Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture

Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MW(r) of about 120 kDa and specific PNPP activity of 1200 U/mg. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 U/mg), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and beta-glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function.     

淫羊藿苷促进体外培养骨髓间充质细胞成骨性分化活性强于蛇床子素

比较研究蛇床子素与淫羊藿苷处理对体外培养的大鼠骨髓间充质细胞(rat bone marrow stromal cell, rBMSC)成骨性分化的影响.从体外分离培养的大鼠骨髓间充质细胞,筛选出最佳的蛇床子素和淫羊藿苷处理的浓度为1×10-5 mol/L, 然后用最佳的浓度对体外培养的大鼠骨髓间充质细胞进行药物干预;在药物干预后的第3、6、9、12和15 d后测定碱性磷酸酶活性（alkaline phosphatase,ALP）和钙含量;第12 d 进行钙化结节茜素红染色;第12 h、24 h、48 h、72 h和96 h 对OXS、Runx-2、骨形态发生蛋白(bone morphogenetic protein,BMP-2)和collagen-I mRNA 表达水平进行real-time RT-PCR检测.结果显示,浓度为1×10-5 mol/L蛇床子素和淫羊藿苷干预均可提高体外培养的骨髓间充质细胞ALP活性,增加Ca含量,提高Runx、OXS、BMP-2和collagen-1 mRNA的表达水平.同时,淫羊藿苷在促进体外培养骨髓间充质细胞成骨性分化活性强于蛇床子素.     

Expression of bone matrix proteins during dexamethasone-induced mineralization of human bone marrow stromal cells

Glucocorticoids have been shown to induce the differentiation of bone marrow stromal osteoprogenitor cells into osteoblasts and the mineralization of the matrix. Since the expression of bone matrix proteins is closely related to the differentiation status of osteoblasts and because matrix proteins may play important roles in the mineralization process, we investigated the effects of dexamethasone (Dex) on the expression of bone matrix proteins in cultured normal human bone marrow stromal cells (HBMSC). Treatment of HBMSC with Dex for 23 days resulted in a significant increase in alkaline phosphatase activity with maximum values attained on day 20 at which time the cell matrix was mineralized. Northern blot analysis revealed an increase in the steady-state mRNA level of alkaline phosphatase over 4 weeks of Dex exposure period. The observed increase in the alkaline phosphatase mRNA was effective at a Dex concentration as low as 10⁻¹⁰ M with maximum values achieved at 10⁻⁸ M. In contrast, Dex decreased the steady-state mRNA levels of both bone sialoprotein (BSP) and osteopontin (OPN) over a 4 week observation period when compared to the corresponding control values. The relative BSP and OPN mRNA levels among the Dex treated cultures, however, showed a steady increase after more than 1 week exposure. The expression of osteocalcin mRNA which was decreased after 1 day Dex exposure was undetectable 4 days later. Neither control nor Dex-treated HBMSC secreted osteocalcin into the conditioned media in the absence of 1,25(OH)₂D₃ during a 25-day observation period. The accumulated data indicate that Dex has profound and varied effects on the expression of matrix proteins produced by human bone marrow stromal cells. With the induced increment in alkaline phosphatase correlating with the mineralization effects of Dex, the observed concomitant decrease in osteopontin and bone sialoprotein mRNA levels and the associated decline of osteocalcin are consistent with the hypothesis that the regulation of the expression of these highly negatively charged proteins is essential in order to maximize the Dex-induced mineralization process conditioned by normal human bone marrow stromal osteoprogenitor cells. © 1996 Wiley-Liss, Inc.     

