摘要
目的:分离、诱导培养中国实验用小型猪成骨细胞作为骨组织工程种子细胞,并进行形态学与生物学性能鉴定,为骨组织工程研究提供新型种子细胞。方法:实验于2002-08/2004-08在天津市口腔医院暨天津市整形外科医院中心实验室组织工程实验室完成。采用胶原酶消化法和诱导条件培养法自新生中国实验用小型猪四肢骨松质分离成骨细胞,经细胞生活形态观察、细胞倍增时间测定、透射电镜超微结构分析、裂解上清碱性磷酸酶检测、矿化结节形成测定等方法进行成骨细胞的生物学性能鉴定,以获取中国实验用小型猪成骨细胞作为骨组织工程种子细胞的必备的增殖活性与成骨活性数据。结果:①原代培养中国实验用小型猪成骨样细胞具有很强的贴壁率,细胞分裂旺盛,2～3d即可达到完全融合。细胞形态呈多角形和星形、胞体大、胞核圆,核仁丰富,通常为两三个核仁;胞浆中核周见散在黑色颗粒。②条件培养液诱导后的猪成骨样细胞培养6d左右可见细胞呈旋涡状复层生长,胞突伸展互相连接并集结成细胞簇团,中心区形成褐色结节;细胞核周围胞浆中出现大量黑色颗粒。③细胞增殖曲线显示猪成骨样细胞在播种3d后进入对数生长期,15d左右达到生长峰值U经细胞倍增时间公式计算猪成骨样细胞倍增时间为67.2h。④透射电镜观察显示:中国实验用小型猪成骨样细胞表面突起极丰富;核大,有较多和较深的内陷U核仁大而多窗孔,胞质内细胞器常常分布于核的一侧,扩张的粗面内质网池内有中等电子密度的蛋白质沉淀,其膜表面的核糖体密集分布。Golgi氏复合体发达,并见胞质内微丝及多量的游离核糖体。核仁特征为高转录活动的核仁。细胞外形、核谱型、核位置以及胞质内细胞器的特征性分布符合成骨细胞的超微结构特征。⑤猪成骨样细胞的碱性磷酸酶活性在β-磷酸甘油和地塞米松刺激后3d开始上升,18d达到高峰,21d时开始下降。⑥VonKossa染色显示从培养6d开始,小型猪成骨样细胞培养细胞层出现散在的小矿化结节,以后数量渐多,体积渐大,21d达高峰,体外成骨活性显著。结论:中国实验用小型猪骨松质成骨细胞具备骨组织工程种子细胞必备的增殖活性与成骨活性,适合作为骨组织工程的种子细胞。
AIM： To separate, induce and culture osteoblasts from Chinese experimental mini-pigs for bone tissue engineering as seed cells, and to carry out performance evaluation of morphology and biology so as to provide new-type seed cell for bone tissue engineering research. METHODS： The experiment was performed in the Laboratory of Tissue Engineering, Central Laboratory, Tianjin Stomatological Hospital, which was Tianjin Orthopedics Hospital from August 2002 to August 2004. Osteoblasts were isolated from spongy bone of four limbs of newborn Chinese experimental mini-pigs with collagenase digestion method and induction and culture method. Biological performance of osteoblasts was evaluated by the observation of cell living formation, assessment of cell doubling time, uhrastructural analysis under transmission electron microscope, measurement of alkaline phosphatase in split supernatant, evaluation of mineralized tuberculation and so on. The data of necessary proliferative activity and osteoplastic activity of osteoblasts, as seed cells, from Chinese mimi-pigs were obtained for bone tissue engineering. RESULTS：① Primary cultured osteoblast-like cells from Chinese experimental mini-pigs had powerful adherence rate. The osteoblast-like cells with productive fission could coalesce completely from 2-3 days. These cells were polygon or starlike, big cell body, round nucleus, abundant nucleolus, usually two or three nucleoli; Black granules were scattered around nucleus in cytoplasm. ② After being induced with conditioned medium, osteoblast-like cells from pigs at about day 6 grew to multiple lays in spiring shape. Cytoplasmic process extended, connected each other, built up cell cluster; Brown node appeared in the center; A mass of black granules appeared around nucleus in cytoplasm.③Cell growth curve showed that osteoblast-like cells from pigs entered logarithmic phase at day 3 after seeding, reached peak growth value at about day 15. The doubling time of osteoblast-like cells from pigs was 67.2 hours calculated with cell doubling time formula. ④ Transmission electron microscope showed that surface processes of osteoblast-like cells from Chinese experimental mini-pigs were abundant; Nucleus was big, with a lot of deep invagination, and nucleolus was big, with many windows. Cellular organ always distributed at one side of nucleus in the cytoplasm. Protein sediment with middle electron density appeared in extended rough endoplasmic reticulum, and the ribosome in its membrane surface distributed closely. Golgi complex became developed, had micmfilament and abundant free ribosome in cytoplasm. Nucleoli were characterized by high transcriptional activity. Characterized distributions of cell shape, nucleus spectral pattern, nucleus position and the cell organs in cytoplasm were accorded with the uhrastrnctural character of osteoblasts. ⑤Alkaline phosphatase activities of osteoblast-like cells from pigs increased from day 3 after being stimulated with β-glycerophosphate and dexamethasone, reached the peak at day 18, and then decreased at day 21. ⑥Von Kossa staining showed that small scattered mineralized nodes appeared in cellular layers of mini-big osteoblast-like cells from day 6 after culture, and then became more, and the volume became larger, reached the peak at day 21; The ossified activity was significant in vitro. CONCLUSION： Osteoblasts from spongy bone of Chinese experimental mini-pigs have the proliferative activity and osteoplastic activity of seed cells for bone tissue engineering, and can be considered as seed cells for bone tissue engineering.
出处
《中国临床康复》
CSCD
北大核心
2006年第45期43-46,I0005,共5页
Chinese Journal of Clinical Rehabilitation
基金
天津市自然科学基金资助(003609011)~~
