摘要
背景:选择适合的生物支架与骨髓间充质干细胞作为新型的种子细胞复合能否构建出理想的组织工程化软骨?目的:观察将扩增的兔骨髓间充质干细胞接种于聚羟基乙酸构建人工软骨的可行性。设计:单一样本观察。单位:泸州医学院附属医院耳鼻咽喉头颈外科。材料:实验于2004-10/2005-10在泸州医学院完成。选用8只日本大耳兔,雌雄不拘,清洁级,二三个月龄,体质量1.5～2.0kg,常温常湿环境饲养。聚羟基乙酸由美国Albany公司生产。方法:分离、获取、扩增实验兔骨髓间充质干细胞,将聚羟基乙酸剪成1cm×1cm×1cm大小,并以多聚赖氨酸包被,通过将扩增的骨髓间充质干细胞接种于聚羟基乙酸上,按4mL/cm3多点播散的方式均匀地接种在预湿的聚羟基乙酸支架上,体外诱导培养3周,作为实验组。将不加入骨髓间充质干细胞的聚羟基乙酸支架作为对照组。将实验组和对照组的标本分别植入4只自体兔腹腔内,进行体内培养6～12周后,分别取出实验组与对照组的标本,对其进行大体观察。同时以100g/L中性甲醛固定,行5μm切片,采用苏木精-伊红染色观察标本的组织形态学特点,进行阿新蓝染色以观察酸性粘多糖形成,进行甲苯胺蓝染色以观察异染性基质的形成,进行免疫组织化学染色检测Ⅱ型胶原蛋白的表达。主要观察指标:两组支架植入实验兔体内后6,12周时标本大体观察结果、不同染色观察结果及Ⅱ型胶原蛋白的表达。结果:纳入8只实验兔均进入结果分析。①支架植入实验兔体内后6及12周标本大体观察结果:支架植入兔腹腔6周后,实验组标本均仍被腹腔大网膜包裹,剥去大网膜后,有3个标本成淡黄色,光滑,质地中,外形与植入前基本一致,1个标本呈灰黑色,质地很软,标本不能成形。12周后,实验组标本的外形仍与植入前基本一致,呈灰白色,光滑,但质地明显较硬。而第6,12周的对照组的标本大部分均被吸收,以第12周的标本更明显,且均未见软骨样组织形成。②支架植入实验兔体内后6及12周不同染色观察结果及Ⅱ型胶原蛋白的表达:苏木精-伊红染色结果显示:6周时,有软骨陷窝样结构开始形成,聚羟基乙酸支架开始降解;12周时,复合物呈现软骨组织样的形态,有较明显的软骨陷窝样结构形成,细胞排列规则,成群存在于软骨陷窝样结构内,边缘的细胞小,中央的细胞大,聚羟基乙酸基本完全消失。阿新蓝染色结果:6周时,组织内有部分区域被染成淡蓝色;12周时组织的基质被广泛染成蓝紫色,说明有大量的酸性粘多糖形成。苯胺蓝染色结果:6周时,组织内可见蓝色异染性基质;12周时组织内呈现很强的蓝色异染。免疫组织化学染色结果:6周时,胞浆内可见棕黄色阳性颗粒,基质内有少量的Ⅱ型胶原蛋白表达;12周时,胞浆、基质内均有较强的阳性表达。Ⅱ型胶原蛋白的表达:6周时,胞浆内可见深棕黄色阳性颗粒,基质内有少量的Ⅱ型胶原mRNA表达;12周时,胞浆、基质内均有强的阳性表达。对照组标本基本上已被吸收,没有成形的组织工程化标本。结论:兔骨髓间充质干细胞在诱导剂作用下,经体外和体内培养后,可生成组织工程化类软骨。
BACKGROUND： Whether choosing a suitable biological scaffold compounding with mesenchymal stem cells （MSCs） can construct an ideal tissue-engineering cartilage or not should be researched further.
OBJECTIVE： To investigate the feasibility of constructing artificial cartilage by using amplified rabbit MSCs which were inoculated on poly-glycolic acid （PGA）.
DESIGN： Single sample observation.
SETTING ： Department of Otolaryngology-Head ＆ Neck Surgery, Affiliated Hospital of Sichuan Luzhou Medical College.
