表面改性及微沟槽技术对肌腱细胞生长与取向的影响

Effects of Micropatterned Surfaces Coated with TypeⅠ Collagen on the Orientation and Growth of Tenocytes

利用光刻技术制作微格式模板,微接触转印法制作微沟槽PDMS表面,微流道技术裱衬不同浓度的Ⅰ型胶原于微沟槽表面上,比较其对细胞生长形态及生长取向的影响。对照组为未裱衬胶原的微沟槽、平板PDMS及裱衬胶原的平板PDMS。种植SD大鼠肌腱细胞在材料上,于37℃孵箱中培养48 h。采用MTT比色法测定不同浓度的胶原对肌腱细胞的生长增殖的影响。通过倒置相差显微镜、扫描电镜、荧光显微镜观察细胞生长的形态,取向情况。结果显示:Ⅰ型胶原修饰的PDMS微沟槽材料比未裱衬胶原的对照组具有明显的促细胞生长的作用(P<0.05),并且随着胶原浓度的增加作用越明显。有微沟槽表面的材料对细胞的生长形态和生长取向有明显影响。提示,Ⅰ型胶原修饰的微沟槽材料不仅能规范肌腱细胞的生长取向,而且能促进肌腱细胞的生长,从而得到理想的细胞生长形态,该结果对工程化肌腱的构建有重要的指导意义。

It is crucial to improve the orientation and growth of cells on substrates in tissue engineering. In this study, we investigated the effects of micropatterned surfaces coated with type Ⅰ collagen （CNI） on the orientation and growth of SD rat tenocytes. Using the technique of microcontact printing and microfluidic channelS, we prepared micropatterned microgrooves with a 10 tan width and 4 tan depth on silicone membrane substrates. The microgrooves were coated with CNI at concentrations 0.25, 0.5, 0.75, 1.0, and 1.25 mg/ml,respectivdy. The rat tenocytes at 1 × 10^5/ ml were seeded onto the CNI-coated substrates and the control substrates （without CNI coating）, and then cultured in a humidified 37℃（2/5% CO2 incubator for 48 hours. Cell proliferation was measured by MTT method. After 1, 12, 24, 48 hrs of incubation, the tenocytes＇ alignment and morphology were observed by means of inverted phase microscope, scan- ning electron microscope and fluorescent microscope. The results showed there was obvious orientation of tenocytes in CNI-modified grooves, and most of the tenocytes spread along the grooves. The tenocyte orientation became more obvious with the increasing CNI concentration over a range from 0.25 to 1.25 mg/ml. This method could find important application in the construct of engineered tendons which need precise spatial organization of cells.