Expression of collagen,osteocalcin, and bone alkaline phosphatase in a mineralizing rat osteoblastic cell culture

Summary Rat calvaria bone cells isolated by collagenase digestion form a bone-like matrix which mineralizes in vitro in the presence of -glycerophosphate, in less than 2 weeks. The purpose of this work was to investigate, in this mineralizing rat osteoblastic cell culture, the synthesis of collagen, osteocalcin, and bone alkaline phosphatase (ALP). The results obtained indicate (1) After 15 days in culture, the extracellular-matrix contains collagen type I, V, and to some extent type III. Metabolic labeling at day 14, during the phase of nodules mineralization as well as new nodules formation, shows that collagen types I and type V are synthesized; (2) During the phase of cell growth, no osteocalcin could be detected in the medium, however, at the point of nodule formation, the osteocalcin level reached values of 3.55±1.39 ng/ml, followed by a 30-fold increase after nodules became mineralized. At day 14, after metabolic labeling, de novo synthesized osteocalcin was chromatographed on an immunoadsorbing column. With urea-SDS PAGE the apparent molecular weight was determined to be 9,000 daltons. (3) Specific activity of ALP was found to be 10 nmol/min/mg of proteins at cell confluence. At day 15, when nodules are mineralized, this activity was increased by 40-fold. The Michaelis constant was 1.58 10^-3 M/L. ALP was inhibited by L-homoarginine and levamisole but not by L-phenylalanine. ALP was shown to be heat sensitive at 56°C with two slopes of inhibition. On SDS-PAGE, apparent molecular weight of ALP showed one band at 116,000 daltons (d) when extracted at cell confluence and two bands at 116,000 and 140,000 d when extracted at the 15th day of culture. ³²P-labeled subunit of the enzyme migrated as one band at 75,000 d. Sialic acid content was demonstrated by neuraminidase treatment either on the dimeric form or on the ³²P-labeled subunit. These data indicate that ALP expressed in this culture is bone specific. The results of the present study show that this mineralizing rat osteoblastic cell culture system synthesizes collagen type I, V, and traces of type III, osteocalcin, and bone ALP isoenzyme. Medium osteocalcin was detected during nodule formation and increased during mineralization. Increase in ALP activity as well as the presence of an additional form of ALP occurred in the mineralization phase. Therefore, this culture may be a useful model for studying the functions of bone-specific proteins during the process of mineralization.     

Matrix metalloproteinases as diagnostic (MMP-13) and prognostic (MMP-2, MMP-9) markers of prostate cancer

Previous studies have detected high levels of matrix metalloproteinases (MMPs) in metastatic prostate cancer. In this study, we recruited 40 patients with prostate cancer (PCa): 20 presented organ-confined carcinoma and 20 had metastatic cancer. We also recruited 40 subjects for control groups, 20 with benign prostate hyperplasia (BPH) and 20 healthy males with similar characteristics. All of the patients were monitored at the beginning (time 0) and after 90 days. We analyzed the plasma concentrations of MMP-2, MMP-9, MMP-13, TIMP-1 and the enzyme activity of MMP-2 and MMP-9,using specific ELISA tests. The plasma concentrations of MMP-2, MMP-9 and MMP-13 were higher in PCa patients with metastasis than in the other groups, and in these patients decreased markedly after therapy began. For MMP-2 and MMP-9, greater differences were observed in enzyme activity than in plasma concentrations. TIMP-1 was reduced in PCa patients with metastasis, even if the intergroup differences were not statistically significant. Our results suggest that the plasma concentration and activity of MMPs, in association with PSA determination, could play a role in diagnosis, monitoring therapy and evaluating malignant progression in PCa.     

