Effects of phospholipase D during cultured osteoblast mineralization and bone formation

Mammalian phospholipase D (PLD) mostly hydrolyzes phosphatidylcholine producing phosphatidic acid. PLD activity was previously detected in different osteoblastic cell models, and was increased by several growth factors involved in bone homeostasis. To confirm possible actions of PLD isoforms during mineralization process, we analyzed their effects in osteoblastic cell models and during bone formation. PLD1 expression, along with PLD activity, increased during differentiation of primary osteoblasts and Saos-2 cells, and peaked at the onset of mineralization. Subsequently, both PLD1 expression and PLD activity decreased, suggesting that PLD1 function is regulated during osteoblast maturation. In contrast, PLD2 expression was not significantly affected during differentiation of osteoblasts. Overexpression of PLD1 in Saos-2 cells improved their mineralization potential. PLD inhibitor Halopemide or PLD1-selective inhibitor, led to a decrease in mineralization in both cell types. On the contrary, the selective inhibitor of PLD2, did not affect the mineralization process. Moreover, primary osteoblasts isolated from PLD1 knockout (KO) mice were significantly less efficient in mineralization as compared with those isolated from wild type (WT) or PLD2 KO mice. In contrast, bone formation, as monitored by high-resolution microcomputed tomography analysis, was not impaired in PLD1 KO nor in PLD2 KO mice, indicating that the lack of PLD1 or that of PLD2 did not affect the bone structure in adult mice. Taken together, our findings indicate that PLD activity, especially which of PLD1 isoform, may enhance the mineralization process in osteoblastic cells. Nonetheless, the lack of PLD1 or PLD2 do not seem to significantly affect bone formation in adult mice.     

Effects of dietary phosphorus intake on bone mineralization and calcium absorption in adult female rats

We investigated the effects of dietary phosphorus (P) intake on the bone mineralization and calcium (Ca) absorption in adult female rats. Fifteen 16-wk-old female Wistar rats were divided into three groups, and respectively fed a low-P diet containing 0.15% P (LP), a control diet containing 0.5% P (C), and a high-P diet containing 1.5% P (HP) for 42 d. The apparent Ca absorption was significantly increased with decreasing dietary P level. The serum parathyroid hormone concentration was significantly lower in the LP group than in the C and HP groups. The serum osteocalcin concentration and urinary excretion of deoxypyridinoline were significantly higher in the HP groups than in the LP and C groups. The bone mineral density of the fifth lumbar vertebra was significantly increased with decreasing dietary P level. These results indicate that the low-P diet increased Ca absorption, this being effective for bone mineralization in adult female rats.     

Intrauterine growth restriction affects bone mineral density of the mandible and the condyle in growing rats

Objectives:To investigate in growing rats the effect of intrauterine growth restriction (IUGR) on the bone mineral density of the mandible and tibia, as well as the quality of the mandibular and condylar bone.Methods:Twelve male rats were born IUGR by mothers sustaining 50% food restriction during pregnancy. Twelve control male rats were born by mothers fed ad libitum. Dual-energy X-ray absorptiometry (DEXA) of the tibia, proximal tibial metaphysis and the mandible, biochemical markers, histology and histomorphometrical analysis on the mandibular and subchondral bone of the condyle were performed.Results:IUGR significantly affected bone mineral density (BMD) of both tibial and mandibular bones. IUGR rats had significantly lower osteocalcin values (p=0.021) and phosphorus (p=0.028), but not 25-OH vitamin D (p=0.352). Bone area percentage in the mandible was significantly lower (51.21±5.54) in IUGR compared to controls (66.00±15.49), and for subchondral bone of the condyle for IUGR (47.01±6.82) compared to controls (68.27±13.37). IUGR had a significant reduction in the fibrous layer, but not the proliferating layer, with the hypertrophic layer significantly increased.Conclusion:Maternal restricted nutrition during gestation can affect BMD of the mandible and the tibia of the offspring animals.     

