胰岛素对糖尿病大鼠下颌骨成骨细胞糖摄取的影响

目的：研究胰岛素对糖尿病大鼠下颌骨成骨细胞糖摄取的影响。方法：按照培养基中胰岛素的浓度（0,10-6mol/L）,将对照组和糖尿病组成骨细胞分为N、N＋Ins、DM、DM＋Ins4组,荧光显微镜观察它们对2-脱氧荧光葡萄糖（2-NBDG）的摄取能力。结果：2-NBDG的摄取量,20min时由大到小依次为N＋Ins〉DM＋Ins〉DM〉N;40min时依次为DM〉N＋Ins〉N及DM＋Ins。结论：胰岛素可以抑制糖尿病大鼠成骨细胞过高的糖摄取。     

Uptake of postprandial lipoproteins into bone in vivo: impact on osteoblast function

Dietary lipids and lipophilic vitamins are transported by postprandial lipoproteins and are required for bone metabolism. Despite that, it remains unknown whether bone cells are involved in the uptake of circulating postprandial lipoproteins in vivo. The current study was performed to investigate a putative participation of bone in the systemic postprandial lipoprotein metabolism in mice, to identify potentially involved cell type populations and to analyze whether lipoprotein uptake affects bone function in vivo. As a model for the postprandial state, chylomicron remnants (CR) were injected intravenously into mice. Next to the liver and compared to other organs, bone appeared to be the second most important organ for the clearance of radiolabeled CR particles from the circulation in vivo. In addition, uptake of radiolabeled CR by primary murine osteoblasts and hepatocytes was quantified to be in a similar range in vitro. A complementary approach with fluorescently labeled CR and immunohistochemical staining for apoE proved that intact CR particles were taken up into bone and liver. Electron microscopy localization studies of bone sections revealed CR uptake into sinusoidal endothelial cells, macrophages and osteoblasts. The relative amount of radiolabeled CR uptake into femoral cortical bone, representing predominantly osteoblasts, and bone marrow, representing predominantly non-osteoblast cells, was within the same range. Most importantly, the injection of vitamin K1-enriched CR resulted in an increase of the degree of osteocalcin carboxylation in vivo while total osteocalcin concentrations remained unaffected, giving functional proof that osteoblasts process CR in vivo. In conclusion, here we demonstrate that bone is involved in the postprandial lipoprotein metabolism in mice. Osteoblasts participate in CR clearance from the circulation, which has a direct impact on the secretory function of osteoblasts.     

The effect of mechanical deformation on the distribution of potassium ions across the cell membrane of sutural cells

Summary Ionic concentrations of potassium, sodium, and chloride were determined in osteocytes of the rat calvarium. The values were determined by fluorescent microscopy of both intra- and extracellular concentrations. Following the baseline determination, the calvaria were placed in tension by retraction of a microelectrode manipulator, and the fluorescence of the cells were measured again. A statistically significant change in the derived ion distribution was found. Thus, the tensile forces affected the distribution of ions across the cell membranes, increasing intracellular sodium and decreasing intracellular potassium. This would have an effect on the resting cell membrane potential with a change of potential of 8 mV. This has implications in the interpretation of clinical findings.     

骨膜成骨细胞的分离培养及其成骨作用的放射自显影研究

取４只家兔胫前骨膜进行成骨细胞分离培养，用３Ｈ－ＴｄＲ标记，然后回植于同一供体的皮下、耳软骨缺损处及挠骨缺损处。分别在２周和４周后处死动物，原位取材，用放射自显影方法观察种植细胞的转归。结果表明，种植的细胞在皮下转化为类骨组织；在软骨缺损处转化为软骨组织；而在骨缺损处则转化为典型的骨组织。提示用骨膜分离培养成骨细胞，回植体内，有可能用于骨缺损和软骨缺损的修复。     

Ultrastructure and Cytochemical Detection of Alkaline Phosphatase in Long-Term Cultures of Osteoblast-like Cells from Rat Calvaria

Two methods of collecting osteoblast-like cells from newborn rat calvaria were tested, either placing individual glass fragments or tipping dense glass beads onto the endocranial surface of periosteum-free bone. Inoculated at high density, cells collected by using these two methods form large mineralized plates after three weeks of culture. The main purpose of our investigation was to analyze the progressive formation of this mineralized structure and to localize alkaline phosphatase activity. At the beginning of the culture, flattened cells gathered into multilayers and synthesized collagen fibers. Cells in the upper layer became rapidly cuboidal in shape and continued to secrete collagen at their basal pole, whereas other cells became progressively embedded in the extracellular matrix. The upper cells featured ultrastructural characters of osteoblasts, whereas the embedded cells resembled osteocytes. After two weeks, the matrix began to mineralize: crystals appeared on collagen fibers, on matrix vesicles, and on cell debris. During the first days of the culture, the alkaline phosphatase activity was localized on the plasma membranes and on the collagen fibers. Thereafter, only the upper cells and collagen fibers that were juxtaposed to these cells showed alkaline phosphatase activity. In addition, the presence of mineralized matrix prevented the reaction product from being visualized on collagen fibers. The ultrastructural analysis reveals large mineralized plates with a structure resembling that of bone in vivo. This culture appears to be an appropriate model to study bone formation and regulation. Received: 30 September 1995 / Accepted: 3 May 1996     

