摘要
目的:利用三维动态诱导的同种异体非软骨来源种植细胞,复合可注射型支架材料聚氧乙烯聚氧丙烯共聚物Pluronic F-127修复关节软骨缺损。方法:实验于2005-06/2006-03在哈尔滨医科大学附属第一医院动物实验中心完成。①选取2个月龄新西兰大白兔27只,3只用于骨髓间充质干细胞的分离,剩余24只随机数字表法分为诱导细胞复合支架组、单纯支架组、空白对照组,8只(16膝)/组。可注射型支架材料Pluronic F-127为白色粉沫状,无味,其水溶液在低温4℃呈液态,37℃形成固态凝胶(sigma公司,产品批号P2443)。②兔麻醉后穿刺抽取胫骨骨髓,分离培养骨髓间充质干细胞,取3代细胞进行实验。③收集细胞,离心成团,分4组不同诱导条件。三维动态培养组:10g/L藻酸钠溶液洗涤并重新悬浮细胞,细胞浓度为5×1010L-1,滴入200mmol/L氯化钙溶液,立即形成藻酸钙凝胶微球,细胞被悬浮固定于球内部,静止5min,取出凝胶微球,置于旋转式细胞培养系统,微重力条件下动态诱导。二维动态培养组:细胞直接接种于旋转式细胞培养系统,微重力条件下动态诱导。三维静态培养组:藻酸钙凝胶微球悬浮细胞接种于培养瓶静态培养。二维静态培养组:细胞直接接种于平面培养瓶静态培养。各组细胞诱导培养2周,制备切片,分别行甲苯胺蓝染色及Ⅰ、Ⅱ型胶原免疫组织化学染色。柠檬酸钠溶液溶解藻酸钙凝胶微球,测定各组胶原和蛋白多糖含量。④各组兔的双膝均制备膝关节全层软骨缺损模型。诱导细胞复合支架组选取诱导分化最佳的骨髓间充质干细胞与25%可注射型支架材料Pluronic F-127在低温下混匀,重悬,细胞终浓度为5×1010L-1,注入股骨内髁软骨缺损区。单纯支架组只将25%Pluronic F-127注入股骨内髁软骨缺损区。空白对照组关节软骨缺损区不作任何处理。各组分别于术后4,8,12,24周取材,大体及镜下观察关节软骨表面缺损修复情况,同时进行组织学评分。结果:24只兔全部进入结果分析。①骨髓间充质干细胞体外诱导培养及鉴定结果:藻酸钠接触钙离子迅速形成透明凝胶微球,有光泽,凝胶微球中的细胞呈球形,核仁清晰,与正常软骨细胞立体结构相似。培养过程中各组细胞甲苯胺兰染色均呈阳性,并表达Ⅱ型胶原,但无明显Ⅰ型胶原表达。②不同体外诱导分化条件下细胞生化指标的表达:三维、二维动态培养组的胶原和蛋白多糖含量均明显高于三维、二维静态培养组,且以三维动态培养组效果最佳[(0.078±0.002),(0.048±0.002),(0.035±0.001)A,P<0.05;(0.111±0.003),(0.069±0.003),(0.058±0.002)A,P<0.05]。③关节软骨缺损修复情况:诱导细胞复合支架组术后12周修复软骨表面光滑,质地坚硬,为透明软骨组织结构,与周围软骨结合紧密,至24周仍保持透明软骨形态;而单纯支架组、空白对照组均未被修复。术后4,8,12,24周,诱导细胞复合支架组关节软骨组织学评分均优于单纯支架组、空白对照组(P<0.05),且术后时间越长,修复效果越佳。结论:微重力条件下三维动态诱导骨髓间充质干细胞可提高分化质量,获得的种植细胞复合可注射型支架材料修复关节软骨缺损能够取得稳定的长期修复效果。
AIM： To repair the articular cartilage defects with the non-cartilage derived plant cells induced from allogenic mesenchymal stem cells （MSCs） in microgravity dynamic culture system of three-dimensional （3D） Pluronic F-127.
