摘要
目的探讨Ⅰ型胶原浓度对胶原水凝胶理化性能的影响,以及在体外组织工程模型中不同浓度的Ⅰ型胶原水凝胶对软骨细胞表型和基因表达的影响。方法制备Ⅰ型胶原浓度为12、8、6 mg/mL的3种胶原水凝胶,记为C12、C8、C6。扫描电镜观察形貌,并通过溶胀性能和抗压性能测试表征其理化性能。取2只新生7日龄新西兰大白兔的关节软骨,酶消化法分离、培养软骨细胞。取第2代软骨细胞与3种胶原水凝胶复合体外培养(C12组、C8组及C6组),1 d后采用二乙酸荧光素(fluorescein diacetate,FDA)/碘化丙啶(propidium iodide,PI)染色,激光共聚焦显微镜观察细胞活性,体外培养14 d后行HE和甲苯胺蓝染色观察组织学形态,实时荧光定量PCR测定软骨细胞相关基因,即Ⅱ型胶原、蛋白聚糖(Aggrecan)、Ⅰ型胶原、Ⅹ型胶原、Sox9 mRNA表达。结果随着Ⅰ型胶原浓度从6 mg/mL提高到12 mg/mL,胶原水凝胶的理化性质出现明显变化,扫描电镜下纤维网络变得致密;192 h时C6、C8、C12溶胀率依次增大,分别为0.260±0.055、0.358±0.072、0.539±0.033,比较差异均有统计学意义(P<0.05);压缩模量逐渐增加,分别为(4.86±0.96)、(7.09±2.33)、(11.08±3.18)kPa,比较差异均有统计学意义(P<0.05)。体外培养1 d,3组凝胶中软骨细胞经FDA/PI染色后,绝大部分被染成绿色,仅有少数被染成红色。14 d后组织学观察示:C12组软骨细胞在组织中聚集明显,并出现明显的甲苯胺蓝异染为紫红色的现象;C8组中也有部分增殖聚集的区域,细胞周围有较明显的甲苯胺蓝异染现象;C6组细胞分布较为均匀,也有较明显的甲苯胺蓝异染现象。实时荧光定量PCR检测显示,3组Ⅱ型胶原mRNA和Aggrecan mRNA表达水平相近;Ⅰ型胶原、Sox9和X型胶原mRNA表达水平C12组最高,C6组最低,C8组介于二者之间,组间差异均有统计学意义(P<0.05)。结论提高Ⅰ型胶原浓度至12 mg/mL后,胶原水凝胶具有更好的理化性质,但软骨细胞纤维化和肥大相关基因表达也有所上调。
Objective 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.Methods Three kinds of collagen hydrogels with concentrations of 12,8,and 6mg/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.Results 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 12mg/mL and were the lowest at 6 mg/mL,showing significant differences among 3 groups（P 0.05）.Conclusion 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.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2012年第11期1356-1361,共6页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家重点基础研究发展计划(973)资助项目(2011CB606201)~~
关键词
软骨组织工程
Ⅰ型胶原
胶原水凝胶
软骨细胞
支架材料
Cartilage tissue engineering Collagen type I Collagen hydrogel Chondrocytes Scaffold material
