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背景:种子细胞的研究是组织工程众多研究领域中最重要的基础环节,骨髓基质干细胞以其诸多优点,成为理想的骨组织工程的种子细胞。目的:观察骨髓基质干细胞在体外培养的生长特点和非诱导条件下的成骨特性。设计:单一样本实验。单位:福建医科大学附属口腔医院种植中心。材料:实验于2003-05/12在福建医科大学附属口腔医院中心实验室完成。骨髓基质干细胞来自4只1岁龄的杂交犬的原代～第3代传代细胞。方法:无菌条件下在杂交犬两侧股骨大转子部做骨髓穿刺,抽取5mL肝素抗凝的骨髓,加入到含有50mL含双抗Dulbecco’s改良的Eagle’s培养液的离心管中分离单个核细胞,进行首次提纯获取骨髓基质干细胞单细胞,置培养箱中培养传代,培养48h后,吸除培养液,以后每3天换液1次。继续传代。采用倒置相差显微镜及苏木精-伊红染色观察骨髓基质干细胞形态;每天进行细胞计数,测定倍增时间,绘生长曲线;用钙钴法检测碱性磷酸酶;用茜素红法染色观察钙化结节生长情况。主要观察指标:①犬骨髓基质干细胞光镜结构。②犬骨髓基质干细胞生长曲线。③成骨分化指标碱性磷酸酶及钙化结节的观察。结果:①形态学观察表明,骨髓基质干细胞贴壁细胞呈集落生长,有成纤维样细胞外观,未加入成骨诱导剂,细胞形态发生变化。②碱性磷酸酶表达原代细胞呈强阳性,第1代细胞呈阳性,第2,3代细胞呈弱阳性。③钙沉积出现,原代细胞染色强于传代细胞。结论:①骨髓基质干细胞在体外培养能大量扩增,具有自然向成骨细胞分化的能力,是骨组织工程理想的种子细胞。②3代内扩增的骨髓基质干细胞有成骨活性,但原代细胞传代活性优于传代后细胞。
Background: Seed cell research is the most important basic link in many fields of tissue engineering. Bone marrow stromal stem cells have become ideal seed cells for bone tissue engineering with many advantages. OBJECTIVE: To observe the growth characteristics of bone marrow stromal stem cells cultured in vitro and osteogenic characteristics under non-induced conditions. Design: Single sample experiment. Unit: Stomatological Hospital Affiliated to Fujian Medical University Planting Center. MATERIALS: The experiment was performed at the Central Laboratory of Stomatology Hospital Affiliated to Fujian Medical University from May to December 2003. Bone marrow stromal cells Primary to 3rd passage cells from four 1-year-old crossbred dogs. METHODS: Under sterile conditions, bone marrow puncture was performed in the greater trochanter of both sides of the crossbred dogs. 5mL heparin anticoagulated bone marrow was sampled and mononuclear cells were isolated by centrifugation into 50mL double-labeled Dulbecco’s modified Eagle’s medium. The first purification of bone marrow stromal stem cells obtained single cell culture incubator subculture, 48h after culture, aspirate the culture fluid, fluid change every 3 days after 1. Continue to pass on. The morphological changes of bone marrow stromal cells were observed by inverted phase contrast microscope and hematoxylin-eosin staining. Cell counts were counted every day. The doubling time and growth curve were measured. Alkaline phosphatase was detected by calcium and cobalt method. Calcification was observed by alizarin red staining Festival growth. MAIN OUTCOME MEASURES: ① light microscopic structure of canine bone marrow stromal cells. ② canine bone marrow stromal cell growth curve. Osteogenic differentiation index alkaline phosphatase and calcified nodules were observed. Results: ① Morphological observation showed that the adherent cells of bone marrow stromal cells showed colony growth with the appearance of fibroblast-like cells, and the morphology of cells was changed without adding osteoinductive agent. ② Alkaline phosphatase expression of primary cells was strongly positive, the first generation cells were positive, the second and third generation cells were weakly positive. ③ calcium deposition, primary cells stained stronger than the passage cells. Conclusion: ①BMSCs can be expanded in vitro and have the natural ability to differentiate into osteoblasts. It is an ideal seed cell for bone tissue engineering. (2) The bone marrow stromal cells expanded within 3 generations had osteogenic activity, but the passaging activity of primary cells was superior to that after passage.