Multiple dexamethasone treatment affects morphometric parameters of gonadotrophic cells in adult female rats

Exposure to glucocorticoids leads to numerous changes in various biological systems including the reproductive system. The aim of the present work was to find out whether dexamethasone (Dx) treatment of adult female rats would influence the histological and morphometric characteristics of the pituitary gonadotrophic cells (luteinizing--LH cells and follicle stimulating--FSH cells). One group of female Wistar rats received Dx injections on three consecutive days in doses 1.0, 0.5 and 0.5 mg/kg b.w. respectively, while the control rats were treated with equivalent volumes of saline. Experimental and control animals were sacrificed 24 h and 72 h after the last injection. The peroxidase-antiperoxidase (PAP) immunocytochemical procedure was used to study the LH and FSH cells. The stereological and morphometric analyses showed that multiple Dx treatments of female rats significantly decreased the volume of LH cells and the volume of their nuclei 24 h and 72 h after the last Dx injection in comparison with control values. At 24 h after Dx treatment, the volume density of LH cells was significantly increased, but at 72 h differences between the experimental and control groups were insignificant. The increase in number of LH cells per unit area (mm2) was significant at both timepoints (24 h and 72 h). Stereologic and morphometric characteristics of FSH cells was changed after Dx treatment in the same manner as that of LH cells, except for the volume density, where a significant increase was established 24 h and 72 h after the last Dx application. These results clearly demonstrate that 24 h and 72 h after the last of three Dx injections there were changes in the immunocytochemical and morphometric features of gonadotrophic cells.     

Dextromethorphan affects ventilation differently in male and female rats

Schlenker, Evelyn H. Dextromethorphan affectsventilation differently in male and female rats. J. Appl.Physiol. 81(5): 1911-1916, 1996.Subcutaneous administrationof aspartic acid results in a long-lasting but reversible depression ofventilation in male but not in female rats. Aspartic acid acts onN-methyl-D-aspartate receptors. The present studytested the hypothesis that a noncompetitive N-methyl-D-aspartate-receptor antagonist,dextromethorphan (Dex), would depress ventilation in female rats andstimulate it in male rats. Moreover, Dex administered prior to asparticacid should prevent the aspartic acid-induced depression of ventilationin male rats. In female rats, Dex caused a 30% depression ofventilation relative to saline at 5 and 10 mg/kg (P < 0.01)but not at the highest dose (20 mg/kg). In male rats, Dex had no effecton ventilation. At a dose of 20 mg/kg, Dex depressed oxygen consumptionto 50% of the saline value at all time points in female rats(P < 0.001) and in male rats 45 and 60 minafter administration. The time points when Dex depressed ventilationand oxygen consumption were different in female rats, suggesting thatthe depression of ventilation was not the result of a depression inoxygen consumption. During a hypercapnic challenge (7%CO₂), female rats treated with 5 and 10 mg/kg of Dexexhibited a smaller increase in ventilatory response relative to salinetreatment. At a dose of 20 mg/kg, the hypercapnic responsiveness ofmale rats was markedly stimulated (85.8 ± 8.95 ml/min) relative tosaline (50.6 ± 9.14 ml/min; P < 0.001). Finally, Dexadministered before aspartic acid prevented the aspartic acid-induced depression of ventilation in male rats. Thus, in rats, Dex has gender-specific effects on ventilation and these effects are not associated with changes in oxygen consumption.

     

The effect of chronic food restriction on immunopositive ACTH cells in peripubertal female rats

In peripubertal female rats, we have previously found that 50% food restriction (FR) increases plasma IL-6, haptoglobin and both alanine transaminase (ALT) and alkaline phosphatase (AST) aminotransferases, indicating the existence of an inflammatory response. To study whether such FR influences the hypothalamic-pituitary-adrenal (HPA) axis, we examined by immunohistochemistry the morphofunctional features of pituitary adrenocorticotroppic (ACTH) cells. In FR rats the volume and volume density of ACTH cells as well as plasma ACTH levels were increased by 17.6%, 12.5% and 13.4%, respectively, in comparison with controls (p<0.05). We concluded that chronic FR is a systemic stressor in young females, capable to stimulate the HPA axis, probably as a result of IL-6 action.     

Further characterization of human fetal osteoblastic hFOB 1.19 and hFOB/ER alpha cells: bone formation in vivo and karyotype analysis using multicolor fluorescent in situ hybridization

We have previously generated an immortalized human fetal osteoblastic cell line (hFOB) using stably transfected temperature sensitive SV40 T-antigen (Harris et al. [1995a] J. Bone. Miner. Res. 10:178-1860). To characterize these cells for phenotypic/genotypic attributes desired for a good cell model system, we performed karyotype analysis by multicolor fluorescent in situ hybridization (M-FISH), their ability to form bone in vivo without developing cell transformation, and finally their ability to form extracellular matrix formation in vitro. The karyotype analysis of hFOB cells revealed structural or numeric anomalies involving 1-2 chromosomes. In contrast, the human osteosarcoma MG63 cells displayed multiple, and often complex, numeric, and structural abnormalities. Subcutaneous injection of hFOB cells in the presence of Matrigel into nude mice resulted in bone formation after 2-3 weeks. Electron microscopic analysis of the extracellular matrix deposited by hFOB cells in culture revealed a parallel array of lightly banded fibrils typical of the fibrillar collagens such as type I and III. These results demonstrate that the hFOB cell line has minimal chromosome abnormalities, exhibit the matrix synthetic properties of differentiated osteoblasts, and are immortalized but non-transformed cell line. These hFOB cells thus appear to be an excellent model system for the study of osteoblast biology in vitro.     

