Li Kuifeng, Guo Likun, Fan Yujiang, Zhang Xingdong
National Engineering Research Center for Biomaterials, Sichuan University, Chengdu Sichuan, 610065, P.R.China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2012 Nov;26(11):1356-61.
To investigate the effect of collagen type I concentration on the physical and chemical properties of the collagen hydrogel, and to analyze the effect of different concentrations of collagen type I hydrogel on the phenotype and gene expression of the chondrocytes in vitro.
Three kinds of collagen hydrogels with concentrations of 12, 8, and 6 mg/mL (C12, C8, and C6) were prepared, respectively. The micro-structure, compressive modulus, and swelling ratio of the hydrogels were measured and analyzed. The chondrocytes at 2nd passage were cocultured with three kinds of collagen hydrogels in vitro, respectively. After 1-day culture, the samples were stained with fluorescein diacetate (FDA)/ propidium iodide (PI) and the cell activity was observed under confocal laser microscope. After 14-day culture, HE staining and toluidine blue staining were carried out to observe the histological morphology, and mRNA expressions of chondrocytes related genes (collagen type II, Aggrecan, collagen type I, collagen type X, Sox9) were determined by real-time fluorescent quantitative PCR.
With the increase of collagen type I concentration from 6 to 12 mg/mL, the physical and chemical properties of the collagen hydrogels changed significantly: the fiber network became dense; the swelling ratios of C6, C8, and C12 were 0.260 +/- 0.055, 0.358 +/- 0.072, and 0.539 +/- 0.033 at 192 hours, respectively, showing significant differences among 3 groups (P < 0.05); and the compression modulus were (4.86 +/- 0.96), (7.09 +/- 2.33), and (11.08 +/- 3.18) kPa, respectively, showing significant differences among 3 groups (P < 0.05). After stained with FDA/PI, most cells were stained green, and few were stained red. The histological observation results showed that the chondrocytes in C12 hydrogels aggregated obviously with strong heterochromia, chondrocytes in C8 hydrogels aggregated partly with obvious heterochromia, and chondrcytes in C6 hydrogels uniformly distributed with weak heterochromia. Real-time fluorescent quantitative PCR results showed that the mRNA expressions of collagen type II and Aggrecan were at the same level in C12, C8, and C6; the expressions of collagen type I, Sox9, and collagen type X were up-regulated with the increase of collagen type I hydrogels concentration, and the expressions were the highest at 12 mg/mL and were the lowest at 6 mg/mL, showing significant differences among 3 groups (P < 0.05).
Increasing the concentration of collagen hydrogels leads to better mechanical properties and higher shrink-resistance, but it may induce the up-regulation of cartilage fibrosis and hypertrophy related gene expression.
研究Ⅰ型胶原蛋白浓度对胶原水凝胶物理化学性质的影响，并分析不同浓度Ⅰ型胶原蛋白水凝胶对体外软骨细胞表型及基因表达的影响。
分别制备浓度为12、8和6mg/mL的三种胶原水凝胶（C12、C8和C6）。对水凝胶的微观结构、压缩模量和溶胀率进行测量和分析。将第2代软骨细胞分别与三种胶原水凝胶进行体外共培养。培养1天后，用双醋酸荧光素（FDA）/碘化丙啶（PI）染色，在共聚焦激光显微镜下观察细胞活性。培养14天后，进行苏木精-伊红（HE）染色和甲苯胺蓝染色以观察组织形态，并通过实时荧光定量PCR测定软骨细胞相关基因（Ⅱ型胶原蛋白、聚集蛋白聚糖、Ⅰ型胶原蛋白、Ⅹ型胶原蛋白、Sox9）的mRNA表达。
随着Ⅰ型胶原蛋白浓度从6mg/mL增加到12mg/mL，胶原水凝胶的物理化学性质发生显著变化：纤维网络变得致密；C6、C8和C12在192小时时的溶胀率分别为0.260±0.055、0.358±0.072和0.539±0.033，三组间差异有统计学意义（P<0.05）；压缩模量分别为（4.86±0.96）、（7.09±2.33）和（11.08±3.18）kPa，三组间差异有统计学意义（P<0.05）。经FDA/PI染色后，大多数细胞染成绿色，少数染成红色。组织学观察结果显示，C12水凝胶中的软骨细胞明显聚集，异染色质强；C8水凝胶中的软骨细胞部分聚集，异染色质明显；C6水凝胶中的软骨细胞均匀分布，异染色质弱。实时荧光定量PCR结果显示，Ⅱ型胶原蛋白和聚集蛋白聚糖的mRNA表达在C12、C8和C6中处于同一水平；Ⅰ型胶原蛋白、Sox9和Ⅹ型胶原蛋白的表达随着Ⅰ型胶原蛋白水凝胶浓度的增加而上调，在12mg/mL时表达最高，在6mg/mL时表达最低，三组间差异有统计学意义（P<0.05）。
增加胶原水凝胶浓度可导致更好的力学性能和更高的抗收缩性，但可能诱导软骨纤维化和肥大相关基因表达上调。
