期的HUEVC接种于96孔板,5 h后计算每孔管腔生成的细胞数目。使用Calcein/PI试剂盒评估细胞与金属样品共同孵育24 h、48 h和72 h后的凋亡情况。利用Actin-Tracker Green荧光染色比较同金属支架共同孵育72 h、正常培养72 h后经胰酶消化液处理种板的MC3T3-E1细胞的微丝含量。结果:本研究利用激光粉末床熔融工艺成功制备出NiTi合金支架,通过扫描电子显微镜观察发现支架表面存在着未熔NiTi颗粒、裂纹等缺陷。利用CCK-8试剂盒进行细胞毒性检测,发现不同浓度的浸出液组与对照组比较,细胞活性的差异无统计学意义(P>0.05)。利用Calcein/PI试剂盒进行细胞活性与细胞毒性检测,发现不同浓度金属浸出液均无生物毒性;不同组别HUEVC在孔板中生成血管管腔的平均分支数目比较,差异无统计学意义(P>0.05)。通过荧光显微镜观察与金属样品共孵育后的MC3T3-E1细胞,发现细胞在72 h后完全长入样品孔隙。Actin-Tracker Green荧光染色结果显示,与金属支架共同孵育的细胞的平均微丝荧光强度为56.69%±6.95%,显著大于正常培养的细胞(36.37%±6.88%,P<0.05),说明细胞接种于NiTi形状记忆合金后细胞结构强度更大。结论:本研究发现激光增材制造骨科用NiTi形状记忆合金具备较小的细胞毒性、良好的生物相容性,并可以通过增加细胞内微丝含量增强细胞结构,为开发新型的激光增材制造NiTi合金骨科植入物提供了基础。

Objective: To evaluate the cytotoxicity and biocompatibility of additively manufactured nickel-titanium shape memory alloys(AM-NiTi). Methods: The AM-NiTi scaffold was fabricated using laser powder bed fusion technique. Scanning electron microscope was used to observe the morphology of the AM-NiTi scaffold. The undiluted, diluted 50% and diluted 10% metal ion leachates were obtained by centrifuging and stirring the metal scaffold in cell culture medium at 37℃ for 7 d. Mouse osteoblast precursor cells(MC3T3-E1 cells) and human umbilical vein endothelial cells(HUVECs) were cultured in different dilutions of medium and divided into control group and dilution group with different dilution concentrations. CCK-8 kit and Calcein/PI kit were used to detect the survival rate and apoptosis of MC3T3-E1 cells after 24 h incubation. HUVECs at the exponential growth stage were inoculated into 96-well plates, and the number of lumen generation per well was counted after 5 h. The apoptosis of cells after coincubation with metal scaffold for 24 h, 48 h and 72 h was detected by the Calcein/PI kit. Microfilament contents of MC3T3-E1 cells treated with trypsin digest after 72-hour incubation with metal scaffolds and after 72-hour normal culture were assessed using ActinTracker Green fluorescent staining. Results: Unmelted NiTi particles and cracks on the surface of the NiTi scaffold was observed by scanning electron microscopy. No statistical difference in cell activity was found between the leachate and control groups by the detection of CCK-8 kit(P>0.05). No biotoxicity was found for different concentrations of metal leachate using the Calcein/PI kit. No statistical difference was found in the mean number of lumens in the different groups(P>0.05). MC3T3-E1 cells co-incubated with metal scaffold were found to be fully grown into the sample pores after 72 h by fluorescence microscopy. Greater average microfilament fluorescence intensity was found in cells inoculated with metal scaffold(56.69%±6.95% vs. 36.37%±6.88%, P<0.05). Conclusions: In this study, AM-NiTi was found to possess low cytotoxicity and good biocompatibility, and could enhance cell structure by increasing intracellular microfilament content.