MATERIALS ： The experiment was carried out in the Luzhou Medical College from October 2004 to October 2005. A total of 8 Japanese large ear rabbits, of both genders, clean grade, aged from 2 to 3 months, weighing 1.5-2.0 kg, were fed in normal temperature and humidity. Poly-glycolic acid was provided by Albany Company, USA.
METHODS： Rabbit MSCs were separated, obtained and amplified. In addition, poly-glycolic acid was sheared into pieces with the size of 1 cm × 1 cm × 1 cm and embedded with poly-L-lysine. Amplified MSCs were inoculated on the surface of poly-glycolic acid, and then, they were averagely grown on pre-wet scaffold of poly-glycolic acid according to 4 mL/cm^2 multi-points spreading style and cultured in vitro for 3 weeks. After the operations mentioned above, samples were regarded as the experimental group. Scaffolds of poly-glycolic acid without MSCs were considered as the control group. Samples in both experimental group and control group were transplanted into abdominal cavity of 4 rabbits, respectively, cultured in vivo for 6-12 weeks, taken out and observed generally. Meanwhile, the samples were fixed with 100 g/L neutrality formaldehyde, cut into sections with the thickness of 5 μm, stained with haematine-eosin （HE） and observed their histomorphological characteristics. Moreover, the samples were stained with alcian blue for observation of glycosaminoglycans formation, with toluidine blue for observation of metachromasia matrix formation, and with immunohistochemical staining for detection of type- Ⅱ collagen expression.
MAIN OUTCOME MEASURES： Generally observational results of two kinds of scaffolds at 6 and 12 weeks after transplanting into experimental rabbits, various staining results and expression of type- Ⅱ collagen.
RESULTS ： A total of 8 experimental rabbits were involved in the final analysis. ① Generally observational results of two kinds of scaffolds at 6 and 12 weeks after transplanting into experimental rabbits： At 6 weeks after transplanting scaffold into abdominal cavity of rabbits, samples in the experimental group were still coated with greater omentum of abdominal cavity. After clearing greater omentum, three samples were light yellow, smooth and moderate quality; meanwhile, their appearances were coincidence with those before transplantation. However, one sample was gray black and soft quality; meanwhile, it was not able to take shape. Twelve weeks later, appearances of samples in the experimental group were still coincidence with those before transplantation. They were gray white, smooth but hard quality. However, samples in the control group were mostly absorbed at 6 and 12 weeks after transplantation, especially, samples were remarkably absorbed at 12 weeks after transplantation. Any tissue like cartilage did not form in both two durations. ② Various staining results and expression of type-Ⅱ collagen at 6 and 12 weeks after transplanting scaffolds into experimental rabbits： Results of HE staining showed that, at 6 weeks after transplantation, structure of cartilage lacuna-like was started form, and PGA scaffold began to be degraded. In contrast, at 12 weeks postoperatively, some cartilage-like tissue were observed in compound, cartilage lacuna-like structure formed obviously; cells arrayed regularly and geminately existed in cartilage lacuna-like atructure; the smaller cell sizes observed at borders and the bigger ones observed at the center; PGA degraded completely. Results of alcian blue staining showed that, at 6 weeks after transplantation, a partial of regions in tissue were light blue; meanwhile, at 12 weeks after transplantation, matrixes in tissue were mostly blue. This suggested that a lot of glycosaminoglycans were formed. Results of toluidine blue staining suggested that, at 6 weeks after transplantation, blue metachromasia matrixes were observed in tissue; meanwhile, at 12 weeks after transplantation, blue metachromasia matrixes were stronger and stronger in tissue. Results of immunohistochemical staining indicated that, at 6 weeks after transplantation, buffy positive granules were observed in plasma and a few of type-Ⅱ collagen expressed in matrix; meanwhile, at 12 weeks after transplantation, powerfully positive expressions were observed in both plasma and matrix. Expression of type- Ⅱ collage showed that, at 6 weeks after transplantation, dark buffy positive granules were observed in plasma and a few of type-Ⅱ collagen mRNA expressed in matrix; meanwhile, at 12 weeks after transplantation, powerfully positive expressions were observed in both plasma and matrix. Samples in the control group were completely absorbed and any tissue-engineering samples did not form.
CONCLUSION： Affecting by osteogenic inducer, rabbit MSCs can generate tissue-engineering cartilage after culture in vitro and in vivo.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第14期2761-2764,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
四川省科技厅科研基金资助(2006J13163)~~
