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背景:载体材料是种子细胞生长微环境的重要组成部分,有实验进行了软骨细胞在藻酸钙凝胶载体中的长期体外培养。目的:观察骨髓基质细胞复合藻酸钠形成的藻酸钙凝珠体外培养扩增后其细胞活力、组织形态学和细胞超微结构的变化。设计:单一样本观察。单位:郑州大学骨科研究所,上海第二医科大学骨科实验室。材料:实验于2001-09/2002-03在上海第二医科大学骨科实验室完成。4～6周龄新西兰大白兔8只用于骨髓基质细胞提取。方法:取白兔8只,麻醉后,用肝素预处理过的16号穿刺针于股骨第3转子下抽取骨髓1.0～2.0mL,用于骨髓基质细胞培养,传代培养的骨髓基质细胞;制备藻酸钠溶液;藻酸钠复合骨髓基质细胞形成藻酸钙凝珠,细胞密度为1×109L-1,体外培养4周。①骨髓基质细胞活力观察采用锥虫蓝染色,倒置相差镜下观察。②骨髓基质细胞组织学检测采用甲苯胺蓝染色。③骨髓基质细胞超微结构观察采用透射电镜检查。主要观察指标:①骨髓基质细胞活力。②骨髓基质细胞组织学观察。③骨髓基质细胞超微结构观察。结果:①骨髓基质细胞活力:倒置相差显微镜下可见细胞为圆形,大小不一,部分细胞呈团状分布,胞核清楚,个别为双核细胞,几乎无蓝染细胞。②骨髓基质细胞组织学观察结果:细胞与材料均匀混合,材料无着色;细胞均匀着色,大小不一,核大浆少,个别为双核或多核细胞,可见细胞分裂相;细胞结构完整、轮廓清楚,周围有少量的异染性反应。③骨髓基质细胞超微结构:结构正常,无线粒体肿大及核糖体脱颗粒现象发生;胞核完整,形态幼稚且较大,呈圆形、椭圆形或不规则形,可见双核。结论:①复合骨髓基质细胞的藻酸钙凝珠亲水性好,营养物质易于渗透,适合细胞生长、增殖。②骨髓基质细胞增生分裂能力活跃,通过体外培养,使体内骨髓基质细胞实现数目扩增是可行的。③藻酸钙复合骨髓基质细胞用于组织工程方法修复骨软骨缺损具有可行性。
BACKGROUND: Carrier material is an important part of the seed cell growth microenvironment. Chondrocytes were cultured in calcium alginate gel for a long time in vitro. OBJECTIVE: To observe the changes of cell viability, histomorphology and ultrastructure of alginate formed by the composite of sodium alginate and bone marrow stromal cells cultured in vitro. Design: Single sample observation. Unit: Zhengzhou University Institute of Orthopedics, Shanghai Second Medical University Orthopedic Laboratory. Materials: The experiment was performed at the Orthopedic Laboratory, Shanghai Second Medical University from September 2001 to March 2002. Eight New Zealand white rabbits, 4-6 weeks old, were used for bone marrow stromal cell extraction. Methods: Eight rabbits were used. After anesthesia, the bone marrow was sampled from 1.0 to 2.0 mL under the third femur using a 16-gauge needle pretreated with heparin for bone marrow stromal cell culture and subcultured bone marrow stromal cells. Sodium alginate solution; sodium alginate composite bone marrow stromal cells to form alginate gel beads, a cell density of 1 × 109L-1, cultured in vitro for 4 weeks. ① Bone marrow stromal cell viability was observed using Trypan blue staining, inverted phase contrast microscope. ② Bone marrow stromal cells histological examination using toluidine blue staining. ③ ultrastructure of bone marrow stromal cells using transmission electron microscopy. MAIN OUTCOME MEASURES: ① Bone marrow stromal cell viability. ② bone marrow stromal cells histological observation. ③ ultrastructure of bone marrow stromal cells observed. Results: (1) Bone marrow stromal cells vitality: The inverted phase contrast microscope showed that the cells were round and of different sizes. Some of the cells were in clusters and the nuclei were clear. (2) histological observation results of bone marrow stromal cells: cells and materials were evenly mixed, the material without coloring; cells uniformly colored, the size of different, large nuclear plasma less, individually for the dual-core or multinucleated cells, showing cell division phase; cell structure integrity, clear outline , Surrounded by a small amount of heterotropic reaction. ③ The ultrastructure of bone marrow stromal cells: normal structure, no enlargement of mitochondria and degranulation of ribosome; complete nucleus, naive morphology and larger, round, oval or irregular shape, showing binucleate. Conclusions: ① The calcium alginate beads of compound bone marrow stromal cells have good hydrophilicity and easy infiltration of nutrients, which is suitable for cell growth and proliferation. ② The proliferation and differentiation of bone marrow stromal cells are active. It is feasible to expand the number of bone marrow stromal cells in vitro through in vitro culture. ③ Calcium alginate composite bone marrow stromal cells for tissue engineering methods to repair osteochondral defects is feasible.