Cyclic stress promotes the proliferation of rabbit condyle chondrocytes

doi:10.3969/j.issn.2095-4344.2014.20.006

Abstract

Abstract:

BACKGROUND: The condyle is one of important areas of lower mandible growth, and has growth remodeling capacity lifetime. The in vivo functional research fails to obtain satisfactory outcome due to the complexity of physiological environment, nondirectiveness of stimulating factor transduction, and difficulty to control experimental conditions. Further studies will explore the effect of stress on condyle chondrocytes in vitro.
OBJECTIVE: To observe the effect of cyclic stress on the proliferation of rabbit condyle chondrocytes cultured in vitro.
METHODS: Rabbit condyle chondrocytes were separated and cultured in vitro. Passage 3 chondrocytes were subject to cyclic tensile stress (10% surface elongation, 6 cycles/min) for 1, 6, 12, 24 hours. While those cells without stress served as the control group. The cell cycle changes were detected with flow cytometry analysis, and the cell proliferation were analyzed by MTT assay.
RESULTS AND CONCLUSION: The flow cytometry analysis showed that S-phase promoting factor exhibited significant differences at 6 and 12 hours compared with control group, and got the maximum value at 24 hours (P < 0.01). MTT assay results showed that the cells proliferation at 6 and 12 hours exhibited significant differences compared with the control group, and got the maximum value at 24 hours (P < 0.01). The cyclic stress can obviously promote the proliferation of condyle chondrocytes, and the stimulative effect can be sustained 24 hours.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: chondrocytes, cyclic stress analysis, mandibular condyle, cell proliferation

CLC Number:

R318

Liu Wen, Wang Yan, Song Ling, Zhang Chun-yan, Zhang Yue, Yuan Xiao. Cyclic stress promotes the proliferation of rabbit condyle chondrocytes[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(20): 3144-3148.

Figures/Tables 2

2.1 兔髁状突软骨细胞的生长状态原代培养的髁状突软骨细胞24 h内可完全贴壁，细胞形态不规则，三角形、多角形均存在。原代培养的软骨细胞7 d可铺满整个培养瓶底，形成单层，软骨细胞在部分区域簇集成团，呈集落样生长。传代培养的软骨细胞一般36 h内可完全贴壁，一般5 d可形成单层。随传代次数增加，细胞中梭形细胞增多，软骨细胞分泌和增殖能力下降，传代周期延长(图1A，B)。 2.2 兔髁状突软骨细胞的甲苯胺蓝染色结果原代培养的髁状突软骨细胞培养7 d后，甲苯胺蓝染色见单层生长区域的细胞正染成浅蓝色，有二至三个核仁，核仁呈深蓝色，集落样生长和多层生长区的中心部位细胞及细胞外基质异染成紫红色，细胞核正染成深蓝色，而其他边缘部位异染不及中心区域(图1C)。 2.3 兔髁状突软骨细胞的透射电镜观察结果可见培养的软骨细胞结构正常细胞核为球形，椭圆形，核形态不规则，核仁边集，常染色质明显，可见核小体，细胞内有大量粗面内质网，线粒体及分泌小泡(图2A)。 2.4 兔髁状突软骨细胞S期细胞比率流式细胞术结果显示，10%形变率、6循环/min的周期性牵张力加载于细胞6 h和12 h后，细胞S期细胞比率开始增加，差异有显著性意义(P < 0.05)；应力加载24 h后，细胞S期细胞比率增加为实验时间内最高值，差异有显著性意义(P < 0.01)。随着应力加载时间的延长，加力组的S期细胞比率出现逐渐增高的变化，提示10%形变率、6循环/min的周期性牵张力在24 h内能够促进髁状突软骨细胞的增殖活性(表1)。 2.5 MTT染色结果在10%形变率、频率6循环/min的周期性张应力作用下，髁状突软骨细胞在加力1，6，12和24 h，与相应对照组比较均有细胞数量上的增加，其中加力24 h时细胞数量增加明显，与对照组比较差异有显著性意义(P < 0.01)，见表2。 MTT染色结果显示，髁状突软骨细胞内形成蓝紫色结晶，加力1，6和12 h组细胞内呈现蓝紫色颗粒状物，结晶物较少；加力组在24 h时间点，细胞内呈现大量松枝状的蓝紫色结晶物，细胞增殖活性增强(图2B，C)。"

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