壳聚糖/Ⅰ型胶原复合支架与滑膜成纤维样细胞构建颞下颌关节盘的研究

Temporomandibular joint disc tissue engineering of synovial fibroblast-like cells cultured in the chitosan/collagen I scaffolds

目的 探讨利用滑膜成纤维样细胞（SFBs）及壳聚糖/Ⅰ型胶原（CS/COL-Ⅰ）复合支架构建颞下颌关节盘软骨的可行性.方法 获取兔颞下颌关节滑膜组织进行SFBs培养,第3～5代SFBs三维培养于通过冷冻干燥法制备的CS/COL-Ⅰ支架材料中7 d,用噻唑蓝（MTT）比色法分别在1、3、5、7 d检测支架材料对细胞增殖的影响.细胞支架复合物在体外经过人重组转化生长因子β1（rhTGF,10μg/L）和碱性成纤维细胞生长因子（bFGF,50 μg/L）诱导后,植入裸鼠体内4、8周后获取标本,进行组织学检测细胞在支架材料上的生长状态及黏多糖（GAGs）形成,免疫组织化学和逆转录-聚合酶链反应（RT-PCR）半定量分析Ⅱ型胶原的表达.结果 支架材料表面及内部均呈多孔隙蜂窝状结构.SFBs在CS/COL-Ⅰ中的增殖要明显高于平板培养（P＜0.05）.细胞支架复合物植入裸鼠体内4、8周后,组织学及免疫组织化学半定量分析显示在8周时GAGs（6.900±0.316）和Ⅱ型胶原（0.0952±0.0248）IA/μm2与4周时GAGs（3.600±0.699）和Ⅱ型胶原（0.0411±0.0127）IA/μm2差异有统计学意义（P＜0.05）.结论 体外诱导后的SFBs/CS-COL-Ⅰ复合物具有应用于组织工程化颞下颌关节盘构建的可能.

Objective To evaluate the potential of synovial fibroblast-like cells （SFBs） cultured in the chitosan/collagen I （ CS/COL- I ） scaffold for the tissue engineering of temporomandibular joint （TMJ） disc. Methods CS/COL-I copolymers were synthesized by the freeze-drying method. The morphological characteristics were evaluated under the scanning electronic microscopy. Synovium was harvested from rabbit TMJs. The third to fifth passage SFBs were cultured onto the CS/COL- I copolymers. MTT assay was employed to evaluate the proliferation and viability of SFBs cultured on the CS/COL- I at the day 1,3,5,7.After being induced by the cocktail application of recombinant human transforming growth factor β1（rhTGF-Bl, 10 μg/L） and basic fibroblast growth factor （bFGF, 50 μg/L） in vitro, SFBs/CS-COL- I constructs were transplanted into nude mice to evaluate the chondrogenic formation through histo-logical and immulohistochemical staining and reverse transcription polymerase chain reaction （PCR） at the week 4 and 8.Results The synthesized CS/COL-1 scaffold was spongy-like structure with a bulk of pores. MTT results showed that the scaffolds could improve SFBs proliferation （P 〈 0.05）. Immulohisto-chemically and histologically, semi-quantitative analysis of the implants revealed that the contents of GAGs and collagen type Ⅲat the week 8 after transplantation were （6.900 ±0.316） and （0.0952 ±0.0248） IA/μm2 respectively and at the week 4 （3.600 ± 0.699 ） and （0.0411 ±0.0127） IA/μm2 respectively. Conclusion It is suggested that the SFBs/CS-COL- I constructs could be a candidate for TMJ disc tissue engineering.