微弧氧化涂层改性对3D打印钛合金支架细胞生物学的影响

摘要

背景:微弧氧化处理可提高钛合金植入体的生物学性能.目前研究多集中于钛合金片材,有关微弧氧化改性处理后3D多孔钛合金支架对细胞生长及分化的影响鲜有报道.目的:研究微弧氧化表面改性对3D打印多孔钛合金支架表面细胞生物学性能的影响.方法:将大鼠骨髓间充质干细胞分别接种于微弧氧化处理Ti6Al4V合金三维支架(实验组)与未经微弧氧化处理Ti6Al4V合金三维支架(对照组)上,培养4,7 d,进行扫描电镜观察、细胞骨架染色分析及细胞活力分析;培养4,7,11 d,检测细胞上清液中碱性磷酸酶和骨钙素水平;培养1,4,7,11 d,MTT法检测细胞增殖.结果与结论:①培养4,7 d时,live/dead染色显示骨髓间充质干细胞在两组材料上生长良好;细胞骨架染色分析显示,实验组细胞骨架呈立体多角形,对照组支架表面的细胞以扁平梭形状沿支架宏观结构铺展;扫描电镜显示,实验组细胞排列较为紧密,细胞伸展状态良好,细胞间的板状伪足与丝状伪足交叉连接,以锚状结构牢固地黏附于材料表面;对照组细胞伪足间的连接较少,细胞外基质较少,平铺在材料表面生长;②随着培养时间的延长,两组细胞上清液中碱性磷酸酶与骨钙素水平逐渐升高;实验组培养7,11 d的碱性磷酸酶水平高于对照组(P < 0.05),培养11 d的骨钙素水平高于对照组(P < 0.05);③MTT检测显示,随着培养时间的延长,两组细胞数量逐渐增加;实验组培养7,11 d的细胞数量明显多于对照组(P < 0.05);④结果表明,微弧氧化处理后的钛合金支架更有利于细胞的黏附、增殖与分化.%BACKGROUND: Micro-arc oxidation (MAO) treatment can improve the biological properties of titanium alloy implants. Previous studies mostly focused on the evaluation of titanium alloy plate, while the effects of the MAO-modified 3D titanium scaffold on the cell growth and differentiation were rarely reported. OBJECTIVE: To investigate the effect of MAO coating on the biological performance of cells seeded onto the 3D-printed porous titanium alloy scaffold. METHODS: Rat bone marrow mesenchymal stem cells were seeded onto the MAO-modified Ti6Al4V alloy scaffolds (experimental group) and unmodified scaffolds (control group). After 4 and 7 days of culture, cell/scaffold constructs were retrieved and processed for the assessment of cell morphology by using scanning electron microscopy, cytoskeletal staining analysis and cell viability assay were also evaluated. At 4, 7 and 11 days of culture, the levels of alkaline phosphatase and osteocalcin in the cell supernatant were detected. At 1, 4, 7 and 11 days of culture, the cell proliferation rate was measured using the MTT assay. RESULTS AND CONCLUSION: (1) At 4 and 7 days of culture, live/dead staining showed that the bone marrow mesenchymal stem cells grew well on the two kinds of scaffolds. The analysis of cytoskeleton staining showed that the cytoskeleton of the experimental group was stereo and polygonal, while the cells on the scaffold surface in the control group were flat and spindle-shaped, spreading along the macro structure of the scaffolds. Under the scanning electron microscopy, the cells in the experimental group arranged closely and spread in a good condition, with interconnected lamellipodia and filopodia that firmly adhered to the scaffold surface in an anchor-shaped structure; in the control group, less filopodia interconnected, less extracellular matrix, and flat and sheet-like cells were observed. (2) With the time increase, the levels of alkaline phosphatase and osteocalcin increased gradually in both groups. The alkaline phosphatase level in the experimental group was significantly higher than that in the control group at 7 and 11 days of culture (P < 0.05), while the osteocalcin level was higher in the experimental group than the control group at 11 days of culture (P < 0.05). (3) With the prolongation of culture time, the number of cells in the two groups increased gradually. The number of cells cultured in the experimental group was significantly higher than that in the control group at 7 and 11 days of culture (P < 0.05). To conclude, the MAO-modified titanium alloy scaffold is favorable for cell adhesion, proliferation and differentiation.