重建胶原纤维对细胞生长的影响

Effects of reconstructive collagen fibers on the growth of cells

背景:胶原种类很多,而不同细胞只在特定类型的胶原环境中才表现出最佳的增殖和表型。目的:观察Ⅰ型胶原和Ⅱ型胶原对小鼠皮肤成纤维细胞、人牙周膜成纤维细胞和兔软骨细胞生长的影响。设计、时间及地点:对比观察,细胞学体外实验,于2007-09/2008-09在四川大学生物材料工程研究中心胶原研究室完成。材料:自制达到中试水平的猪皮Ⅰ型胶原和猪软骨Ⅱ型胶原。方法:分别将3种细胞在猪皮Ⅰ型胶原和猪软骨Ⅱ型胶原环境中进行体外培养,比较不同细胞在材料上的行为差异。主要观察指标:傅里叶变换红外光谱、示差扫描热分析和凝胶动力学分析比较2种胶原的热变性温度及在结构和性质上的差异;MTT法观察细胞在2种胶原环境中的黏附、增殖情况。结果:猪软骨Ⅱ型胶原和猪皮Ⅰ型胶原的热变性温度分别为105.8,87.5℃,2种胶原的官能团区相似。凝胶化曲线的对比中猪皮Ⅰ型胶原的成纤维过程较猪软骨Ⅱ型胶原迅速。MTT值显示细胞生长初期,小鼠皮肤成纤维细胞在软骨Ⅱ型胶原环境表现了较好的黏附性,但是更适于在猪皮Ⅰ型胶原上生长;人牙周膜成纤维细胞与小鼠皮肤成纤维细胞对胶原纤维的反应恰好相反。兔软骨细胞第2天在两种胶原上的黏附相似,第4天时在猪软骨Ⅱ型胶原环境中表现出较大的细胞生长密度。结论:不同细胞对Ⅰ,Ⅱ型胶原纤维表现了不同的时间依赖性,但猪软骨Ⅱ型胶原环境更接近于软骨生长的真实环境,更有利于软骨细胞朝特定方向分化,使软骨细胞维持稳定的表型。

BACKGROUND： Considering the variety types of collagen, different cells tend to express diverse behaviors on different type of collagen. OBJECTIVE： To investigate the growth of mouse fibroblasts, human periodontal ligament fibroblasts and rabbit chondrocytes on the type Ⅰ collagen and type Ⅱ collagen. DESIGN, TIME AND SETTING： In vitro controlled experiment was performed in Biomaterials Engineering Research Center of Sichuan University from September 2007 to September 2008. MATERIALS： Type Ⅰ collagen and type Ⅱcollagen were prepared for pilot test. METHODS： Three kinds of cells namely L92, human periodontal ligament fibroblasts and rabbit chondrocytes were cultured in type Ⅰ collagen and type Ⅱ collagen, and then the behaviors of different cells were compared. MAIN OUTCOME MEASURES： Fourier transform infrared spectroscopy, differential scanning calorimeter and gel formation kinetics analysis were performed to compare the thermal denaturation, structure and character of two types of collagen; cell adhesion and proliferation in different collagen were observed using MTT method. RESULTS： The thermal denaturation of type Ⅱcollagen and type Ⅰ collagen was 105.8 ℃ and 87.5 ℃, respectively; The two types of collagen had the similar functional group. Gelation curve demonstrated the fibroblasts formed more rapidly in type Ⅰ collagen than type Ⅱ collagen. MTT method had presented that mouse skin flbroblasts were adhesive to type Ⅱ collagen, but were suitable to grow in type Ⅰ collagen. Human periodontal ligament fibroblasts were contrary to the mouse skin flbroblasts regarding the reaction to the collagen fibers. Rabbit chondrocytes were identical at 2 days and type Ⅱ collagen showed a bigger celt growth density than type Ⅰ collagen at 4 days. CONCLUSION： Different cells show diverse time dependence on different collagen fibers, however, type Ⅱ collagen is more close to the environment that cartilage grow and more beneficial for the differentiation chondrocytes, thus maintaining the phenotype of phenotype.