丹仙康骨胶囊对培养成骨细胞影响的观察

目的：为了解补肾活血中药丹仙康骨胶囊对体外培养成骨细胞的作用。方法：应用透射电镜、MTT、对硝基苯磷酸盐法(PNPP)及骨钙素(BGP)含量放免测定法，观察丹仙康骨胶囊对成骨细胞超微结构、增殖与分化作用的影响。结果：丹仙康骨胶囊刺激(20mg／ml)的成骨细胞，ALP活性提高及骨钙素含量增多；透射电镜观察其细胞线粒体致密、游离核糖体增多、内质网丰富扩大增粗呈中等电子密度，而糖原溶解与脂肪空泡均较少。结论：丹仙康骨胶囊具有促进成骨细胞的代谢、增殖和分化的作用。     

复方仙贞汤抑制成骨细胞凋亡的实验研究

目的 观察不同浓度的复方仙贞汤对TNF-α诱导的成骨细胞凋亡的影响。方法 TNF-α培养液诱导体外培养成骨细胞凋亡后，分别加入不同浓度的复方仙贞汤，用流式细胞仪检测其对成骨细胞的凋亡率。结果 复方仙贞汤1μg／ml组和500μg/ml组细胞凋亡率明显降低，与模型组和空白对照组相比，差异非常显著，其中1μg/ml组作用更强。500μg／ml可能是通过影响成骨细胞周期中的G0-G1和S期而达到抑制凋亡作用的，而1μg／ml组还能增加G2-M期成骨细胞。结论 复方仙贞汤能抑制TNF—α诱导的成骨细胞凋亡，以低浓度(1μg／ml)抑制作用较好。     

Dexamethasone-treated ROS 17/2.8 rat osteosarcoma cells are responsive to human carboxylterminal parathyroid hormone peptide hPTH (53–84): Stimulation of alkaline phosphatase

Summary We tested the effects of various parathyroid hormone (PTH) peptides on alkaline phosphatase (ALP) activity in the osteoblastic cell line ROS 17/2.8. In dexamethasonetreated ROS 17/2.8 cells there was a dose-related increase in ALP activity due to treatment with hPTH (53–84). ALP activity was stimulated by 10 nM hPTH (53–84) by a mean of 1.51±0.07-fold (P<0.001) in nine experiments, whereas the same dose of bPTH (1–34) and bPTH (1–84) inhibited enzyme activity to 0.36±0.02-fold (P<0.001) and 0.37±0.03-fold (P<0.001), respectively. Significant stimulation of ALP activity occurred with doses of hPTH (53–84) as low as 0.01 nM. There was no stimulation of enzyme activity by hPTH (53–84) in the absence of dexamethasone; the maximum ALP response to hPTH (53–84) occurred between 96 and 144 hours, and no significant effect was seen at time periods less than 96 hours. The optimum dose of dexamethasone required to enable the response to hPTH (53–84) was 10 nM. Carboxylterminal PTH fragments had a specific stimulatory effect on ALP activity in dexamethasone-treated ROS 17/2.8 cells, but the aminoterminal PTH effect appeared to be dominant, as the equimolar combination of bPTH (1–34) and hPTH (53–84) resulted in inhibition of ALP activity. Thus, in order for the effects of carboxylterminal fragments to be manifest, the cells would have to be stimulated under conditions in which the aminoterminal receptor is unoccupied; this could occur under somein vivo conditions. The physiological significance of these observations is unknown, but the enabling effect of dexamethasone on hPTH (53–84) stimulation of ALP suggests that osteoblastic cells are responsive to this hormonal peptide at a certain stage of differentiation.     

Effect of ternary phosphate-based glass compositions on osteoblast and osteoblast-like proliferation, differentiation and death in vitro

There is currently a need to expand the range of graft materials available to orthopaedic surgeons. This study investigated the effect of ternary phosphate-based glass (PBG) compositions on the behaviour of osteoblast and osteoblast-like cells. PBGs of the formula (in mol.%) P(2)O(5)(50)-CaO(50-X)-Na(2)O(X), where X is either 2, 4, 6, 8 or 10, were produced and their influence on the proliferation, differentiation and death in vitro of adult human bone marrow stromal cells (hBMSCs) and human fetal osteoblast 1.19 (HFOB 1.19) cells were assessed. Tissue culture plastic (TCP) and hydroxyapatite (HA) were used as controls. Exposure to PBGs in culture inhibited cell adhesion and proliferation and increased cell death in both cell types studied. There was no significant difference in percentage cell death between the PBGs, which was significantly greater than the controls. However, compared with other PBGs, a greater number of cells were found on the 48mol.% CaO which may have been due to either increased adherence or proliferation, or both. This composition was capable of supporting osteogenic proliferation and early differentiation, and supports the notion that chemical modification of the glass could lead to a more biologically compatible substrate with the potential to support osteogenic grafting. Realisation of this potential should lead to the development of novel grafting strategies for the treatment of problematic bone defects.