Shared oxidative pathways in response to gravity-dependent loading and gamma-irradiation of bone marrow-derived skeletal cell progenitors

Astronauts are exposed to radiation during space travel under conditions of dramatically reduced weightbearing activity. However, we know little about how gravity-dependent loading affects tissue sensitivity to radiation. We hypothesize gravity-dependent loading and irradiation share common molecular signaling pathways in bone cell progenitors that are sensitive to stress-induced reactive oxygen species (ROS), species capable of impacting skeletal health. To address this, progenitor cells with potential to differentiate into bone-forming osteoblasts were extracted from bone marrow, then cells were centrifuged (from 5-gravity (g) to 50-g for 5-180 min) on day 2 in culture, or were exposed to a single dose (1-5 Gy) of irradiation (137Cs 1 Gy/min) on day 3 or 4. Production of ROS was measured via fluorescence-activated cell sorting (FACS) using an oxidation-sensitive dye. Cell numbers were assessed by measurement of DNA content (CyQUANT). Osteoblastogenesis was estimated by measurement of alkaline phosphatase (ALP) activity and production of mineralized matrix (Alizarin Red staining). Transient centrifugation was a potent stimulus to bone marrow stromal cells, increasing production of ROS (1.2-fold), cell number (1.5-fold to 2.2-fold), and ALP activity (2.7-fold). Radiation also caused dose- and time-dependent increases in ROS production (1.1-fold to 1.4-fold) by bone marrow stromal cells, but inhibited subsequent osteoblast differentiation. In summary, gravity-dependent loading by centrifugation stimulated ROS production and increased numbers of osteoblasts. Although radiation increased production of ROS by bone marrow stromal cells, cell number and differentiation of osteoprogenitors appeared reduced. We conclude gravity-dependent loading and radiation both stimulate production of ROS and affect critical bone cell functions including growth and differentiation.     

Localization of 1,25-(OH)2D3-responsive alkaline phosphatase in osteoblast-like cells (ROS 17/2.8, MG 63, and MC 3T3) and growth cartilage cells in culture

Previous studies have shown 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-responsive alkaline phosphatase in cultured growth zone cartilage chondrocytes is localized in extracellular matrix vesicles (MV). Since osteoblast-like cells also have 1,25-(OH)2D3-responsive alkaline phosphatase, this study determined whether the 1,25-(OH)2D3-responsive enzyme activity is localized to MV produced by these cells as well. Osteoblast-like cells from rat (ROS 17/2.8), mouse (MC 3T3), human (MG 63), and rat growth zone cartilage were cultured in Dulbecco's modified Eagle's medium containing 10(-7)-10(-12) M 1,25-(OH)2D3. Alkaline phosphatase total activity and specific activity were measured in the cell layer, MV, and plasma membrane (PM) fractions. MV and PM purity were verified by electron microscopy and MV alkaline phosphatase specific activity compared to PM (MV versus PM: ROS 17/2.8 6 x; MG 63, 5.5 x; MC 3T3, 33 x; GC, 2 x). There was a dose-dependent stimulation of MV alkaline phosphatase (5- to 15-fold increase at 10(-7)-10(-9) M) in all cell types in response to the 1,25-(OH)2D3. The PM enzyme was stimulated in a parallel fashion in the osteoblast cultures. No effect of 1,25-(OH)2D3 was observed in growth cartilage PM. Although MV accounted for less than 20% of the total activity they contributed 50% of the increase in alkaline phosphatase activity in the cell layer in response to 1,25-(OH)2D3 and MV specific activity was enriched 10 times over that of the cell layer. These are common features of MV produced by cells which calcify their matrix and suggest that hormonal regulation of MV enzymes may be important in primary calcification.     

Streptozotocin-induced diabetes influences the activity of ecto-nucleoside triphosphate diphosphohydrolase 1 of rat osseous plate membranes

We report the kinetic characterization of an ecto-nucleosidetriphosphate diphosphohydrolase 1 from rat osseous plate membranes in streptozotocin-induced diabetic rats, which arises during ectopic mineralization twenty days after a subcutaneous implantation of demineralized bone matrix, Insulin deficiency decreased the ecto-nucleoside triphosphate diphosphohydrolase activity from 1293.1 +/- 39.8 (control rats) to 556.0 +/- 8.2 nmol Pi/(min mg). Two families of ATP hydrolyzing sites showed cooperative effects with specific activities of 256.2 +/- 7.7 nmol Pi/(min mg) and 299.8 +/- 8.9 nmol Pi/(min mg), and studies on the stimulation of the enzyme by magnesium and calcium ions showed that the decrease in enzyme activity results from changes in the affinity of the enzyme for these ions. To our knowledge this is the first study associating the effects of type I diabetes with an ecto-nucleoside triphosphate diphosphohydrolase activity from rat osseous plate membranes.     

