Cytobiological effect of micro-arc oxidation coating on 3D-printed titanium alloy scaffold

doi:10.3969/j.issn.2095-4344.0717

Abstract

Abstract:

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.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

Key words: Alloys, Biology, Stem Cells, Tissue Engineering

CLC Number:

R318

Li Jiong-jiong, Zou Qin, Hu Fu,Chen Jie, Li Ji-dong, Li Shu-jun, Hao Yu-lin, Li Yu-bao, Zuo Yi. Cytobiological effect of micro-arc oxidation coating on 3D-printed titanium alloy scaffold[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(10): 1565-1572.

Figures/Tables 8

2.5 接触角分析实验组和对照组表面接触角结果分别为(76.57±2.37)°和(68.02±5.39)°。结果表明，接触角虽有小幅度增大，仍低于90°，钛合金支架在表面微弧氧化处理后仍可称之为亲水性材料。接触角在10°-80°之间的表面属于中等润湿表面，细胞在中等润湿的材料表面黏附能力相对较强[25]。与文献[26]中钛合金平板表面微弧氧化后出现接触角下降不同，此次实验三维支架表面微弧氧化后的接触角增加。原因可能是相对于平板结构，支架的大孔占比远高于微孔，液滴更易从光滑表面的未改性支架进入大孔实现浸润，表现为接触角比改性支架的更小。 2.6 细胞活性测试和形态学分析细胞接种培养4 d以后，在两种材料的荧光图片上(图4A，B，G，H)，均能看到明亮的绿色，反映出细胞较强的生命活力，说明细胞在材料上生长状态良好，几乎看不到红色荧光响应，可能因支架孔隙相对细胞较大，黏附性差的凋亡细胞在染色冲洗时从支架上脱落流失。细胞在支架材料上分布较为均匀。真核细胞细胞质中错综复杂的纤维网构成细胞骨架，细胞的黏附状态与细胞骨架纤维紧密相关。研究中的钛合金支架多孔结构尺寸为400-500 μm，远大于细胞直径，微弧氧化处理后支架表面形成了直径为0.5-5 μm的微孔，这些微米和亚微米尺寸的多孔结构对细胞超微结构产生更为显著的影响。在实验组支架表面，细胞骨架结构立体分明，被染成红色的细胞肌动蛋白呈多边形形态(图4C)。在未对照组支架表面的细胞，肌动蛋白呈片状荧光响应(图4I)。由扫描电镜图片可看到，在实验组支架材料表面黏附的细胞伸展成细长形或多角形不一，丝状伪足结构明显，细胞伸展状态良好，形态多样化(图4D-F)。不同于对照组支架上的细胞形态(图4J-L)，在实验组支架上的细胞有更多板状伪足且部分伪足直接锚进表面微孔内，与材料黏附更加牢固。"

细胞接种培养7 d以后，细胞数目明显增多。在两种材料的荧光图片上，较密集地布满代表细胞活性良好的绿色(图5A，B，G，H)，说明材料具有较好的细胞相容性。在实验组支架表面，被染成红色的蛋白微丝呈网状结构，由微丝组成的骨架结构呈立体多角形(图5C)。而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，肌动蛋白红色响应较强(图5I)。由此可见，细胞骨架的结构和铺展状态等超微结构与支架微表面粗糙度有关。当细胞黏附于较粗糙的表面时，通过伪足可锚接于微弧氧化表面的沟和孔洞，无需排列良好的肌动蛋白网架。而在未改性支架的光滑表面上黏附时，细胞需伸展出肌动蛋白纤维，才能将自己稳定于材料的表面，因此观察到在未改性支架表面的肌动蛋白粗大清晰(红色荧光响应更强)。此时，由于应力纤维的强力伸展，细胞被施加一定张力，表现为未改性支架表面扁平伸展的细胞骨架结构。由扫描电镜图片可看到，在实验组支架材料表面黏附的细胞排列较为紧密，开始出现细胞间的重叠，细胞伸展状态良好，支架表面细胞突出材料表面较为明显，胞浆饱满，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(图5D-F)；对照组支架材料上的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(图5J-L)。李玉梅等[27]以微弧氧化处理的纯钛圆片进行相关研究也表明，微弧氧化改性钛表面黏附的细胞形状较不规则，无一定的方向性，而未处理的圆片微表面相对光滑，成骨细胞易被牵伸，并沿材料的二维平面伸展。由此可见，无论基材是三维支架还是二维平面体，微表面的粗糙度才是细胞黏附的关键。相对于未改性支架的光滑微表面黏附的细胞，微弧氧化改性后形成的粗糙微表面，不仅更适于细胞黏附，还利于细胞以三维立体状态生长增殖，细胞超微结构形态表现更好。"

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