成人骨髓源成骨细胞体外培养条件下的生物学特性(英文)

Biological characteristics of adult marrow-origin osteoblast in culture in vitro

背景:骨髓基质干细胞作为种子细胞构建组织工程化骨组织,具有广泛的应用前景,可作为首选的骨组织工程种子细胞来源。尽管目前对于动物骨髓基质干细胞向成骨细胞诱导分化的研究已不断深入,但对于成人骨髓基质干细胞大量诱导分化为成骨细胞的研究尚处于探索阶段。目的:研究成人骨髓基质干细胞在体外培养条件下的生物学特性及成骨能力,探讨简便易行的成人成骨细胞体外培养方法,为骨组织工程选择理想的种子细胞来源。设计:采用完全随机实验对照单盲设计进行横断面研究。地点和对象:实验在解放军第一军医大学南方医院全军创伤骨科中心完成,对象为健康成年志愿者骨髓组织。干预:抽取骨髓组织,在100mL/L胎牛血清的DMEM培养基中,置于37℃,50mL/LCO2湿化空气孵箱中培养,传代后改用含地塞米松、β-甘油磷酸钠和维生素C的条件培养基培养,用倒置相差显微镜、光镜(HE染色)、扫描与透射电镜观察细胞的形态特征和增殖分化情况,并测定培养细胞的生长曲线(MTT法)、碱性磷酸酶活性和钙结节形成能力。主要观察指标:形态学观察和生物学特性检测。结果:原代和传代培养的细胞具有活跃的增殖能力,诱导培养2周后,细胞呈多角形或梭形,具有多个突起可互相联结,透射电镜下观察细胞核较为幼稚,细胞器丰富。诱导分化后的细胞可分泌碱?

BACKGROUND:It can be widely used as a preferred origin of bone tissue seeding cell to employ bone marrow stromal cells(BMSCs) as seeding cells in the establis hment of bone tissue engineering.Although the researches in the induction and di fferentiation of animal BMSCs into osteoblast have entered into a certain depth, the researches in human are still in the exploring phase.OBJECTIVE:To study the biological characteristics and the osteogenous abilitie s of adult BMSCs cultured in vitro as well as to discuss an easy and practicable method of culture adult osteoblast in vitro and to select an ideal origin of se eding cell for bone tissue engineering.DESIGN:A randomized controlled single-blind cross section study was performed .SETTING and PARTICIPANTS:The study was completed in the Department of Orthopae dics and Traumatology,Nanfang Hospital,First Military Medical University.All bon e marrows were from healthy adult volunteers.INTERVENTIONS:Bone marrow was aspirated and put into complete DMEM with 100 mL /L fetal bovine serum, and incubated in an air wetting with 50 mL/L CO2 under 37 ℃.Bone marrows were both cultured in a the complete EMDM and conditional DMEM, which contained dexamethasone,β-glycerophosphate sodium and vitamin C after pa ssage.Cellular characteristics and proliferation were observed by an inversion p hase-contrast microscope,optical microscope(HE staining), and scanning and tran smission electron microscope.Cellular growth curve(MTT method), the activities o f ALP and the abilities in calcium tuberculation were assayed.MAIN OUTCOME MEASURES:Morphological observations and biological characteristic assays.RESULTS:There were active proliferation abilities in primary and passage cells .After 2-week culture, cells were in polygon or shuttle shapes with multiple um bos that could connect with each other.Cells were comparatively puerile under tr ansmission electron microscope with abundant of organells. Cells could excrete A LP after induction and differentiation with a positive rate of 74.2% by modifi ed Gomori Ca-Co method and with calcium tuberculation by Von Kossa, which had m orphological characteristics and biological functions similar to typical osteobl asts.CONCLUSION:It is safe and convenient to aspirate adult BMSCs,which can be easi ly differentiated into osteoblast.Our method can be used as a routine method in seeding cell culture for bone tissue engineering.