不同强度静电磁场对体外培养骨髓间充质干细胞增殖与分化的影响

本实验研究不同强度静电磁场对体外培养大鼠骨髓间充质干细胞增殖与分化作用. 体外分离培养大鼠骨髓间充质干细胞,传代后随机分为6组,分别用强度为0（对照组）、0.9、1.2、1.5、1.8和2.1 mT的静电磁场处理,每d每次处理30 min. 在磁场处理后的9~10 d ,骨髓间充质干细胞开始出现钙化小颗粒. 0.9 mT组抑制骨髓间充质干细胞增殖,1.5到2.1mT组促进骨髓间充质干细胞增殖. 在磁场处理后的12 d和15 d ,1.5和1.8 mT组极显著地增加了碱性磷酸酶(AKP)活性. 采用AKP组织化学染色和钙化结节染色对骨髓间充质干细胞成骨性分化进行鉴定,AKP组织化学染色和钙化结节染色都呈现了极强的阳性结果,尤以1.5 mT和1.8 mT阳性染色面积最大. 在SEMFs处理后的48 h 和96 h ,1.5 mT和1.8 mT组胶原I(collagen-Ⅰ)和骨形态发生蛋白2(bone morphogenetic protein-2, Bmp-2) 基因表达水平显著高于对照组.在SEMFs处理后的12 d, BMP-2蛋白表达量高于对照组. 研究表明,0.9 mT 组抑制骨髓间充质干细胞增殖,1.5 mT到2.1 mT组不同强度静电磁场促进体外培养骨髓间充质干细胞的增殖. 磁场组能促进骨髓间充质干细胞成骨性分化,其中尤以1.5 mT和1.8 mT组促进大鼠骨髓间充质干细胞分化作用效果最为明显.     

Isolation and characterization of rat alveolar bone cells.

Samples of rat alveolar bone were first treated by collagenase digestion and then used as explants for cell culture. The cells obtained were subcultured and characterized by morphological and functional criteria. Their alkaline phosphatase activity was increased after incubation in 1,25-(OH)2 vitD3 10(-8) M whereas with gingival cells it did not change. The bone derived-cells organized nodular structures, synthesized type I collagen, Gla-protein, few type III collagen, and fibronectin. In the defined culture conditions no mineralization was observed. However, the method used allows to obtain cells from rat alveolar bone displaying some features of the osteoblastic phenotype.     

Enhanced superoxide anion generation but reduced leukotriene B4 productivity in thioglycollate-elicited peritoneal macrophages

