Effect of type I or type II collagen on biological characteristics of human chondrocytes

doi:10.3969/j.issn.2095-4344.2014.30.014

Abstract

Abstract:

BACKGROUND: Experiments have shown that the collagen substrate has the capability of stimulating cartilage generation, but the stimulating role of different types of collagen substrates remains controversial.
OBJECTIVE: To investigate the effect of type I and type II collagen on the biological characteristics of human chondrocytes cultured in vitro.
METHODS: Human chondrocytes at passage were cultured onto the ordinary culture plates (ordinary plate), type I collagen-coated culture plates (type I plate), and type II collagen-coated culture plates (type II plate). Cell growth curves were determined by MTT method after cells were cultured for 10 days. By ELISA, PCR, and 1,9-dimethyl methyleneblue technology, type I and type II collagen and glycosaminoglycan contents were quantitatively detected in cartilage cells 28 days after culture.
RESULTS AND CONCLUSION: The number of cartilage cells was the highest in type II plate, which was twice of that in type I plate and five times of that in ordinary plate. Cartilage cells in type II plate secreted the least amount of type I collagen, which showed significant differences compared with the ordinary plate (P < 0.01) and had no statistically significant difference with type I plate (P > 0.01). Cartilage cells in type II plate secreted the most amount of type II collagen and glycosaminoglycan, showing significant differences compared with the other two plates (P < 0.01). The cartilage cells cultured in collagen plates are better than that cultured in ordinary culture plate, type II collagen culture plate is better than type I collagen culture plate in maintaining cell shape, extending the dedifferentiation pattern, and promoting cell 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

全文链接：

Key words: collagen type I, collagen type II, chondrocytes

CLC Number:

R318

Jiang Ping, Wei Peng, Zhao Ming-cai, Chen Qiong, Wang Zi . Effect of type I or type II collagen on biological characteristics of human chondrocytes[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(30): 4845-4850.

Figures/Tables 2

2.1 倒置相差显微镜观察软骨细胞形态软骨细胞培养：贴壁的原代软骨细胞呈椭圆形、多角形；传代软骨细胞较原代细胞稍大，呈椭圆形、多角形或者不规则形；P3代软骨细胞和P1、P2代细胞相似，但细胞逐渐伸展，变成以长椭圆形为主(图1A，B)；传至P7代软骨细胞明显类似于成纤维细胞(图1C，D)。不同培养板培养的软骨细胞形态：培养10 d后，普通板中软骨细胞数量少，形态多为梭形，较细长(图2A)；细胞出现较多分叉，细胞核不明显，无明显细胞分裂增殖相(图2B)。Ⅰ型板细胞表面被胶原蛋白纤维覆盖，细胞外观立体，细胞数多且细胞短于普通板(图2C)，具有分叉的细胞较普通板少，分叉的细胞分支较少，60%细胞核明显，具有分裂增殖能力(图2D)。Ⅱ型板软骨细胞数明显较多，约长满培养板90%，且多为短小细胞，同Ⅰ型板细胞一样，细胞外观立体，表面胶原蛋白覆盖(图2E)，短小细胞占80%，无明显分叉细胞，80%细胞核明显，具有较强的分裂增殖能力(图2F)。 2.2 不同培养板中软骨细胞的增殖速度观察细胞生长曲线(图3)，第2天即进入快速生长的对数生长期。Ⅰ、Ⅱ型板中的软骨细胞增殖快于普通板，其中Ⅱ型板中的软骨细胞增殖速度最快，为Ⅰ型板的2倍、普通板的5倍。在培养第8天时，Ⅰ、Ⅱ型板中的软骨细胞增殖缓慢，进入平台期，考虑细胞增殖快，接触抑制。由于Ⅱ型板中软骨细胞个体小于Ⅰ型和普通板，故培养第10天时Ⅱ型板中软骨细胞仍多于另外培养板。 2.3 不同培养板对软骨细胞生物学特性的影响软骨细胞分泌Ⅰ、Ⅱ型胶原蛋白水平及定量检测：3种培养板中软骨细胞Ⅰ型胶原蛋白分泌量的差异均有显著性意义(P < 0.01，表1)，再使用q检验进行两两比较显示，普通板中软骨细胞分泌Ⅰ型胶原蛋白量高于Ⅰ、Ⅱ型板，但不能认为Ⅰ、Ⅱ型板中软骨细胞分泌Ⅰ型胶原蛋白量不同。3种培养板中软骨细胞Ⅱ型胶原蛋白分泌量差异有显著性意义(P < 0.01)，使用q检验进行两两比较后，显示Ⅰ、Ⅱ型板中软骨细胞分泌Ⅱ型胶原蛋白量均高于普通板，其中Ⅱ型板中软骨细胞分泌Ⅱ型胶原蛋白量最高。根据半定量PCR检测也得出Ⅱ型板中软骨细胞Ⅱ型胶原蛋白基因表达最高，Ⅰ型板其次，最少的为普通板(图4)。软骨细胞分泌糖胺多糖的量：3种培养板中软骨细胞糖胺多糖分泌量差异有统计学意义(P < 0.01)，使用q检验进行两两比较后显示，Ⅰ、Ⅱ型板中软骨细胞分泌糖胺多糖都高于普通板，其中Ⅱ型培养板中软骨细胞分泌量最高(表1)。"

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