Three-dimensional culture of marrow mesenchymal stem cells under microgravity on injectable Pluronic F-127 in the repair of articular cartilage defects
- VernacularTitle:微重力三维动态诱导骨髓间充质干细胞复合可注射型支架材料Pluronic F-127修复关节软骨缺损
- Author:
Hc WANG
;
Zy ZHANG
;
Wgfirst XIN
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2007;0(14):-
- CountryChina
- Language:Chinese
-
Abstract:
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 3rd passage cells were 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?1010 L-1; 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, collogenⅠ, 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 were mixed at 4℃ and suspended, until the final concentration of MSCs was 5?1010 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. ①The results of culture and examination of MSCs: Algin contacted Ca2+ 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 collagenⅠwas 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
