动态和静态压力作用对山羊颞下颌关节盘细胞凝胶组织基质代谢的影响
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摘要
目的：
采用酶消化结合组织块培养法对山羊颞下颌(temporomandibular joint, TMJ)关节盘细胞进行体外培养和扩增,探索TMJ关节盘细胞体外培养及扩增的新方法。对山羊关节盘细胞进行体外琼脂糖凝胶三维培养,探讨动态压力和静态压力对凝胶组织细胞外基质分泌的影响。
方法：
1.在无菌条件下,切取一月龄山羊TMJ关节盘,剪至1.0 mm3的碎块,用0.25%胰酶、0.1%Ⅰ型胶原酶消化关节盘组织块,将消化好的组织块置入6孔板中培养。在倒置相差显微镜下连续观察细形态变化及贴壁率,甲苯胺蓝染色、Ⅰ型胶原免疫组化染色进行细胞鉴定,测定其生长曲线。
2.将关节盘细胞培养至第2代时,转入琼脂糖凝胶中进行三维培养。仿照Shram系统构建体外立体培养的细胞动态压力加载模型。培养三天后将组织块随机分为三组：一组为动态加力组,即参照Shram等压力加载条件给予关节盘细胞凝胶组织0.2Mpa,频率0.1Hz的周期性压力；另一组为静态加力组,即关节盘细胞凝胶组织在0.2MPa静态压力作用下培养；对照组凝胶组织在常规条件下静态培养。加力完成后对琼脂糖凝胶内的关节盘细胞进行组织学染色和免疫组化染色,Ⅰ型胶原免疫组化染色结果使用图像分析系统做半定量分析。
结果：
1.原代培养的关节盘纤维软骨细胞4天可观察到贴壁细胞,7天贴壁细胞逐渐增多,第10天时细胞彼此相连,铺满平底,细胞以梭形为主,部分为多角形。传代后12小时贴壁率达90%,大部分为多角形,4-5天即可长满瓶底。甲苯胺蓝染色可见异染颗粒,Ⅰ型胶原免疫组化染色胞浆内可见棕黄色颗粒。
2.在琼脂糖凝胶中培养的关节盘细胞能够维持其表型,能够正常合成细胞外基质。受0.2MPa,频率0.1Hz的周期性压力和0.2 MPa的静态压力刺激后,GAG和Ⅰ型胶原的表达均增加,且两者无显著性差异。
结论：
1.酶消化结合组织块培养法培养的山羊TMJ关节盘细胞具有较强的增殖能力,可作为TMJ关节盘组织工程中获取大量原代细胞的实用方法。
2.周期性动态压力和间断静态压力都对关节盘细胞的细胞外基质合成具有促进作用,力学刺激同细胞外基质的分泌密切相关。在本实验条件下,两种压力模式产生的结果无显著性差异。
Objective:
The purpose of this study was to search a novel method suitable for proliferation of temporomandibular joint (temporomandibular joint, TMJ) disc cells, to observe the biological characteristics of cultured TMJ disc cells and to investigate how dynamic and static compression influences extracellular matrix synthesis of goat temporomandibular joint disc cells cultured in agrose gel.
Methods:
1. The small pieces (1.0 mm3 in size) of isolated and chopped TMJ disc tissues, which were dissected from two one-month-goats under sterile conditions, were digested with 0.25% trypsin and 0.1% typeⅠcollagenase, then put into 6-well plate to be cultured with DMEM. The phase contrast microscope was used to observe the morphological changes and attachment efficiency of TMJ cells. Immunohistochemical staining for type I collagen and toluidine blue staining for proteoglycans were used for identifying extracellular matrix characterization. The cell growth curve was plotted.
2. Temporomandibular joint disc cells were mixed with agarose, serum and medium to form agrose-gel constructs. After cultured for 3d in static state, the constructs were divided into three groups:dynamic compression group, static compression group and static comparison group. The compression unit was set up followed the method of Sharam et al. The constructs were exposed to 0.2MPa, 0.1Hz cyclic loading and 0.2MPa static loading. Each group was cultured for 2 weeks.2 weeks later, the TMJ disc cells were identified with Safranin-O,Toluidine blue and immunohistochemistry of collagen type I staining,and the histochemistry analysis was used to examine distribution of extracellular matrix in constructs.
Results:
1. On the 4th day of culture, primary TMJ disc cells (fibrochondrocytes) could be seen in adherence to the dishes. After the 7th days, the number of adhesive fibrochondrocytes was increased. On the day 10, the cells were spindle-shaped and some of them were polygon-shaped, started to contact with each other to form a monolayer on which cells covering the bottom of the 6-well plate. At the 12th hour after passage, the seeding efficiency of the fibrochondrocytes was 90%. From the 5th to 6th day after passage, the bottom of the culture bottle was filled with these cells. Immunohistochemical staining for type I collagen and toluidine blue staining for GAGs exhibited positive results.
2. Agarose gel was found to provide a good microenvironment for the formation of extracellular matrix. The amount of the extracellular matrix in dynamic compression group and static compression group increased compared with the static comparison group, however, there was no significant difference between the dynamic compression group and the static compression group.
Conclusions:
1. The goat TMJ disc cells present proliferous ability in vitro culture using enzyme digestion with explant culture method. It shows that the method is a practical way to obtain numberous primary cells for the TMJ disc tissue engineering.
2. The compression has an important effect on metabolism of constructs composed of TMJ disc cells and agarose gel. Both the dynamic and static compression can promote the production of main extracellular matrix in native TMJ disc tissue such as GAGs and type I collagen.