Effects of feed restriction on fertility in female rats

BACKGROUND: Feed restriction with its resultant body weight loss impacts the rodent estrous cycle; however, the manifestation of these changes in a regulatory study design has not been documented. This study reports the effects of feed restriction in the context of an FDA regulatory submission. METHODS: Adult female rats (n = 20/group; weighing approximately 200 g each) were provided rodent chow ad lib (control) or at 20, 15, 10, or 7.5 g/rat/day (g/day) during a 2-week pre-mating phase, throughout the mating phase, and up to gestation day (GD) 7. On GD 8, all animals were provided ad lib feed until necropsy on GD 14. Estrous cyclicity, mating, and fertility parameters were evaluated. RESULTS: Ad lib rats consumed approximately 20 and 28 g/day during the pre-mating and gestation phases, respectively. All measured fertility parameters in the 20 g/day group were similar to control values. In the 15 g/day group, body weight was reduced by 16% at 2 weeks, prolonged diestrus occurred, and fertility was compromised due to reductions in corpora lutea. Within 2 weeks, mean body weight in groups receiving < or = 10 g/day was reduced by > or = 29% compared to ad lib values, and overt changes in estrous cyclicity, mating, and fertility occurred. The 7.5 g/day group was not sustainable beyond the pre-mating phase. CONCLUSIONS: For this study type, feed intake at < or = 50% ad lib values (< or = 10 g/day) was inadequate due to the magnitude and rapidity of body weight effects. Estrous parameters appeared slightly more sensitive than functional measures, as body weight changes of approximately 16% appeared near the threshold of changing routinely calculated estrous cycle parameters and were later associated with reduced fertility. In general, body weight differences of 10-15% by themselves were not adverse to normal reproduction (20 g/day).     

Chitosan enhances mineralization during osteoblast differentiation of human bone marrow-derived mesenchymal stem cells, by upregulating the associated genes

Objectives: Chitosan is widely used as a scaffold for bone tissue engineering. However, up‐to‐date, no previous detailed study has been conducted to elucidate any mechanism of osteogenesis by chitosan itself. Here, we have evaluated effects of chitosan‐coated tissue culture plates on adhesion and osteoblast differentiation processes of human mesenchymal stem cells (hMSCs), isolated from adult bone marrow. Materials and methods: Tissue culture plates coated with chitosan at different coating densities were used to evaluate the effects on hMSC adhesion and osteoblast differentiation. hMSCs were induced to differentiate into osteoblasts on the chitosan‐coated plates and were evaluated using established techniques: alkaline phosphatase assay, demonstration of presence of calcium and real time PCR. Results: The cells adhered to plates of lower coating density of chitosan, but formed viable cell aggregates at higher coating density (100 μg/sq.cm). Coating density of 25 μg/sq.cm, supporting cell adhesion was chosen for osteoblast differentiation experiments. Differentiating hMSCs showed higher mineral deposition and calcium content on chitosan‐coated plates. Chitosan upregulated genes associated with calcium binding and mineralization such as collagen type 1 alpha 1, integrin‐binding sialoprotein, osteopontin, osteonectin and osteocalcin, significantly. Conclusions: We demonstrate for the first time that chitosan enhanced mineralization by upregulating the associated genes. Thus, the study may help clinical situations promoting use of chitosan in bone mineralization, necessary for healing non‐union fractures and more.     

染料木黄酮对去势大鼠骨骼矿化的影响

目的: 研究染料木黄酮对去势大鼠骨骼矿化的影响.方法: 雌性Wistar大鼠47只随机分为假手术组,去势对照组、去势 雌激素组(己烯雌酚20 μg.kg bw-1.d-1)、去势 染料木黄酮组(剂量分别为25、50、100 mg.kg bw-1.d-1).饲养三个月后处死,测定骨密度、骨矿化相关参数、骨钙、磷、锌、镁、锰、血清甲状旁腺激素、降钙素和雌激素含量.结果: 大鼠去势后,股骨骨密度降低,平均类骨质宽度增大,骨矿化延迟时间和类骨质成熟时间延长,骨中钙、磷、锌、镁和血清雌激素含量降低,与假手术组相比均有显著性差异(P＜0.05);补充染料木黄酮后,股骨骨密度有改善的趋势,平均类骨质宽度变窄,骨矿化延迟时间和类骨质成熟时间缩短,骨中钙、磷、镁含量升高.结论: 染料木黄酮通过促进类骨质矿化,减少骨中钙、磷、镁丢失,预防骨质疏松的发生.     