Comparative effects of a novel vitamin D analogue MC-903 and 1,25-dihydroxyvitamin D3 on alkaline phosphatase activity, osteocalcin and DNA synthesis by human osteoblastic cells in culture

MC-903 is a novel vitamin D analogue which has been shown to promote epidermal cell differentiation but is 100 times less active than 1,25 dihydroxyvitamin D3 (1,25(OH)2D) in causing hypercalcemia. In order to determine the activity of this compound on bone cells, we have compared the effects of MC-903 and 1,25 dihydroxyvitamin D3 (1,25(OH)2D) on parameters of cell proliferation and differentiation in cultured normal human osteoblastic cells derived by migration from trabecular bone fragments. Dose response curves showed that MC-903 was 10 to 100 times less effective than 1,25(OH)2D in stimulating the synthesis of the osteoblast specific protein osteocalcin by human bone cells depending on the basal osteocalcin production. In cells showing high basal osteocalcin synthesis, 1,25(OH)2D (10(-8) M) was 2- to 3-fold more potent than MC-903 (10(-8) M) in inducing osteocalcin from 48 to 96 h of treatment. The greater activity of 1,25(OH)2D over MC-903 was observed in human bone cell cultures with elevated basal osteocalcin levels, indicating that the response to 1,25(OH)2D but not to MC-903 was amplified in cells with the higher osteoblastic characteristics. The effects of MC-903 and 1,25(OH)2D on alkaline phosphatase activity were not markedly different. Transforming Growth Factor-beta (TGF beta) (0.5 ng/mL, 48 h) was found to completely suppress the osteocalcin synthesis induced by 1,25(OH)2D (10(-8) and 10(-9) M), whereas the MC-903-induced osteocalcin synthesis was not affected, suggesting a negative interaction between TGF beta and 1,25(OH)2D but not MC-903 on osteocalcin synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

男性骨特异性碱性磷酸酶、骨钙素、I型胶原氨基末端肽与骨密度的关系

目的探讨男性人群血清骨特异性碱性磷酸酶(BAP)、血清骨钙素(sOC)和血清Ⅰ型胶原氨基末端肽(sNTX)与BMD的相互关系。方法用ELISA方法测定309名20~80岁男性志愿者的血清骨特异性碱性磷酸酶(sBAP)、血清骨钙素(sOC)和血清Ⅰ型胶原氨基末端肽(sNTX),用DEXA(双能X线吸收法)测定腰椎正位(AP)L1-L4总体、腰椎侧位、股骨颈、Wards区(华氏区)及髋部总体的面积BMD。结果(1)直线相关分析显示,sOC、sNTX与腰椎正位总体BMD呈负相关,r分别为-0.007,-0.100。BAP与腰椎正位总体、腰椎侧位、髋部总体、股骨颈及Wards区BMD均负相关,r分别为-0.190、-0.087、-0.175、-0.128、-0.128(P<0.05)。(2)校正年龄、体重指数和吸烟的影响后,sOC和各部位BMD相关性消失;sNTX与腰椎正位总体BMD;BAP与腰椎正位总体、髋部总体、股骨颈及Wards区BMD相关性仍存在,r分别为-0.164、-0.171、-0.148、-0.191、-0.105(P<0.05)。(3)以50岁为切点,将所有样本按年龄分两段,偏相关分析显示50岁以前sOC、sNTX和BAP与各部位BMD无显著相关;50岁以后除腰椎侧位外,BAP与腰椎正位总体、髋部总体、股骨颈及Wards区BMD负相关,偏相关系数分别为-0.206、-0.256、-0.183、-0.126(P<0.05)。sOC与各部位BMD无显著相关,sNTX与腰椎正位总体显著负相关,偏相关系数为-0.202(P<0.05)。(4)按BMD分组,方差分析显示50岁以上年龄匹配男性骨质疏松组BAP高于正常对照组与低骨量组,NTX高于正常对照组(P<0.05)。(5)分别以各部位BMD为应变量,年龄、BMI、吸烟(每日吸烟数量×烟龄)、BAP、sOC和sNTX为自变量,进行多元逐步线性回归分析。年龄、体重指数为各部位BMD的独立决定因子;吸烟为腰椎正位总体、髋部总体及Wards区BMD的独立决定因子。BAP为腰椎正位总体,髋部总体,股骨颈及Wards区BMD的独立决定因子,解释其BMD变化的百分数分别为16.5%、18.0%、13.4%、10.8%。(P均<0.05);sNTX为腰椎正位总体BMD的独立决定因子,解释腰椎正位总体BMD变化的15.7%。结论(1)校正年龄、体重指数和吸烟后,50岁以上男性BAP与腰椎正位总体、髋部总体、股骨颈及Wards区BMD,sNTX与腰椎正位总体BMD均呈负相关,BAP与sNTX均为50岁以上男性BMD的独立决定因子。(2)50岁以上男性骨质疏松组BAP显著高于正常对照组与低骨量组,NTX高于正常对照组,较高的骨代谢转换水平与较低的BMD相关联。(3)年龄、体重指数与吸烟均为各部位BMD的独立影响因素。     

