不同种子细胞复合壳聚糖水凝胶构建可注射组织工程髓核的比较研究  

COMPARISON STUDY ON INJECTABLE TISSUE ENGINEERED NUCLEUS PULPOSUS CONSTRUCTED BY DIFFERENT CELLS AND CHITOSAN HYDROGEL

作　　者：王建[1] 刘杰[1] 田华科[1] 李长青[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》2010年第7期806-810,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：目的比较兔髓核细胞(nucleus pulposus cells,NPCs)和BMSCs在温敏性壳聚糖水凝胶支架中的生长及细胞外基质合成,筛选用于构建可注射组织工程髓核的种子细胞。方法取3周龄新西兰乳兔,体重150～200g,雌雄不限,分离培养兔NPCs;取1月龄新西兰白兔,体重1.0～1.5kg,雌雄不限,穿刺抽取骨髓分离培养BMSCs。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。取第2代NPCs和第3代BMSCs分别与壳聚糖水凝胶混匀,构建可注射组织工程髓核。复合培养2d,观察NPCs和BMSCs在壳聚糖水凝胶中的存活情况;复合培养1周,扫描电镜观察两种细胞在支架中的生长分布;复合培养3周,行组织学、免疫组织化学检查,并用RT-PCR检测组织工程髓核中聚集蛋白聚糖、Ⅱ型胶原mRNA的表达。结果温敏性壳聚糖水凝胶室温呈液态,37℃放置15min发生交联反应成为半固态凝胶。吖啶橙/碘化丙啶染色示壳聚糖水凝胶中多数NPCs和BMSCs存活,少数死亡,细胞存活率均>90%。扫描电镜示NPCs和BMSCs分布于网状支架中,细胞表面有分泌的细胞外基质。HE染色、番红O染色及免疫组织化学染色显示细胞具有分泌细胞外基质的功能。RT-PCR检测示NPCs在壳聚糖水凝胶支架中复合培养3周,Ⅱ型胶原、聚集蛋白聚糖mRNA表达量分别为0.564±0.071、0.725±0.046,BMSCs复合培养物分别为0.713±0.058、0.852±0.076,两种细胞比较差异均有统计学意义(P<0.05)。结论温敏性壳聚糖水凝胶具有良好的细胞相容性,与NPCs比较,BMSCs复合壳聚糖水凝胶培养保持了更好的形态、增殖及细胞外基质合成功能。Objective To compare the growth and extracellular matrix biosynthesis of nucleus pulposus cells（NPCs） and bone marrow mesenchymal stem cells（BMSCs） in thermo-sensitive chitosan hydrogel and to choose seed cells for injectable tissue engineered nucleus pulposus.Methods NPCs were isolated and cultured from 3-week-old New Zealand rabbits（male or female,weighing 150-200 g）.BMSCs were isolated and cultured from bone marrow of 1-month-old New Zealand rabbits（male or female,weighing 1.0-1.5 kg）.The thermo-sensitive chitosan hydrogel scaffold was made of chitosan,disodium β glycerophosphate,and hydroxyethyl cellulose.Then,NPCs at the 2nd passage or BMSCs at the 3rd passage were mixed with chitosan hydrogel to prepare NPCs or BMSCs-chitosan hydrogel complex as injectable tissue engineered nucleus pulposus.The viabilities of NPCs and BMSCs in the chitosan hydrogel were observed 2 days after compound culture.The shapes and distributions of NPCs and BMSCs on the scaffold were observed by scanning electron microscope（SEM） 1 week after compound culture.The histology and immunohistochemistry examination were performed.The expressions of aggrecan and collagen type Ⅱ mRNA were analyzed by RT-PCR 3 weeks after compound culture.Results The thermo-sensitive chitosan hydrogel was liquid at room temperature and solidified into gel at 37℃（after 15 minutes） due to crosslinking reaction.Acridine orange/propidium iodide staining showed that the viability rates of NPCs and BMSCs in chitosan hydrogel were above 90%.The SEM observation demonstrated that the NPCs and BMSCs distributed in the reticulate scaffold,with extracellular matrix on their surfaces.The results of HE,safranin O histology and immunohistochemistry staining confirmed that the NPCs and BMSCs in chitosan hydrogel were capable of producing extracellular matrix.RT-PCR results showed that the expressions of collagen type Ⅱ and aggrecan mRNA were 0.564 ± 0.071 and 0.725 ± 0.046 in NPCs culture with chitosan hydrogel,and 0.713 ± 0.058 and 0.852 ±

关 键 词：组织工程髓核 髓核细胞 BMSCS 温敏性壳聚糖水凝胶 兔 

分 类 号：R318[医药卫生—生物医学工程]