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.     

Bioactivity studies and adhesion of human osteoblast (hFOB) on silicon-biphasic calcium phosphate material

Surface reactivity of bioactive ceramics contributes in accelerating bone healing by anchoring osteoblast cells and the connection of the surrounding bone tissues. The presence of silicon (Si) in many biocompatible and bioactive materials has been shown to improve osteoblast cell adhesion, proliferation and bone regeneration due to its role in the mineralisation process around implants. In this study, the effects of Si-biphasic calcium phosphate (Si-BCP) on bioactivity and adhesion of human osteoblast (hFOB) as an in vitro model have been investigated. Si-BCP was synthesised using calcium hydroxide (Ca(OH)₂) and phosphoric acid (H₃PO₄) via wet synthesis technique at Ca/P ratio 1.60 of material precursors. SiO₂ at 3 wt% based on total precursors was added into apatite slurry before proceeding with the spray drying process. Apatite powder derived from the spray drying process was pressed into discs with Ø 10 mm. Finally, the discs were sintered at atmospheric condition to obtain biphasic hydroxyapatite (HA) and tricalcium phosphate (TCP) peaks simultaneously and examined by XRD, AFM and SEM for its bioactivity evaluation. In vitro cell viability of L929 fibroblast and adhesion of hFOB cell were investigated via AlamarBlue® (AB) assay and SEM respectively. All results were compared with BCP without Si substitution. Results showed that the presence of Si affected the material’s surface and morphology, cell proliferation and cell adhesion. AFM and SEM of Si-BCP revealed a rougher surface compared to BCP. Bioactivity in simulated body fluid (SBF) was characterised by pH, weight gain and apatite mineralisation on the sample surface whereby the changes in surface morphology were evaluated using SEM. Immersion in SBF up to 21 days indicated significant changes in pH, weight gain and apatite formation. Cell viability has demonstrated no cytotoxic effect and denoted that Si-BCP promoted good initial cell adhesion and proliferation. These results suggest that Si-BCP’s surface roughness (164 nm) was significantly higher than BCP (88 nm), thus enhancing the adhesion and proliferation of the osteoblast.     

Chronic exposure to constant light affects morphology and secretion of adrenal zona fasciculata cells in female rats

The effect of chronic exposure to light of adult Wistar rats on growth and function of adrenal zona glomerulosa (ZG) and zona fasciculata (ZF) were examined. The females were exposed to continuous light of 600 lux for 95 days, starting on day 30 of age. The controls were kept under a 12:12 h light-dark cycle, at ambient temperature. The rats were sacrificed by decapitation and the left adrenal gland of each animal was dissected out and prepared for morphometric analyses. In animals exposed to chronic lighting, the absolute and relative volume of ZG were insignificantly increased by 5% (p>0.05) compared to controls. The volume of ZG cells and their nuclei were insignificantly changed by 1% (p>0.05) in comparison with corresponding controls. The absolute and relative volume of ZF were significantly increased (by 14 and 9%, respectively; p<0.05), as compared to controls. The volume of ZF cells and their nuclei were significantly increased (by 12 and 9%, respectively; p<0.05). Serum concentration of corticosterone was also significantly (p<0.05) increased by 13% in light-exposed group in comparison with control rats. These findings suggest that continuous exposure of female rats to constant light increased growth and secretory activity of ZF cells.     

Calcium metabolism and bone mineralization in female rats fed diets marginally sufficient in calcium: effects of increased dietary calcium intake

Experiments were carried out to determine the ability of female rats with poorly mineralized skeletons to increase bone mineralization in response to increased dietary Ca consumption. We specifically addressed this question with regard to two different periods of the life cycle: the period of sexual maturation (6–9 weeks of age), and in animals that had attained adult rates of skeletal mineralization (100 days of age). We found that at both stages, increased dietary Ca consumption resulted in increased trabecular bone volume and total bone Ca. In the younger animals, it was found that dietary history influenced the disposition of bone mineral. Animals that were initially Ca-deprived exhibited increased trabecular bone and decreased cortical thickness compared to animals continuously fed 0.5% Ca. Ovariectomy of mature animals reduced but did not eliminate the response to increased Ca intake.     