Immature osteoblastic cells express the pro-alpha2(XI) collagen gene during bone formation in vitro and in vivo.

Type XI collagen is predominantly found in cartilage. However, expression of the pro-alpha2(XI) collagen gene (COL11A2) has recently been detected in various non-cartilaginous tissues. We identified the differentiation stage at which COL11A2 was expressed in cultured fetal rat calvarial (FRC) cells and in rat femoral fracture calluses in order to investigate the involvement of COL11A2 during bone formation in vitro and in vivo. We also studied the alternative splicing of exons 6-8 in FRC cells and fracture calluses. In FRC cells, mineralized nodules stained with von Kossa stain were observed from day 9 after confluence. COL11A2 was highly expressed on days 0 and 5, but the expression levels were rapidly decreased on day 9 by Northern blot analysis. During rat femoral fracture repair, intramembranous ossification proceeded and newly formed woven bone was observed on the cortex on day 7 after fracture. In situ hybridization showed that COL11A2 signals were detected in osteoblastic cells in the newly formed woven bone. According to the maturation and remodeling of the woven bone into the trabecular bone, the distribution of the signal for COL11A2 mRNA was limited to the superficial osteoblastic cells of the newly formed trabecular bone. These results demonstrated that COL11A2 was expressed in relatively immature osteoblastic cells during bone formation in vitro and in vivo. RT-PCR showed that the shortest band corresponding to mRNA lacking exons 6-8 was clearly detected when using RNA from soft calluses. In contrast, the largest band corresponding to mRNA with exons 6-8 was predominant when using RNA from FRC cells or from hard calluses on days 7 and 14. These results indicate that the splicing pattern of exons 6-8 in osteoblastic cells is different from the pattern in chondrocytes.     

Adherence of osteoblast-like cells on calcospherites developed on a biomaterial combining poly(2-hydroxyethyl) methacrylate and alkaline phosphatase.

The polymer poly(2-hydroxyethyl) methacrylate (pHEMA) can copolymerize with alkaline phosphatase (AlkP) to form a hybrid material. The enzyme retains its biological activity and forms hydroxyapatite nodules (calcospherites) when polymer pellets are incubated with a synthetic body fluid. Osteoblast-like cells (ROS 17/2.8) were seeded on pellets of pHEMA and pHEMA-AlkP on which calcospherites were grown. They were examined by scanning electron microscopy (SEM) with backscattered electron imaging. Cell surface and shape were measured by image analysis combining the SEM images. Cells grown on pHEMA-AlkP had an increased surface area (449 +/- 216 microm(2) vs. 204 +/- 80 microm(2)). The number of filopodia anchoring the cells on the free polymer surface was reduced on pHEMA-AlkP, but numerous thick pseudopodia permitted a direct anchorage on the calcospherites. Pseudopodia were wider and longer than the filopodia. The backscattered images revealed that each cell was seated on 7.1 +/- 1.5 calcospherites and partially covered 10.3 +/- 1.9 others. Antifibronectin and anti-bone sialoprotein antibodies were used to investigate cell attachment. With confocal microscopy, both molecules were located at the interface between the cells and the mineral, inside the cells, and as free molecules on the calcospherites. Immunogold labeling was done with the same antibodies and examined with transmission electron microscopy (TEM). Adsorption of fibronectin and bone sialoprotein was noticeable at the cell/calcospherite interface and on the surface of the hydroxyapatite crystals. Immunogold studies revealed adhesion proteins (bone sialoprotein, fibronectin) to be present at the surface of crystals and at focal points of cell contact.     