METHODS： The experiment was conducted in the Experimental Animal Center, First Affiliated Hospital of Harbin Medical University from June 2005 to March 2006. ①Twenty-seven two-month New Zealand rabbits were selected, of which 3 were used as the donors of mesenchymal stem cells, and the left rabbits were randomly divided into induced cell and Pluronic group, pluronic group and control group with 8 rabbits （16 knees） in each group. Pluronic F-127 （Sigma Company, No. P2443） was white power with no odour, and its water solution was liquidness at 4 %, and solid gelatum at 37 %. ②After the rabbits were anesthetized, the bone marrow was taken from superior extremity of tibia to isolate and culture the MSCs; the 3^rd passage cells ware used in the experiment. ③The MSCs were collected and centrifuged, then divided into four groups with different inducing conditions： 3D dynamic culture group： The cells were washed with 10 g/L algin solution and renewed to suspend cells with density of 5×10^10 L^-l; 200 mmol/L calcium Chloride solution was dropped into and calcium alginate gelatum microballoons formed immediately, and the cells were floated and fixed in the microballoons. Five minutes later, the gelatum microballoons were dislodged and put in Revolve Cell Culture System （RCCS） for dynamic culture under microgravity. 2D dynamic culture group： The cells were given inoculation directly in RCCS for dynamic culture under microgravity. 3D static culture group： The cells in the calcium alginate gelatum were inoculated directly in a culture flask for static culture. 2D static culture group： The cells were inoculated directly in a plane culture flask for static culture. Each group was cultured for 2 weeks, following by slices preparation and toluidine orchid dyeing, collogen Ⅰ, Ⅱ immunohistochemical staining. Citrate sodium solution was adopted to dissolve the calcium alginate gelatum microballoons to determine the collogen and proteoglycan contents of the four groups. ④The models of full-thickness defects of articular cartilage were prepared. Induced cells and Pluronic group： The best induced MSCs compounded with 25% Pluronic F-127 ware mixed at 4% and suspended, until the final concentration of MSCs was 5×10^10 L^-1, which poured into the articular cartilage defects. Pluronic group： 25% Pluronic F-127 was poured into the articular cartilage defects. The articular cartilage defects of the control group were not given any treatment. The animals of each group were killed at 4, 8, 12 and 24 weeks to observe the repair effect of the defects grossly and microscopically. Meanwhile, the histological examinations were performed.
RESULTS： All 24 animals entered the result analysis. ②The results of culture and examination of MSCs： Algin contacted Ca^2＋ formed quickly transparent gelatum microballoons, which was lustered; the cells in the microballoons were globular, with clear nucleoles, and the stereochemical structure was similar with normal chondrocyte. Toluidine blue dyeing was positive, and expressed collagen Ⅱ but no collagen Ⅰ was found. ②Expressions of cell biochemical indicator of the four groups under different inducing conditions： Collagen Ⅱ and proteoglycan production of the 3D and 2D dynamic culture groups were higher than the two static culture groups, and the 3D dynamic culture group was the best [（0.078±0.002）, （0.048±0.002）, （0.035±0.001）A, P 〈 0.05; （0.111±0.003）, （0.069±0.003）, （0.058±0.002）A, P 〈 0.05]. ③Repair effect of the articular cartilage defects： In the induced cells plus Pluronic group, the cartilage defects were smoothly repaired by hard hyaline-cartilage like tissue after 12 weeks, and sustained the histological appearance to 24 weeks. No obvious repair was observed in the pluronic groups and control group. After 4, 8, 12 and 24 weeks, the articular cartilage histological score of induced cells plus Pluronic group was better than the pluronic groups and control group （P 〈 0.05）, and the repaidng effect was better with time.
CONCLUSION： MSCs differentiation quality is improved in 3D microgravity environment, and the cultured cells combined the injectable scaffold could repair the cartilage defects with a long-term and stable effect.
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2609-2612,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
黑龙江省自然科学基金~~