Effect of dietary protein level and source on bone mineralization in rats

Bone mineralization was studied in rats. Animals were divided into three feeding groups: LCP - diet with 13.5% crude protein in DM (5% of gluten, 10% of casein), HCP - diet with 21.2% CP in DM (8% of gluten, 10% of casein), and LSM - diet based on grain meals and meat-bone meal (21% CP in DM). After 28 days feeding, animals were euthanased by cervical dislocation and femur bones were collected, weighed and kept frozen until analyses. Diets with 21% protein (HCP, LSM) significantly increased weight of femur bones. Despite of the substantially higher ash level (7.1%) in the LSM diet than in the LCP diet (3.4%), rats of both groups had the similar bone concentration of Ca (15.7 +/- 1.1 vs. 17.4 +/- 1.1 g/kg) and Zn (178.7 +/- 7.9 vs. 173.0 +/- 8.5 mg/kg). However bone density in LSM rats was significantly higher than in LCP ones. Although rats fed HCP diet had intermediate bone density, the bone concentration of Ca (11.4 +/- 0.5 g/kg) and Zn (145.1 +/- 2.9 mg/kg) was significantly lower, than in animals fed LCP and LSM diets. This was related to the very wide protein/calcium (37:1 g/g) and protein/zinc (5.3:1 g/mg) ratios in HCP diet. Those ratios were narrowest in the LSM diet: 16.2:1 (CP/Ca) and 2.6:1 (CP/Zn). It can be conluded that protein/mineral ratio in a diet is a very important factor in bone development, besides dietary protein and ash contents itselves.     

Length of prolactin priming differentially affects maternal behavior in female rats

Prolonged exposure to prolactin (Prl) or to ectopic pituitary grafts that secrete Prl has been shown to stimulate maternal behavior in steroid-treated, hypophysectomized female rats. Since Prl levels in the blood of pregnant rats increase beginning 2-3 days prepartum, it was of interest to determine whether acute Prl priming prior to exposure to rat young would also stimulate full maternal behavior. Hypophysectomized, ovariectomized nulliparous rats were assigned to one of three treatments: Group 1, prolonged Prl; Group 2, acute Prl; or Group 3, controls/no Prl. All groups were implanted with 3 X 30 mm progesterone (P)-filled Silastic capsules s.c. at the time of ovariectomy (ovx) on Treatment Day 1. After ovx, Group 1 rats (prolonged Prl) were injected twice daily with 0.5 mg Ovine (o) Prl throughout the course of the study. Group 2 (acute Prl) and 3 (controls/no Prl) females were injected with vehicle alone or noninjected from Day 1-10. On Day 11 of Treatment, P implants were removed from all rats and each female was given a 2 mm estradiol-17 beta (E2)-filled Silastic implant. Starting on Day 11, Group 2 females were injected twice daily with oPrl. Group 3 rats continued to receive vehicle only. Behavioral testing began on Day 12 and was conducted daily through Day 22. Prolonged Prl priming (Group 1) stimulated a rapid onset of all aspects of maternal behavior (latencies less than 1 day, all p less than 0.05-0.001 vs. Group 3 controls). Control latencies ranged from 4-10 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

TAK-778 enhances osteoblast differentiation of human bone marrow cells

TAK-778 has been shown to induce bone growth in in vitro and in vivo models. However, there are no studies evaluating the effect of TAK-778 on human cells. Thus, the aim of this study was to investigate osteogenesis induced by TAK-778 on human bone marrow cells. Cells were cultured in 24-well culture plates at a cell density of 2 x 10(4) cells/well in culture medium containing TAK-778 (10(-7), 10(-6), and 10(-5) M, each) or vehicle. During the culture period, cells were incubated at 37 degrees C in a humidified atmosphere of 5% CO(2) and 95% air. For attachment evaluation, cells were cultured for 4 and 24 h. After 7, 14, and 21 days, cell proliferation, cell viability, total protein content, alkaline phosphatase (ALP) activity, and bone-like formation were evaluated. Data were compared by ANOVA and Duncan's multiple range test. TAK-778 did not affect cell attachment and viability. Cell number was reduced by TAK-778 in all time period evaluated in a dose-dependent way. The effect of TAK-778 on total protein content, ALP activity and bone-like formation was a dose-dependent increase. The present results suggest that initial cell events such as cell attachment are not affected by TAK-778 while events that indicate osteoblast differentiation including reduced cell proliferation, and increased both ALP activity and bone-like formation are enhanced by TAK-778 in a time and dose-dependent way. It means that TAK-778 could be a useful drug to enhance new bone formation in clinical situations that require rapid restoration of physiologic function, such as orthopedic and maxillofacial surgery.