Enhancement of recombinant human bone morphogenetic protein-2 (rhBMP-2)-induced new bone formation by concurrent treatment with parathyroid hormone and a phosphodiesterase inhibitor,pentoxifylline

We investigated the enhancement of new bone |formation elicited ectopically by recombinant human bone morphogenetic protein-2 (rhBMP-2), using parathyroid hormone (PTH) and a phosphodiesterase inhibitor (PDEi), pentoxifylline (PTX), in an animal model. Collagen sponge sheet discs containing rhBMP were implanted onto the back muscles of mice. PTX alone (200mg/kg body weight [BW]), PTH(1–34) (10µg/kg BW), PTX plus PTH (200mg/kg BW and 10µg/kg BW, respectively), or vehicle (control) were injected subcutaneously daily for 3 weeks after implantation. At the end of this period, rhBMP-2-induced ectopic ossicles were harvested from each group of animals. Ossicles from the PTX-treated group were significantly larger in size, with unchanged bone mineral density (BMD), as compared with the ossicles from the controls. In contrast, the ossicles from the PTH-treated group had significantly higher BMD, but showed no difference in size when compared with those from the control animals. The ossicles of the PTX + PTH treatment group were significantly larger than those of the control and PTH treatment groups. In addition, the BMD of the harvested tissues from the PTX + PTH treatment group was signifi-cantly higher than that of tissues from the control and PTX treatment groups. Although the calcium content of ossicles was significantly higher in the PTX-, PTH-, and PTX + PTH-treated groups than in the control group, the Ca content of ossicles from the PTH + PTX-treated group was highest (two times that of controls), followed by the PTH- and PTX-treated groups.     

Neuroendocrine regulation of cyclic AMP formation in osteoblastic cell lines (UMR-106-01, ROS 17/2.8, MC3T3-E1, and Saos-2) and primary bone cells.

The effect of four different neuropeptides and norepinephrine (NE) on cyclic AMP formation in four different osteoblastic cell lines and in isolated neonatal mouse calvarial bone cells has been examined. In the rat osteosarcoma cell line UMR-106-01, vasoactive intestinal polypeptide (VIP, 0.001-1 microM), calcitonin gene-related peptide (CGRP, 0.3-30 nM), and NE (0.1-300 microM), but not neuropeptide Y (NPY, 0.001-1 microM) or substance P (SP, 0.1-10 microM), caused a dose-dependent stimulation of cyclic AMP formation. The stimulatory effects were synergistically potentiated by forskolin (0.1-3 microM). The effects of NE and VIP were time dependent, with an optimal effect seen at 5 minutes. The amount of cyclic AMP accumulated in cells stimulated with NE and VIP was in the same range. The amplitude of the cyclic AMP response induced by CGRP was smaller than that caused by VIP and NE. In the human osteosarcoma cell line Saos-2, NE (0.1 microM) and VIP (0.3 microM) stimulated cyclic AMP formation, and the effect was synergistically potentiated by forskolin. In the absence of forskolin, no effect of CGRP (30 nM) could be seen in the Saos-2 cells, but in the presence of forskolin (3 microM) a stimulatory effect was observed. SP and NPY did not change basal cyclic AMP levels in Saos-2 cells. In the osteoblastic osteosarcoma cell line of rat, ROS 17/2.8, NE (0.1 microM) caused a significant stimulatory action on cyclic AMP formation that was synergistically potentiated by forskolin (3 microM), VIP, CGRP, and SP did not affect the cellular content of cyclic AMP in ROS 17/2.8.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcitonin (but not calcitonin gene-related peptide) increases mouse bone cell proliferation in a dose-dependent manner,and increases mouse bone formation,alone and in combination with fluoride