Lipoxygenase metabolism of arachidonic acid was compared between peritoneal macrophages from untreated rats and those from rats on day 7 after intraperitoneal injection of thioglycollate broth (TG). Resident macrophages (M phi) from untreated rats produced mainly LTB4 (303 +/- 25 pmol/5 x 10(6) cells) and 5-HETE (431 +/- 56 pmol/5 x 10(6) cells) when stimulated with 5 micrograms/ml calcium ionophore A23187 for 20 min at 37 degrees C. On the other hand, TG-elicited M phi generated less amounts of lipoxygenase metabolites (157 +/- 10 pmol LTB4 and 319 +/- 19 pmol 5-HETE/5 x 10(6) cells) with the same stimulus. Then, leukotriene productivity was examined by using subcellular fractions of each M phi lysate and an unstable epoxide intermediate, leukotriene A4. LTA4 hydrolase activity was mainly contained in soluble fractions from the both groups of M phi. The cytosol fraction from the resident M phi exhibited the following specific and total activity; 2.2 +/- 0.1 nmol LTB4/mg protein/5 min and 12.2 +/- 0.5 nmol LTB4/5 min per 10(8) cells. On the contrary, the cytosol fraction from the TG-elicited M phi showed 1.9 +/- 0.1 nmol LTB4/mg protein/5 min and 9.6 +/- 0.3 nmol LTB4/5 min per 10(8) cells. The resident M phi, however, generated 0.14 +/- 0.04 nmol O2-/min/4 x 10(5) cells whereas the TG-elicited M phi did 0.49 +/- 0.13 nmol O2-/min/4 x 10(5) cells when stimulated with wheat germ lectin. These results suggest that the TG-elicited macrophages show enhanced superoxide production but generate less lipoxygenase metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)     

CTP:phosphocholine cytidylyltransferase is a substrate for cAMP-dependent protein kinase in vitro

The role of phosphorylation/dephosphorylation in the regulation of CTP:phosphocholine cytidylyltransferase activity was investigated. Incubation of post mitochondrial supernatant with cAMP-dependent protein kinase (50 units) led to an increased (28%) recovery of the cytidylyltransferase in the cytosolic fraction, while incubation with an intestinal alkaline phosphatase (20 units) led to an increased (61%) recovery in the microsomal fraction. When pure cytidylyltransferase was incubated with washed microsomes in the presence of cAMP-dependent protein kinase (133 units), the enzyme associated with the supernatant fraction increased (3.12 +/- 0.02 to 3.77 +/- 0.03 nmol/min/ml) while that of the microsomal fraction decreased (1.36 +/- 0.01 to 0.56 +/- 0.05 nmol/min/ml) by 2.5-fold. The increase in the cytidylyltransferase activity in the supernatant corresponded to an increase in 32P incorporation into the cytidylyltransferase. Treatment with alkaline phosphatase (40 units) decreased the cytidylyltransferase activity in the supernatant (3.61 +/- 0.08 to 2.88 +/- 0.07 nmol/min/ml) while the activity in the microsomal fraction increased (0.56 +/- 0.08 to 1.16 +/- 0.06 nmol/min/ml) by 2-fold. The decrease in the cytidylyltransferase activity in the supernatant corresponded to a decrease in 32P incorporation into the cytidylyltransferase. Incubation of cytidylyltransferase with phosphatidylcholine vesicles in the presence of cAMP-dependent protein kinase (110 units) decreased the cytidylyltransferase activity by 30%. The decrease in cytidylyltransferase activity corresponded to an increase in 32P incorporation into the cytidylyltransferase. Treatment with alkaline phosphatase (20 units) resulted in a 41% increase in the cytidylyltransferase activity. The increase in cytidylyltransferase activity corresponded to a decrease in 32P incorporation into the cytidylyltransferase. Incubation of the cytidylyltransferase with [gamma-32P] ATP and cAMP-dependent protein kinase led to incorporation of 32P into the serine residues of cytidylyltransferase. If the cytidylyltransferase were preincubated with alkaline phosphatase prior to incubation with cAMP-dependent protein kinase, 2-fold more 32P (0.2 mol P/mol cytidylyltransferase) was incorporated into the cytidylyltransferase. Collectively, this data is in agreement with a role for reversible phosphorylation in the regulation of cytidylyltransferase.     