采用酶消化结合组织块培养法对山羊颞下颌(temporomandibular joint, TMJ)关节盘细胞进行体外培养和扩增,探索TMJ关节盘细胞体外培养及扩增的新方法。对山羊关节盘细胞进行体外琼脂糖凝胶三维培养,探讨动态压力和静态压力对凝胶组织细胞外基质分泌的影响。
方法：
1.在无菌条件下,切取一月龄山羊TMJ关节盘,剪至1.0 mm3的碎块,用0.25%胰酶、0.1%Ⅰ型胶原酶消化关节盘组织块,将消化好的组织块置入6孔板中培养。在倒置相差显微镜下连续观察细形态变化及贴壁率,甲苯胺蓝染色、Ⅰ型胶原免疫组化染色进行细胞鉴定,测定其生长曲线。
2.将关节盘细胞培养至第2代时,转入琼脂糖凝胶中进行三维培养。仿照Shram系统构建体外立体培养的细胞动态压力加载模型。培养三天后将组织块随机分为三组：一组为动态加力组,即参照Shram等压力加载条件给予关节盘细胞凝胶组织0.2Mpa,频率0.1Hz的周期性压力；另一组为静态加力组,即关节盘细胞凝胶组织在0.2MPa静态压力作用下培养；对照组凝胶组织在常规条件下静态培养。加力完成后对琼脂糖凝胶内的关节盘细胞进行组织学染色和免疫组化染色,Ⅰ型胶原免疫组化染色结果使用图像分析系统做半定量分析。
结果：
1.原代培养的关节盘纤维软骨细胞4天可观察到贴壁细胞,7天贴壁细胞逐渐增多,第10天时细胞彼此相连,铺满平底,细胞以梭形为主,部分为多角形。传代后12小时贴壁率达90%,大部分为多角形,4-5天即可长满瓶底。甲苯胺蓝染色可见异染颗粒,Ⅰ型胶原免疫组化染色胞浆内可见棕黄色颗粒。
2.在琼脂糖凝胶中培养的关节盘细胞能够维持其表型,能够正常合成细胞外基质。受0.2MPa,频率0.1Hz的周期性压力和0.2 MPa的静态压力刺激后,GAG和Ⅰ型胶原的表达均增加,且两者无显著性差异。
结论：
1.酶消化结合组织块培养法培养的山羊TMJ关节盘细胞具有较强的增殖能力,可作为TMJ关节盘组织工程中获取大量原代细胞的实用方法。
2.周期性动态压力和间断静态压力都对关节盘细胞的细胞外基质合成具有促进作用,力学刺激同细胞外基质的分泌密切相关。在本实验条件下,两种压力模式产生的结果无显著性差异。
Objective:
The purpose of this study was to search a novel method suitable for proliferation of temporomandibular joint (temporomandibular joint, TMJ) disc cells, to observe the biological characteristics of cultured TMJ disc cells and to investigate how dynamic and static compression influences extracellular matrix synthesis of goat temporomandibular joint disc cells cultured in agrose gel.
Methods:
1. The small pieces (1.0 mm3 in size) of isolated and chopped TMJ disc tissues, which were dissected from two one-month-goats under sterile conditions, were digested with 0.25% trypsin and 0.1% typeⅠcollagenase, then put into 6-well plate to be cultured with DMEM. The phase contrast microscope was used to observe the morphological changes and attachment efficiency of TMJ cells. Immunohistochemical staining for type I collagen and toluidine blue staining for proteoglycans were used for identifying extracellular matrix characterization. The cell growth curve was plotted.
2. Temporomandibular joint disc cells were mixed with agarose, serum and medium to form agrose-gel constructs. After cultured for 3d in static state, the constructs were divided into three groups:dynamic compression group, static compression group and static comparison group. The compression unit was set up followed the method of Sharam et al. The constructs were exposed to 0.2MPa, 0.1Hz cyclic loading and 0.2MPa static loading. Each group was cultured for 2 weeks.2 weeks later, the TMJ disc cells were identified with Safranin-O,Toluidine blue and immunohistochemistry of collagen type I staining,and the histochemistry analysis was used to examine distribution of extracellular matrix in constructs.
Results:
1. On the 4th day of culture, primary TMJ disc cells (fibrochondrocytes) could be seen in adherence to the dishes. After the 7th days, the number of adhesive fibrochondrocytes was increased. On the day 10, the cells were spindle-shaped and some of them were polygon-shaped, started to contact with each other to form a monolayer on which cells covering the bottom of the 6-well plate. At the 12th hour after passage, the seeding efficiency of the fibrochondrocytes was 90%. From the 5th to 6th day after passage, the bottom of the culture bottle was filled with these cells. Immunohistochemical staining for type I collagen and toluidine blue staining for GAGs exhibited positive results.
2. Agarose gel was found to provide a good microenvironment for the formation of extracellular matrix. The amount of the extracellular matrix in dynamic compression group and static compression group increased compared with the static comparison group, however, there was no significant difference between the dynamic compression group and the static compression group.
Conclusions:
1. The goat TMJ disc cells present proliferous ability in vitro culture using enzyme digestion with explant culture method. It shows that the method is a practical way to obtain numberous primary cells for the TMJ disc tissue engineering.
2. The compression has an important effect on metabolism of constructs composed of TMJ disc cells and agarose gel. Both the dynamic and static compression can promote the production of main extracellular matrix in native TMJ disc tissue such as GAGs and type I collagen.
引文
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