Summary Previousin vitro studies have shown that salmon calcitonin had direct effects to increase parameters associated with embryonic chicken bone formation and to increase mouse and chicken osteoblast-line cell proliferation. The current studies demonstrate increased cell proliferation (i.e., [³H]-thymidine incorporation into DNA and tetrazolium salt reduction/deposition) in the osteoblastic murine cell line MC-3T3-E1 in response to salmon calcitonin (P<0.005) and to human calcitonin (P<0.005), but not to human calcitonin gene-related peptide. The current studies also show that salmon calcitonin increased several indices of murine bone formation. We found that 72 hours of exposure to salmon calcitonin [at 5 mU/ml—about 0.37 nM; mU/ml = milliunits of calcitonin activity/ml incubation medium (at 4,000 U/mg protein)] increased net⁴⁵Ca deposition (121% of control,P<0.05), net [³H]-proline incorporation 149% of control,P<0.001), and alkaline phosphatase activity (146% of control,P<0.01), in neonatal mouse half-calvaria. The calcitonin-dependent increase in alkaline phosphatase activity was not affected by co-incubation with 1 nM parathyroid hormone. Co-incubation with fluoride (which also increased net [³H]-proline incorporation and alkaline phosphatase activity in neonatal mouse half-calvaria,P<0.05, for each) enhanced the osteogenic response to low-dose calcitonin, (i.e., co-incubation with fluoride shifted the biphasic calcitonin dose-response curve to a range of lower calcitonin concentrations). The calcitonin-fluoride combinations had proportional effects on net [³H]-proline incorporation and alkaline phosphatase in the treated mouse calvaria (r=0.78,P<0.005).     

The impact of bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) on osseointegration,degradation and biomechanical properties of a synthetic bone substitute

INTRODUCTION: It was the purpose of this investigation to test the osteointegrative capacity and the degradation rate of a neutralized glass ceramic within an animal model after loading with different growth factors. MATERIALS AND METHODS: The glass ceramic GB9N was implanted in the medial tibial head of mature merino sheep (n = 48) after being loaded with either Bone Morphogenetic Protein-2 (BMP-2, 100 micro g/100 micro l), Vascular Endothelial Growth Factor (VEGF, 100 micro g/100 micro l), basic Fibroblast Growth Factor (b-FGF, 60 micro g/100 micro l) or unloaded. At 4 weeks (n = 24) and 9 months (n = 24) after operation histomorphological, histomorphometrical and biomechanical investigations were performed. RESULTS: The amount of newly formed bone was different within the study groups after 4 weeks. BMP-2 induced a significant increase in bone formation and a significant decrease of residual substitute. b-FGF and VEGF induced no such effects in comparison to the controls. Biomechanical investigations could only demonstrate slight increases of the maximal fracture load in the BMP-2 group after 4 weeks in comparison to the others. At 9 months after operation no differences concerning newly formed bone could be found in the study groups comparing to the controls. Only the amount of residual substitute was still significantly lower in the BMP-2 group. Biomechanical data could demonstrate maximal fracture loads of all study samples achieving the level of the controls. CONCLUSION: This investigation has demonstrated that by combining synthetic bone substitutes with growth factors certain parameters of the bone remodelling process can intitially be influenced in a positive way.     