Respiratory activity of isolated rat hepatocytes following cold storage and subsequent rewarming: a comparison of sucrose-based and University of Wisconsin solutions

Investigations were carried out on the respiratory function of isolated rat hepatocytes after cold storage alone for periods up to 48 h in either sucrose-based solution (SBS) or University of Wisconsin (UW) solution and after subsequent normothermic preincubation. In both SBS and UW, cold storage for 24 h depressed respiratory function (to 21 +/- 3 and 23 +/- 3 nmol O(2)/min/10(6) cells, respectively) compared to control cell values (31 +/- 3 and 33 +/- 5 nmol O(2)/min/10(6) cells; P < 0.01 in each case). However, normothermic preincubation for 60 min returned respiratory activity to control values (for SBS and UW storage: 41 +/- 6 and 40 +/- 5 nmol O(2)/min/10(6) cells; for control cells: 43 +/- 5 and 46 +/- 6 nmol O(2)/min/10(6) cells). Storage for 48 h in both SBS and UW allowed further depression of respiratory activity, with no recovery after preincubation. Stimulation of respiration by succinate in hepatocytes stored for longer periods was suggestive of increased membrane permeability. Both SBS and UW are effective storage solutions for isolated hepatocytes for up to 24 h as judged by aerobic metabolism, but significant damage was expressed in both solutions when preservation was extended.     

Stimulation of plasma membrane and matrix vesicle enzyme activity by transforming growth factor-beta in osteosarcoma cell cultures

Transforming growth factor-beta (TGF beta) serves an important role in extracellular matrix formation by stimulating the production of numerous extracellular matrix proteins by connective tissue cells and by osteoblasts or bone-forming cells. TGF beta has been shown to stimulate alkaline phosphatase (ALPase) activity in the rat osteoblast-like osteosarcoma cell line ROS 17/2.8. Previous studies have shown that this enzyme is elevated during calcification of bone and that it is enriched in matrix vesicles, an extracellular organelle associated with initial hydroxyapatite formation. To test the hypothesis that TGF beta plays a role in regulating mineral deposition in the matrix, the effects of TGF beta on ALPase and phospholipase A2, two enzymes associated with mineralization, were examined. ROS 17/2.8 cells were cultured at high and low density with recombinant human TGF beta (0.1-10 ng/ml) to examine the influence of cell maturation on response to TGF beta. Maximal stimulation of ALPase activity in the low density cultures was seen at 5 ng/ml; in high-density cultures, there was further stimulation at 10 ng/ml. There was a dose-dependent increase in ALPase activity seen in the matrix vesicles and plasma membranes in both types of cultures. Matrix vesicle ALPase exhibited a greater response to factor than did the plasma membrane enzyme. However, in low-density cultures, the two membrane fractions exhibited a parallel response with greatest activity consistently in the matrix vesicles. There was a dose-dependent increase in phospholipase A2-specific activity in the plasma membranes and matrix vesicles of both high- and low-density cultures. In agreement with previous studies, TGF beta inhibited cellular proliferation 50%. The results show that addition of TGF beta stimulates the activity of enzymes associated with calcification. The effect of TGF beta is dependent on the stage of maturation of the cell. This study indicates that TGF beta may play an important role in induced bone formation, calcification, and fracture repair in addition to its role in promoting chondrogenesis.     

Mineralization of adult mouse bone marrow in vitro

Murine adult bone marrow exhibits mineralizing capacity in vitro as is demonstrated by the new in vitro assay we report here. In less than 2 weeks after the onset of the cultures, mineralization is obtained in more than 80% of the marrow cultures. Moreover, morphological studies reveal that during incubation phenotypic changes related to osteogenic differentiation occur at the extracellular matrix as well within cell populations. Well banded collagen is synthesized. Matrix vesicles and needles of hydroxy-apatite crystals are observed via transmission electron microscopy. Osteoblast-like cells are present with membrane-associated alkaline phosphatase activity. The mineralization is specific for cultured bone marrow and is not observed in cultured spleen fragments as is shown via 85Sr uptake, calcein uptake and histomorphology. No inducing agent is added to the tissue culture medium except for 10% fetal calf serum, beta-glycerophosphate (10(-2) M) and ascorbic acid. However, the prerequisite for obtaining mineralization is the three-dimensional structure of the marrow in culture. The in vitro organ culture we developed may provide the opportunity to identify which marrow cells have osteogenic potential and to investigate the mechanisms triggering differentiation towards osteogenesis.     

