人胚胎骨骨膜来源成骨细胞的分离培养与生物学特性

背景：胚胎骨来源成骨细胞因其低免疫原性和高增殖与成骨活性有可能成为适用于骨组织工程方法异体骨组织缺损修复的种子细胞。冻存复苏后的生物学特点是否能进行进一步临床应用研究有待证实。 目的：建立人胚胎骨骨膜来源成骨细胞的分离培养、保存方法，观察骨膜来源成骨细胞生物学特点。 设计、时间及地点：细胞形态学与生物学特性实验研究，于2005-04/2006-05在天津市口腔医院细胞与组织工程实验室完成。 材料：细胞来源为5月龄自然流产胎儿，产妇及家属知情同意，无菌条件下取长骨骨膜组织。 方法：采用组织块法对成骨细胞进行原代培养，传代培养细胞液氮冻存3~6个月后，选择不同代次细胞复苏培养。 主要观察指标：通过形态学、超微结构，细胞增殖曲线，钙结节Von kossa法染色以及细胞内碱性磷酸酶定量检测与钙钴法染色确定其增殖与成骨活性。 结果：组织块法培养骨膜来源成骨细胞第6天即可获得原代细胞增殖，传代扩增细胞具有典型成骨细胞形态学和生物学活性。苏木精-伊红染色密集融合的成骨细胞与细胞外基质显示转化为脊样骨组织结构排列。经统计学分析定量检测成骨细胞碱性磷酸酶活性与细胞密度有线性正相关关系。液氮冷冻保存后复苏培养复层成骨细胞钙钴法碱性磷酸酶染色阳性率在45%以上，超微结构显示为高分化功能活跃成骨细胞。 结论：胚胎骨骨膜来源成骨细胞具有良好的增殖与成骨活性。深低温液氮冻存成骨细胞复苏后仍能连续传代增殖并具有良好的成骨代谢活性，能够体外借助细胞外基质形成骨样结构。

Isolation, culture and biological characteristics of human periosteal osteoblasts

BACKGROUND: Because of lower immunogenicity and higher proliferation and osteogenic activity, human periosteal osteoblasts are suitable for seeding cells to repair bone defect by allograft tissue engineering. Whether characteristics of periosteal osteoblasts after cryopreservation and thawing can be maintained and used in clinic need further explored. OBJECTIVE: To investigate isolated culture and conservation method of human periosteal osteoblasts, in addition, to observe the biological characteristics. DESIGN, TIME AND SETTING: The experiment of cell morphology and biological characteristics was performed at the Cytology and Tissue Engineering Laboratory of Tianjin Stomatological Hospital from April 2005 to May 2006. MATERIALS: Under aseptic condition, cells were obtained from spontaneous abortion human embryo with 5-months. Informed consent was obtained from parturient and family members. METHODS: Primary periosteal osteoblasts was cultured with tissue block method, followed by 3-6 months liquid nitrogen cryopreservation, then preserved cells was selective thawed and cultured. MAIN OUTCOME MEASURES: Proliferation and osteogenic activity of cells were measured by morphology, ultrastructure, and cell proliferation curves, analyzed by calcification nodi Von Kossa staining, and determend by quantitative analysis of contents of cytoplasmic alkaline phosphatase (ALP) and amendable calcium-cobalt staining. RESULTS: Primarily cultured periosteal osteoblasts could proliferate from the 6th day. The amplification cells showed representative morphological and biological characteristics of osteoblasts. The ridge-like bone structure cristae appearance was formed after F4 passage human periosteal osteoblasts grew with extracellular matrix by hematoxylin-eosin staining. There was a linear positive correlation between the alkaline phosphatase activities and cell density. Gomori ALP staining showed that the positive rate of osteoblasts in anabiosis cultured was over 45%, which was demonstrated as high differentiation osteoblasts by transmission electron microscope. CONCLUSION: Osteoblast isolated from human fetal bone periosteal has good proliferation and osteogenic activity, which can be maintained and form bone -like structures after thawing from liquid nitrogen cryopreservation.