In vitro cytotoxicity of the hydroxyapatite/barium titanate biological piezoelectric ceramic coating

doi:10.3969/j.issn.2095-4344.2014.34.010

Abstract

Abstract:

BACKGROUND: In order to optimize the biological activity of hydroxyapatite, previous experiments have used plasma spraying technique to prepare a piezoelectric ceramic coating on the surface of hydroxyapatite, but the cytotoxicity of this new material is not clear.

OBJECTIVE: To evaluate the cytotoxicity of hydroxyapatite/barium titanate biological piezoelectric ceramic coating in vitro.

METHODS: The 3^rd generation beagle bone marrow mesenchymal stem cells were seeded on hydroxyapatite/barium titanate piezoelectric ceramic specimens and hydroxyapatite specimens, respectively. After 5 days, the cell adhesion was detected by scanning electron microscopy. The 3^rd generation bone marrow mesenchymal stem cells were also co-cultured with hydroxyapatite/barium titanate piezoelectric ceramic specimen extract, hydroxyapatite specimen extract, low-glucose Dulbecco’s modified Eagle’s medium containing 5% dimethylsulfoxide and 15% fetal bovine serum, and low-glucose Dulbecco’s modified Eagle’s medium containing 15% fetal bovine serum, respectively. The cytotoxicity was tested by Cell Counting Kit-8 assay at days 1, 3, 5 after co-culture.

RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells on the surface of hydroxyapatite/barium titanate piezoelectric ceramic specimens and hydroxyapatite specimens grew proliferatively and presented with multi-layer growth. The connection between cells and pseudopodia was very close, which indicates that the two kinds of materials both have good cytocompatibility. Cell Counting Kit-8 assay showed that the cells cultured in the extracts of hydroxyapatite/barium titanate biological piezoelectric ceramic and hydroxyapatite specimens proliferated more than 80%, and the toxicity was grade 1 that meant no cytotoxicity.

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

全文链接：

Key words: ceramics, materials testing, hydroxyapatites

CLC Number:

R318

Kang Wen, Xu Guo-qiang, Diilnur Aji, Wei Qng, Ma Hui, Li Zhao-peng, Song Qian, Rufiya Zulati, Alina Abdujilil, Pahirdin Kaisar. In vitro cytotoxicity of the hydroxyapatite/barium titanate biological piezoelectric ceramic coating[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(34): 5466-5472.

Figures/Tables 1

2.1 犬骨髓间充质干细胞的原代培养、传代及鉴定最初接种的骨髓间充质干细胞呈圆形，大小不一；6 h后部分细胞贴壁，变形，混有大量造血细胞及部分细胞碎片；第3天更换培养液时，可见大量散在分布的贴壁梭形细胞和悬浮的红细胞，换液时弃去红细胞，换液后贴壁细胞基本纯化大多呈长梭形，少数呈圆形或类圆形(图1A)。第7天数量加速生长，已形成成纤维细胞样集落，大部分细胞成梭形，少数呈多角形。培养第9，10天可达到90%融合。二三天便可传代1次，传代后细胞增殖速度增快。第3代比格犬骨髓间充质干细胞见图1B所示。本实验通过细胞形态及免疫组化方法来鉴定犬骨髓间充质干细胞，显微镜下观察细胞并照相，大部分细胞免疫细胞化学染色后CD44、CD29为阳性(图1C，D)，CD34为阴性，提示所获取的细胞成分为骨髓间充质干细胞。 2.2 不同成分培养液培养的细胞形态观察实验组：培养第1，3，5天，细胞一直保持良好的生长状态，贴壁生长情况良好，细胞均牢固黏附在培养孔底，细胞形态良好，呈典型梭形和多角形，细胞饱满。而且随着时间的延长，细胞数目也逐渐增多，第5天时细胞已经长满，未出现细胞脱落、裂解等细胞病变。对照组：培养第1，3，5天时此组细胞大部分形态正常，与实验组细胞形态无太大差异，都呈梭形，未见死亡的圆形细胞漂浮，细胞胞浆中无气泡，也无细胞核皱缩等病变情况。与实验组一样，细胞培养时间延长，细胞数目变多。阴性对照组：细胞保持优良的生长状态，贴壁生长状态非常好，细胞与培养孔底紧密结合细胞形态正常，呈典型梭形或多角形，细胞胞浆中无气饱，无细胞核皱缩，细胞胞质很饱满，未见死亡圆形细胞漂浮。阳性对照组：细胞数量非常少，胞体极度皱缩或细胞溶解呈空泡状，细胞胞浆中有气泡，见大量死亡细胞漂浮。 2.3 扫描电镜观察不同试件上的细胞黏附如图2所示，两种材料分别与骨髓间充质干细胞复合培养5 d后，可见两组细胞的突起均伸入材料孔隙内，细胞的突起形成胞间连接，大量细胞形成片状包绕材料表面，部分细胞密集区域还可见到细胞外基质的形成，且细胞大部分伸展良好。在两组表面都可观察到细胞增殖旺盛，达到复层生长，伪足与细胞之间连接很紧密，胞体丰富。由扫描电镜观察和显微镜观察均可得出等离子喷涂钛酸钡生物压电陶瓷试件与羟基磷灰石试件和比格犬骨髓间充质干细胞都有很好的细胞相容性。 2.4 CCK-8法检测羟基磷灰石/钛酸钡压电陶瓷材料的体外细胞毒性 4组浸提液对比格犬骨髓间充质干细胞作用1，3，5 d后的A值见表1，细胞相对增殖率、毒性分级见表2。对原始数据分析，方差不齐，所以将A值做了秩变换后进行析因设计的方差分析。在不同时间点，A值有统计学差异；不同组别，A值有统计学差异；时间与组别之间有交互作用。不同时间点之间两两比较，相互都具有统计学差异，结合各时间点的均数，可以发现随着时间推移，A值变大。不同组之间两两比较，实验组和对照组之间没有统计学差异，其余各组之间互相比较具有统计学差异，结合各组均数可以发现，阴性对照组A值最大，实验组和对照组次之，阳性对照组A值最小。因此，可以得出对照组与实验组表现出的细胞毒性无太大差异，但其两组细胞毒性大于阴性对照组，且随着培养时间的增长，实验组和对照组的细胞毒性基本趋于0级。"

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