Influence of dichloromethylene diphosphonate (Cl2MDP) and calcitonin on bone resorption,lactate production and phosphatase and pyrophosphatase content of mouse calvaria treated with parathyroid hormonein vitro

The effect of parathyroid hormone, calcitonin and dichloromethylenediphosphonate (Cl₂MDP) on bone resorption, lactate production and the content of alkaline and acid phosphatases and alkaline neutral and acid pyrophosphatases in mouse calvaria in culture was investigated. Parathyroid hormone increased all measured variables. Calcitonin prevented the increase in⁴⁵Ca release and lactate production but not the increase in the various enzymes. The diphosphonate prevented the increase in⁴⁵Ca release and lactate production but in addition prevented the increase in acid phosphatase and pyrophosphatase.

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Zusammenfassung Die Wirkung von Parathormon, Calcitonin und Dichloromethylen-Diphosphonat auf Knockenresorption, Lactatbildung und den Gehalt an alkalischer und saurer Phosphatase sowie an alkalischer, neutraler und saurer Pyrophosphatase in gezüchteten Mäuse-Calvarien wurde untersucht. Parathormon erhöhte alle diese Parameter, Calcitonin verhinderte die Erhöhung von⁴⁵Calcium-und Lactatfreisetzung, aber nicht die Zunahme der verschiedenen Enzyme. Das Diphosphonat jedoch verhinderte zudem noch die Erhöhung der sauren Phosphatase und Pyrophosphatase.

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Résumé L'effet de la parathormone, de la calcitonine et du dichloromethylenediphosphonate sur la résorption osseuse, la production de lactate et le contenu en phosphatases alcaline et acide aussi bien que celui en pyrophosphatases alcaline, neutre et acide dans une culture de calvaria de souris a été étudié. La parathormone augmente touts ces paramètres. La calcitonine prévient l'augmentation de la libération du⁴⁵Calcium et du lactate, mais ne prévient pas l'augmentation des différents enzymes. Le diphosphonate lui prévient en plus l'augmentation de la phosphatase et pyrophosphatase acide.

     

Inhibition of 1,25-(OH)2D3- and 24,25-(OH)2D3-dependent stimulation of alkaline phosphatase activity by A23187 suggests a role for calcium in the mechanism of vitamin D regulation of chondrocyte cultures.

This study used the ionophore, A23187, to examine the hypothesis that the regulation of alkaline phosphatase and phospholipase A2 activity by vitamin D3 metabolites in cartilage cells is mediated by changes in calcium influx. Confluent, fourth-passage cultures of growth zone and resting zone chondrocytes from the costochondral cartilage of 125 g rats were incubated with 0.01-10 microM A23187. Specific activities of alkaline phosphatase and phospholipase A2 were measured in the cell layer and in isolated plasma membranes and matrix vesicles. There was an inhibition of alkaline phosphatase specific activity at 0.1 microM A23187 in resting zone cells and at 0.1 and 1 microM in growth zone chondrocytes. At these concentrations of ionophore, the 45Ca content of the chondrocytes was shown to increase. Both the plasma membrane and matrix vesicle enzyme activities were inhibited. There was no effect of ionophore on matrix vesicle or plasma membrane phospholipase A2 in either cell type. In contrast, alkaline phosphatase activity is stimulated when growth zone chondrocytes are incubated with 1,25-(OH)2D3 and in resting zone cells incubated with 24,25-(OH)2D3. Phospholipase A2 activity is differentially affected depending on the metabolite used and the cell examined. Addition of ionophore to cultures preincubated with 1,25-(OH)2D3 or 24,25-(OH)2D3 blocked the stimulation of alkaline phosphatase by the vitamin D3 metabolites in a dose-dependent manner. The effects of ionophore were not due to a direct effect on the membrane enzymes since enzyme activity is isolated membranes incubated with A23187 in vitro was unaffected. These results suggest a role for calcium in the action of vitamin D metabolites on chondrocyte membrane enzyme activity but indicate that mechanisms other than merely Ca2+ influx per se are involved.     