Inhibition of osteoblastic cell differentiation by conditioned medium derived from the human prostatic cancer cell line PC-3 in vitro

Human prostatic carcinoma frequently metastasizes to bone tissue and activates bone metabolism, especially bone formation, at the site of metastasis. It has been reported that an extract of prostatic carcinoma and conditioned medium (CM) of a human prostatic carcinoma cell line, PC-3, established from a bone metastastic lesion, stimulate osteoblastic cell proliferation. However, there is little information about the effect of PC-3 CM on the differentiation of osteoblastic cells. In this study, we investigated the effect of PC-3 CM on the differentiation of two types of osteoblastic cells, primary fetal rat calvaria (RC) cells containing many undifferentiated osteoprogenitor cells, and ROS 17/2.8, a well-differentiated rat osteosarcoma cell line. PC-3 CM inhibited bone nodule formation and the activity of alkaline phosphatase (ALPase), an osteoblastic marker enzyme, on days 7, 14, and 21 (RC cells) or 3, 6, and 9 (ROS 17/2.8 cells) in a dose-dependent manner (5–30% CM). However, the CM did not affect cell proliferation or cell viability. PC-3 CM was found to markedly block the gene expression of ALPase and osteocalcin (OCN) mRNAs but had no effect on the mRNA expression of osteopontin (OPN), the latter two being noncollagenous proteins related to bone matrix mineralization. These findings suggest that PC-3 CM contains a factor that inhibits osteoblastic cell differentiation and that this factor may be involved in the process of bone metastasis from prostatic carcinoma. J. Cell. Biochem. 67:248–256, 1997. © 1997 Wiley-Liss, Inc.     

Simvastatin promotes osteoblast differentiation and mineralization in MC3T3-E1 cells

The cholesterol-lowering drug, simvastatin, is a pro-drug of a potent 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor and inhibits cholesterol synthesis in humans and animals. In addition, the bone effects of statins including simvastatin are being studied. We assessed the effects of simvastatin on osteoblastic differentiation in nontransformed osteoblastic cells (MC3T3-E1) and rat bone marrow cells. Simvastatin enhanced alkaline phosphatase (ALP) activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the statin was observed at relatively low doses (significant at 10(-8) M and maximal at 10(-7) M). Northern blot analysis showed that the statin (10(-7) M) increased in bone morphogenetic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. Simvastatin (10(-7) M) slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 14 and 22 of culture. These results indicate that simvastatin has anabolic effects on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases such as osteoporosis.     

Effect of the neuropeptide substance P on the rat bone marrow-derived osteogenic cells in vitro

Substance P containing, thin, sensory nerve fibres have been demonstrated in bone and bone marrow. However the role of substance P in bone tissue is not fully understood. We investigated the effects of substance P on the growth and development of rat bone marrow-derived osteogenic cells in vitro. To examine this, the marrow-derived osteogenic cells were treated from 3rd to 6th day of subculture with substance P at concentrations 10(-10), 10(-9) and 10(-8)M. [(3)H]-thymidine, L-2,3-[(3)H]-proline incorporation, protein accumulation, alkaline phosphatase activity, and calcium deposition were measured in cultures. Substance P slightly stimulated [(3)H]-thymidine incorporation at 10(-10) M. Protein accumulation and L-2,3-[(3)H]-proline incorporation were enhanced in a dose dependent manner. Simultaneous application of spantide, a substance P receptor antagonist, could not block substance P-induced L-2,3-[(3)H]-proline incorporation probably because of statistically significant effect of spantide itself. Calcium deposits were significantly lower (about 30%) in cultures treated with SP. This effect was probably due in part by the fall in alkaline phosphatase activity which in substance P treated cultures was decreased about 17%. Our results indicate that substance P could be one of the factors modulating bone metabolism.     

