用多次酶消化法体外培养的大鼠成骨细胞及其鉴定

目的 针对目前体外培养成骨细胞方法的不足,建立一种理想的原代培养成骨细胞的方法,为骨替代材料的研究提供成骨细胞.方法 本实验用新生1～2天大鼠颅盖骨,采用多次酶消化法进行细胞体外培养.倒置显微镜观察细胞形态,并对其碱性磷酸酶（ALP）活性及矿化能力进行鉴定.结果 所培养细胞具有成骨细胞的形态学特征及体内成骨细胞的生物学行为.结论 本实验体外培养成骨细胞的方法切实可行,可为骨替代材料的研究提供种子细胞,也可为骨细胞生物学和骨组织工程的研究提供一种客观而有效的实验手段.     

促甲状腺素及其受体与骨质疏松关系的研究进展

骨质疏松症(OP)是严重威胁老年人身心健康的常见疾病。随着人口老年化,老年性骨质疏松症发病率逐年增加。近年来内分泌性骨质疏松日见增多,促甲状腺激素(TSH)及促甲状腺激素受体(TSHR)与骨量减少、骨质疏松的关系倍受关注。成骨细胞和破骨细胞上的促甲状腺激素受体的作用是造成这一作用的关键。而此过程还会受到2型碘化甲腺氨酸脱碘酶、肿瘤坏死因子(TNF)等的影响。同时促甲状腺激素受体存在基因多态性,对其功能存在不同程度的影响,也可能是造成骨质疏松的重要原因。通过对这个领域的深入研究为内分泌性骨质疏松的防治提供依据。     

脐血来源的间充质干细胞生物学特性与向成骨细胞分化能力的研究

目的研究人脐血来源的间充质干细胞（UCBMSC）的免疫表型、生物学特性及其向成骨细胞分化的能力。方法分离脐血单个核细胞，以干细胞培养液培养间充质干细胞（MSC），观察其体外生长特性；用流式细胞术进行免疫表型测定和细胞周期分析；以含地塞米松、β-磷酸甘油和维生素C的诱导液定向诱导向成骨细胞分化；以碱性磷酸酶染色、矿化结节染色、骨桥蛋白mRNA的表达以及I型胶原表达鉴定MSC向成骨细胞分化的潜能。结果从脐血中可以培养出MSC，为成纤维细胞样的贴壁细胞。免疫表型分析显示CD45、CD34、CD11a、CD14、HLA．DR阳性细胞占1％～3％；CD166、CD44、CD106、CD29、CD105、CD49e、CD90、CD73表达率达91％～98％。细胞周期分析显示95％以上的细胞处于G0／G1期。定向成骨细胞诱导后，可以检测到碱性磷酸酶、I型胶原的表达，矿化结节的形成和骨桥蛋白mRNA的表达。结论UCBMSC是基质细胞的祖细胞，在合适的条件下可被诱导向成骨细胞分化，因此有可能作为有潜力的骨组织工程的种子细胞来源。     

Chloride current activated by cyclic AMP and parathyroid hormone in rat osteoblasts

In primary cultures of rat osteoblasts, studied with the whole-cell configuration of the patch-clamp technique, 8-bromo-cyclic AMP (8BrcAMP) forskolin (FS) and 1–34 parathyroid hormone (PTH) were shown to activate a Cl conductance. This conductance shows a pronounced outward rectification, even with symmetrical Cl concentrations. It is blocked partially and reversibly by 4,4-diisothiocyanatostilbene 2,2-disulfonic acid (DIDS) or diphenylcarboxylate (DPC). The blockade induced by DIDS is time-and voltage-dependent. The Cl responses to FS and PTH develop slowly, after a delay of several seconds and are very slowly reversible. These responses were observed only in a fraction of the cells tested and their detection was favoured by cell dialysis. This Cl current should be taken into account for studying possible modulations of the voltage-gated Ca currents of osteoblasts. It is suggested that its physiological role may be related to the well-known morphological changes induced by PTH in osteoblasts. The cyclic AMP-sensitivity, the outward rectification and the sensitivity to dialysis of this Cl current are reminiscent of the properties of the cystic fibrosis-sensitive Cl channels of epithelial cells.     

Flow Perfusion Culture of Marrow Stromal Cells Seeded on Porous Biphasic Calcium Phosphate Ceramics

Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% β-tricalcium phosphate) scaffolds, cultured for up to 16 days in static or flow perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. At all culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow perfusion bioreactor for bone tissue engineering applications.     

Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cells Cultured onto Three Different Polymers <Emphasis Type="Italic">In Vitro</Emphasis>

In this study, the osteoinductive and cell-binding properties of three different resorbable polymers were evaluated by human mesenchymal stem cells (MSCs). MSCs were isolated, expanded, and cultivated onto resorbable D,D,L,L-polylactide (PLLA), collagen I/III, and polygalactin-910/polydioxanone (PGPD) scaffolds in vitro. To evaluate the influence of dexamethasone, ascorbic acid, and beta-glycerolphosphate (DAG) on osteoblast differentiation, MSCs were incubated in a DAG-enriched medium. After a 28-day period in vitro, the cellular loaded polymers were digested enzymatically by papain and HCl. The Ca(2+) content of the biomembranes was evaluated by an o-kresolphthalein-complexon reaction via photometer. A PicoGreen assay was performed for dsDNA quantification. Significant differences between the number of adherent MSCs were documented (collagen > PLLA > PGPD). Compared to the initial number of adherent cells, all biomaterials induced a significant decrease in cellular adherence after 28 days in vitro. The presence of DAG-enriched culture medium stimulated the cellular proliferation for PLLA and slightly for PGPD, whereas cell proliferation was inhibited when MSCs were cultivated onto collagen I/III. In comparison with the control groups, all biomaterials (PLLA, PGPD, and collagen I/III) showed a significant increase in local Ca(2+) accumulation under DAG stimulation after 28 days in vitro. Furthermore, collagen I/III and PLLA scaffolds showed osteoinductive properties without DAG stimulation. These results were verified by immunocytochemical stainings against osteoblast-typical markers (osteopontin and alkaline phosphatase) and completed by calcified matrix detection (von Kossa staining). MSCs were identified by CD105 and CD13 antigen expression. Corresponding to an absence of CD34, CD45, and collagen II expression, we found no chondrogenic or hematopoietic cell differentiation. The results indicate significant differences for the proliferation, differentiation, adherence, and Ca(2+) accumulation between the tested polymers in a MSC culture.     