Clinical usefulness of cross-linked N-telopeptide of type I collagen (NTx) as a bone metastatic marker in patients with prostate cancer--comparison with serum PICP, PINP and ICTP

Type I collagen cross-linked N-telopeptide (NTx) in urine, the degraded form of type I collagen cross-linked in bone, has been evaluated as a marker of bone resorption. In this study, the clinical usefulness of NTx as a marker of bone metastasis of prostate cancer was compared with that the carboxyterminal propeptide of type I procollagen (PICP), the aminoterminal propeptide of type I procollagen (PINP), and the pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP) in serum. We assessed 37 cases of prostatic cancer in which the diagnosis had been confirmed pathologically. The patients were 15 patients with prostatic cancer with bone metastasis (before treatment or during a relapse) (Group 1); 11 patients, with bone metastasis, but for whom treatment was effective and condition had stabilized (Group 2); and 11 patients, with localized prostatic cancer and no evidence of bone metastasis (Group 3). The serum PICP, PINP, and ICTP levels and concentration of NTx in urine were compared among the three groups with the Mann-Whitney U test, with p values less than 0.05 considered significant. Urine NTx concentrations in Groups 1, 2 and 3 were 539.3 +/- 202.9, 160.6 +/- 97.6 and 48.6 +/- 7.6 nMBCE/mMCr, respectively. The differences between the Group 1 and Group 2 and between Group 1 and Group 3 were significant (p < 0.01 and p < 0.001). The differences between Group 1 and Group 3 and between Group 2 and Group 3 were significant for serum PICP, PINP and ICTP concentrations (p < 0.05). The correlation coefficient between urine NTx and each serum bone metabolic marker was 0.8 for PICP, 0.4 for PINP and 0.5 for ICTP. These bone metabolic markers are promising clinical markers of bone metastatic and may be useful for prediction of therapeutic efficacy and recurrence in bone and quantification of the extent of bone metastates.     

Effect of doxercalciferol (1alpha-hydroxyvitamin D2) on PTH,bone turnover and bone mineral density in a hemodialysis patient with persistent secondary hyperparathyroidism post parathyroidectomy

The efficacy and safety of the vitamin D analog, doxercalciferol (1alpha-hydroxyvitamin D2, 1alphaD2) in the treatment of secondary hyperparathyroidism in hemodialysis patients has been previously reported. We report these effect of 16-week 1alphaD2 treatment on mineral metabolism and bone mineral density (BMD) in a hemodialysis patient with persistent secondary hyperparathyroidism post parathyroidectomy, resistant to previous calcitriol treatment. Levels of iPTH, bone-specific alkaline phosphatase and serum type I collagen C telopeptide were above normal at baseline and were substantially decreased with 1alphaD2 treatment (-92%, -63% and -53%, respectively). BMD increased in all areas: total skeleton (+6.5%), lumbar spine (+6.9%) and total femur (+4.3%). The patient showed no hypercalcemia, and phosphorus levels remained between 3.3 and 6.2 mg/dl.     

rhIGF-1对成骨细胞增殖、BMP-2及Cbfa1基因表达的影响

目的通过重组人胰岛素样生长因子(rhIGF-1)对体外成骨细胞增殖、骨形态蛋白-2(BMP-2)及核心结合因子1(Cbfa1)基因表达的影响,探讨rhIGF-1对骨代谢影响的可能机制。方法不同浓度的rhIGF-1(0、10、50、100ng/ml)刺激体外培养的大鼠成骨细胞,采用噻唑蓝(MTT)法检测细胞增殖能力,用半定量RT-PCR法检测成骨细胞BMP-2、Cbfa1基因的表达。结果rhIGF-1可明显促进成骨细胞增殖(P0.05),并促进成骨细胞BMP-2、Cbfa1基因的表达(P0.01),具有浓度依赖性。结论rhIGF-1可促进成骨细胞的增殖、分化及成熟,可能是通过增强BMP-2、Cbfa1基因的表达来实现的。     

Increased engraftment and GVHD after in utero transplantation of MHC-mismatched bone marrow cells and CD80low, CD86(-) dendritic cells in a fetal mouse model.