620nm低能量红光对骨髓间充质干细胞成骨分化的影响

为研究发光二极管(LED)发射的红光对大鼠骨髓间充质干细胞的成骨分化的影响,体外培养SD大鼠骨髓间充质干细胞,620 nm波长的LED置于细胞上方2 cm处,照射剂量分别为0,1,2和4J/cm2.采用CCK-8法检测照射后第2、4天的增殖活性,碱性磷酸酶(ALP)活性试剂盒与von kossa矿化结节染色法检测骨髓间...     

Haemopoietic long-term bone marrow cultures from adult mice show osteogenic capacity in vitro on 3–dimensional collagen sponges

Abstract. Adult murine bone marrow cells, cultured under conditions for long-term haemopoietic marrow cultures, produce bone matrix proteins and mineralized tissue in vitro , but only after the adherent stromal cells were loaded on a 3-dimensional collagen sponge. Provided more than 8 × 10⁶ cells are loaded, mineralization as measured by ⁸⁵Sr uptake from the culture medium, occurred in this 3-dimensional configuration (3-D) within 6 days. In contrast if undisrupted marrow fragments (containing more than 10⁷ cells) are placed directly on a collagen sponge, then it requires more than 10 days before significant mineralization can similarly be detected. The 2-dimensional (2-D) long-term marrow culture system allows prior expansion of the stromal cells and some differentiation in an osteogenic direction within the adherent stromal layer. This is suggested by the presence of type I collagen and alkaline phos-phatase positive cells. However, synthesis of osteonectin and a bone specific protein, osteocalcin, as well as calcification are only observed in 3-D cultures. Electron microscopy demonstrated hydroxyapatite mineral on collagen fibres, osteoblast-like cells, fibroblasts, cells which accumulated lipids, and macrophages which were retained on the collagen matrices. Irradiation of confluent long-term bone marrow cultures, prior to their loading on the collagen sponge showed that haemopoietic stem cells are not necessary for the mineralization.     

Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures

We have examined the ability of dexamethasone, retinoic acid, and vitamin D3 to induce osteogenic differentiation in rat marrow stromal cell cultures by measuring the expression of mRNAs associated with the differentiated osteoblast phenotype as well as analyzing collagen secretion and alkaline phosphatase activity. Marrow cells were cultured for 8 days in primary culture and 8 days in secondary culture, with and without 10 nM dexamethasone or 1 microM retinoic acid. Under all conditions, cultures produced high levels of osteonectin mRNA. Cells grown with dexamethasone in both primary and secondary culture contained elevated alkaline phosphatase mRNA and significant amounts of type I collagen and osteopontin mRNA. Addition of 1,25-dihydroxyvitamin D3 to these dexamethasone-treated cultures induced expression of osteocalcin mRNA and increased osteopontin mRNA. The levels of alkaline phosphatase, osteopontin, and osteocalcin mRNAs in Dex/Dex/VitD3 cultures were comparable to those of 1,25-dihydroxyvitamin D3-treated ROS 17/2.8 osteosarcoma cells. Omitting dexamethasone from either primary or secondary culture resulted in significantly less alkaline phosphatase mRNA, little osteopontin mRNA, and no osteocalcin mRNA. Retinoic acid increased alkaline phosphatase activity to a greater extent than did dexamethasone but did not have a parallel effect on the expression of alkaline phosphatase mRNA and induced neither osteopontin or osteocalcin mRNAs. In all conditions, marrow stromal cells synthesized and secreted a mixture of type I and III collagens. However, dexamethasone-treated cells also synthesized an additional collagen type, provisionally identified as type V. The synthesis and secretion of collagens type I and III was decreased by both dexamethasone and retinoic acid. Neither dexamethasone nor retinoic acid induced mRNAs associated with the chondrogenic phenotype. We conclude that dexamethasone, but not retinoic acid, promotes the expression of markers of the osteoblast phenotype in cultures of rat marrow stromal fibroblasts.