Transmission electron microscopic demonstration of vimentin in rat osteoblast and osteocyte cell bodies and processes using the immunogold technique

Background: The immunogold labeling technique and transmission electron microscopy were used to demonstrate the expression and position of the intermediate filament vimentin in rat osteoblast and osteocyte cell bodies and cell processes. Conventional light and transmission electron microscopic studies of bone cells demonstrated adjacent cell linkage to be mediated by osteoblast and osteocyte processes present within the canalicular system traversing the bone matrix. The cell processes were filled with densely packed filaments, many of which have been shown previously to be actin microfilaments. The appearance, however, of 10 nm diameter filaments in some cell processes and the fact that the intermediate filament vimentin has been defined in many cells of mesenchymal origin raised the possibility that some of these filaments might be vimentin. The ultrastructural colloidal gold immunochemical technique allowed for demonstration in situ of the expression of vimentin filaments plus accurate definition of their position. Methods: The studies were performed in newborn rat femoral and tibial diaphyseal cortical bone and in 1-week-old repair bone from 2.4 mm diameter defects made through the lateral cortex in 6-week-old rat femurs and tibias. The bone tissues for the immunochemical study were fixed in 1% glutaraldehyde, 4% paraformaldehyde, and 0.1 M phosphate buffer (pH 7.4) for 2 days. Decalcification was performed in 6% EDTA for 2–3 days. Infiltration involved use of Lowicryl resin K4M, and the embedding and curing processes were performed in a cryostat with temperatures ?30°C. An antivimentin monoclonal antibody was used for labeling using the postembedding technique. Effective antibody dilutions ranged from 1:10 to 1:200, with the dilutions of 1:25 and 1:100 showing the best combination of filament labeling with the least matrix background. The grids were exposed to 10 nanometer gold colloid conjugated goat anti-mouse IgM for demonstration of binding. Results: Vimentin immunolabeling was defined clearly in relation to filaments within the osteoblast and osteocyte cell body cytoplasm, throughout the entire length of the osteoblast and osteocyte cell processes, and in close relationship to the intercellular gap junctions which were present within the cell processes both close to the cell bodies and within the canaliculi well away from them. Conclusions: Immunogold labeling demonstrates the presence of the intermediate filament vimentin in osteoblast and osteocyte cell bodies and processes of rat bone. Vimentin distribution is not concentrated to specific areas, is present throughout the extent of the bodies and processes, and is seen immediately adjacent to gap junctions. © 1995 Wiley-Liss, Inc.     

隔膜条件下骨再生成骨细胞来源的实验观察

目的:研究在隔膜条件下引导性骨再生过程中成骨细胞的来源,进一步认识引导性骨再生的机制.方法:以成年新西兰兔为研究对象,在双侧挠骨中段制作标准骨缺损不愈合模型,用硅胶膜成管状包裹一侧骨缺损,另一侧无特殊处理为对照.术后分别进行X线检查、常规HE染色以及SP方法BMP、BGP抗体的免疫组化染色.结果:硅胶膜在骨缺损处形成隔离密闭的腔室,将周围组织阻挡于骨缺损之外.早期骨端骨内膜、骨髓基质细胞大量增殖,形成肉芽组织占据骨缺损.骨再生过程中表现出明显的组织学特征:骨痂表面为2～3层成骨细胞,骨缺损中央为肉芽组织,两者之间为数层细胞形成的移行区,细胞排列疏松.早期骨端骨内膜、骨髓基质细胞BMP、BGP呈强阳性染色,骨痂生长过程中,移行区部分细胞呈阳性染色.结论:结果表明在隔膜条件下骨再生的成骨细胞在早期来源于髓内的骨内膜和骨髓基质细胞,骨痂形成后,成骨细胞则来源于骨内膜、骨髓、骨膜增殖细胞共同形成的肉芽组织中的间质细胞或成纤维细胞.     

白细胞介素1刺激破骨细胞性骨吸收作用的实验研究

目的：了解白细胞介素1β（IL－1β）在破骨细胞性骨吸收中的刺激作用及其相应的作用机制。方法：分别将新生大鼠破骨细胞单独以及和成骨细胞联合接种于预置象牙片的培养板中。24h后培养液中加入不同浓度的IL－1β。继续培养48h,然后取出象牙片,超声处理后行甲苯胺蓝染色,光镜下观察骨吸收陷窝的数目并计算其部面积。结果：IL－1β能够明显增加坡骨细胞和成骨细胞联合培养组象牙片上吸收陷窝的数目和面前,且刺激作用呈剂量依赖性,但对单独培养的破骨细胞无明显刺激作用。结论：IL－1β的刺激骨吸收作用由成骨细胞所介导,而非直接作用于破骨细胞。