BACKGROUND: Bone marrow transplantation (BMT) is the only known cure for a variety of inherited diseases and requires the administration of high doses of immunosuppressive and myeloablative therapy. Because the fetus is immunoincompetent early in gestation, in utero stem cell transplantation (IUT) could avoid the need for this toxic conditioning. A major limitation to date of IUT is the low level of engraftment and failure to induce tolerance. Dendritic cells (DC) are considered very potent antigen-presenting cells, but DC progenitors (pDC) are strongly tolerogenic. METHODS: We examined the effect of donor pDC on the degree of engraftment and tolerance induction after IUT. Bone marrow-derived pDC (CD80low, CD86-) from male C57BL/6 mice (H2b) were injected with and without donor bone marrow (BM) intraperitoneally into 13 to 15-day BALB/c (H2d) fetuses. Engraftment was determined by flow cytometry and quantitative polymerase chain reaction and tolerance by skin grafts and the mixed lymphocyte reaction. RESULTS: At 1-month posttransplant, mice that received BM+pDC showed a higher degree of engraftment (29+/-46%) than mice that received pDC-enriched cells or BM cells alone (0.11+/-0.70% and 1.71+/-1.66%, respectively, P<0.001). However, 5/19 recipients of BM+pDC died within 6 weeks; 4/5 had significant donor cell engraftment in blood and/or tissues. Also, these mice had evidence of graft-versus-host disease (GVHD). Two mice out of 15 long-term survivors in the BM+pDC group had virtually complete replacement of host with donor hematopoietic cells. Skin grafts and mixed lymphocyte reaction studies showed no durable tolerance induction other than in the two fully engrafted recipients of BM+pDC. CONCLUSIONS: These results suggest that donor pDC, along with donor BM, can have a significant impact on engraftment of MHC-mismatched donor cells associated with an increased incidence of GVHD. However, marrow-derived pDC do not result in an increase in tolerance induction in utero even when microchimerism is present.     

Vector-averaged gravity-induced changes in cell signaling and vitamin D receptor activity in MG-63 cells are reversed by a 1,25-(OH)2D3 analog,EB1089

Skeletal unloading in an animal hindlimb suspension model and microgravity experienced by astronauts or as a result of prolonged bed rest causes site-specific losses in bone mineral density of 1%-2% per month. This is accompanied by reductions in circulating levels of 1,25-(OH)(2)D(3), the active metabolite of vitamin D. 1,25-(OH)(2)D(3), the ligand for the vitamin D receptor (VDR), is important for calcium absorption and plays a role in differentiation of osteoblasts and osteoclasts. To examine the responses of cells to activators of the VDR in a simulated microgravity environment, we used slow-turning lateral vessels (STLVs) in a rotating cell culture system. We found that, similar to cells grown in microgravity, MG-63 cells grown in the STLVs produce less osteocalcin, alkaline phosphatase, and collagen Ialpha1 mRNA and are less responsive to 1,25-(OH)(2)D(3). In addition, expression of VDR was reduced. Moreover, growth in the STLV caused activation of the stress-activated protein kinase pathway (SAPK), a kinase that inhibits VDR activity. In contrast, the 1,25-(OH)(2)D(3) analog, EB1089, was able to compensate for some of the STLV-associated responses by reducing SAPK activity, elevating VDR levels, and increasing expression of osteocalcin and alkaline phosphatase. These studies suggest that, not only does simulated microgravity reduce differentiation of MG-63 cells, but the activity of the VDR, an important regulator of bone metabolism, is reduced. Use of potent, less calcemic analogs of 1,25-(OH)(2)D(3) may aid in overcoming this defect.