Effects of nanonetwork topography on bone marrow mesenchymal stem cell bioactivity

doi:10.3969/j.issn.2095-4344.2014.25.009

Abstract

Abstract:

BACKGROUND: Many studies have shown that different nanostructures produce different influences on cell bioactivity, but the nanonetwork structure is not reported yet.
OBJECTIVE: To study the influence of the nanonetwork topography on the bioactivity of bone marrow mesenchymal stem cells.
METHODS: The nanonetwork topography was fabricated on biomedical titanium surface by alkali-heat treatment, and pure titanium served as control group. Bone marrow mesenchymal stem cells were co-cultured with the above two types of samples. Cell morphology and cytoskeleton were observed using scanning electron microscope and immunofluorescence method. The cell adhesion, proliferation and osteogenic differentiation were detected by measurement of absorbance values at different culture time.
RESULTS AND CONCLUSION: The nanonetwork topography had significant advantage on the number of adherent cells at 30, 60 and 120 minutes of co-culture. The cell proliferation was significantly accelerated by the nanonetwork topography at days 1, 3, 5 of co-culture, and the absorbance values in the nanonetwork group were significantly higher than those in the pure titanium group (P < 0.05). The alkaline phosphatase activity in the nanonetwork group was also significantly higher than that in the pure titanium group at 14 days of osteogenic induction (P < 0.05). The cell shape and cytoskeleton on the nanonetwork surface were better than those on the titanium surface. These findings indicate that the nanonetwork topography has better effects on cell bioactivity compared with pure titanium.

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: titanium, bone marrow mesenchymal stem cells, cytoskeleton, alkaline phosphatase

CLC Number:

R318

Ruan Zheng, Yin Qing-shui, Zhang Yu . Effects of nanonetwork topography on bone marrow mesenchymal stem cell bioactivity[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(25): 3987-3992.

Figures/Tables 2

2.1 材料表面特性变化利用扫描电镜分析纳米化表面形貌的材料后发现，在碱热处理中将温度控制为60 ℃后获得了纳米网状结构，见图1。利用X射线衍射检测获得了纳米网状结构材料表面后发现，生物活性医用钛表面形成的纳米形貌的晶体结构主要由锐钛矿构成，晶体的结晶度因锻造时不同的调控温度而不同，能谱分析结果显示此4种纳米化表面材料的表面元素组成及元素的比例，结果提示此4种材料表面组成一样，都完全是钛元素。 2.2 小鼠骨髓间充质干细胞与纯钛材料和纳米网状结构材料分别共培养后细胞形态及细胞骨架结构变化将纳米网状结构材料和纯钛分别与细胞悬液共培养24 h后，通过扫描电镜观察细胞形态，见图2。通过图片实验可以发现，纳米网状结构材料表面细胞形态较好，细胞帖服效果最好，细胞铺展面积最大，可见板状伪足。在纯钛组材料表面仅可见有丝状伪足伸出，细胞明显纤细，呈长条状，有隆起部分，贴服不够充分。图3为细胞与两种材料共培养24 h后利用TRITC染色在正置荧光显微镜下观察到的细胞骨架情况。与扫描电镜结果相似的是，纳米网状结构材料表面细胞较纯钛组材料表面细胞铺展的面积更大，纯钛组材料表面细胞虽有丝状伪足突出，但整体形态较细长，或为不规则形。 2.3 小鼠骨髓间充质干细胞与纯钛材料和纳米网状结构材料分别共培养后材料与细胞黏附情况图4为骨髓间充质干细胞与两种材料分别共培养30，60，120 min后，在材料表面黏附的情况。图4C显示，无论是在在最初的 30 min，还是在60，120 min时，纳米网状结构材料表面黏附的细胞数量较纯钛组都有明显的增多(P < 0.05)。同时实验还发现，两组材料表面黏附的细胞数量随时间增长而增加，呈时间依赖性(P < 0.05)，表明材料对细胞无毒不良反应。 2.4 小鼠骨髓间充质干细胞与纯钛材料和纳米网状结构材料共培养后细胞增殖情况将两组材料与骨髓间充质干细胞共培养，用MTT法分别检测共培养第1，3，5 天的吸光度值，并采用统计学方法分析细胞增殖情况，见图5。由统计图实验可以看见，在共培养第1，3，5天，纳米网状结构组材料的吸光度值都明显高于纯钛组(P < 0.05)。同时，实验还可以看出，在共培养第1，3，5天的时间里，纳米网状结构组材料的吸光度值呈一上升趋势(P < 0.05)，这表明随时间的延长，纳米网状结构可以促进更多的细胞增殖。 2.5 小鼠骨髓间充质干细胞与纯钛材料和纳米网状结构材料分别共培养后细胞碱性磷酸酶活性变化将两组材料与细胞共培养14 d后，检测各组材料的碱性磷酸酶值及胞内蛋白含量，计算碱性磷酸酶与胞内蛋白的比值来比较各组材料的成骨效应，见图6。由图可以看出，纳米网状结构组材料较纯钛组材料有显著性的促成骨功能(P < 0.05)。"

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