体外构建可注射式组织工程髓核的初步研究  

CONSTRUCTION OF INJECTABLE TISSUE ENGINEERED NUCLEUS PULPOSUS IN VITRO

作　　者：田华科[1] 王建[1] 陈超[1] 刘杰[1] 周跃[1] 

机构地区：[1]第三军医大学新桥医院骨科,重庆400037

出　　处：《中国修复重建外科杂志》2009年第2期173-177,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30772186);重庆市自然科学基金资助项目(2007BB5056)~~

摘　　要：目的研究兔髓核细胞在温敏性壳聚糖水凝胶支架上的生长情况,探讨其构建可注射式组织工程髓核的可行性。方法取3周龄新西兰乳兔,体重150～200 g,雌雄不限,分离培养兔髓核细胞。以壳聚糖、β-甘油磷酸钠水溶液和羟乙基纤维素溶液为原料制备壳聚糖水凝胶支架,并行物理性状及大体观察。将支架与兔髓核细胞复合构建组织工程髓核。复合培养2 d,观察髓核细胞在壳聚糖水凝胶中的存活情况;复合培养1周,扫描电镜观察髓核细胞在支架上的生长情况;复合培养3周后,行组织学、免疫组织化学检测,并用RT-PCR检测其分泌的聚集蛋白聚糖、ColⅡmRNA表达。结果制备的温敏性壳聚糖水凝胶室温呈液态,37℃放置15 min可发生交联反应成为固态凝胶。吖啶橙/碘化丙啶染色示壳聚糖水凝胶中大多数髓核细胞存活,少数死亡,细胞存活率>90%;扫描电镜示髓核细胞分布于网状支架中,细胞表面有分泌的ECM;HE、甲苯胺蓝、番红O染色及免疫组织化学染色结果显示软骨细胞具有分泌ECM的功能;RT-PCR检测示髓核细胞在壳聚糖水凝胶支架中立体培养3周后ColⅡ、聚集蛋白聚糖mRNA分别为0.631±0.064、0.832±0.052,较单纯培养(0.528±0.039、0.773±0.046)分泌基质的能力更强,差异均有统计学意义(P<0.05)。结论温敏性壳聚糖水凝胶材料具有良好的细胞相容性,髓核细胞与其复合培养后可保持正常形态及分泌功能,有望成为髓核细胞载体构建组织工程髓核。Objective To investigate the feasibility of using thermo-sensitive chitosan hydrogen as a scaffold to construct tissue engineered injectable nucleus pulposus （NP）. Methods Three-month-old neonatal New Zealand rabbits （male or female） weighing 150-200 g were selected to isolate and culture NP cells. The thermo-sensitive chitosan hydrogel scaffold was made of chitosan, disodium β-glycerophosphate and hydroxyethyl cellulose. Its physical properties and gross condition were observed, The tissue engineered NP was constructed by compounding the scaffold and rabbit NP cells. Then, the viability of NP cells in the chitosan hydrogel was observed 2 days after compound culture and the growth condition of NP cells on the scaffold was observed by SEM 7 days after compound culture. NP cells went through histology and immunohistochemistry detection and their secretion of aggrecan and expression of Col II mRNA were analyzed by RT-PCR 21 days after compound culture. Results The thermo-sensitive chitosan hydrogel was liquid at room temperature and solidified into gel at 37℃ （15 minutes） due to crosslinking reaction. Acridine orange-propidiumiodide staining showed that the viability rate of NP cells in chitosan hydrogel was above 90%. Scanning electron microscope observation demonstrated that the NP cells were distributed in the reticulate scaffold, with ECM on their surfaces. The results of HE, toluidine blue, safranin O and histology and immunohistochemistry staining confirmed that the NP cells in chitosan hydrogel were capable of producing ECM. RT-PCR results showed that the secretion of Col Ⅱ and aggrecan mRNA in NP cells cultured three-dimensionally by chitosan hydrogen scaffold were 0.631 ± 0.064 and 0.832 ±0.052, respectively, showing more strengths of producing matrix than that of monolayer culture （0.528 ± 0.039, 0.773± 0.046） with a significant difference （P 〈 0.05）. Conclusion for NP cells to maintain their normal With good cellular compatibilities, the thermo-sensitive chitosan hydrogel ma

关 键 词：组织工程髓核 温敏性壳聚糖水凝胶 支架材料 兔 

分 类 号：R318[医药卫生—生物医学工程]