An in vitro Experimental Insight into the Osteoblast Responses to Vitamin D3 and Its Metabolites

Pharmacology ◽  
2018 ◽  
Vol 101 (5-6) ◽  
pp. 225-235 ◽  
Author(s):  
Dong Wang ◽  
Jiang Song ◽  
Huasong Ma
Keyword(s):  
Vitamin D3 ◽  
Osteoblast Differentiation ◽  
Cell Types ◽  
Cell Counting ◽  
Dependent Manner ◽  
Dihydroxyvitamin D3 ◽  
Osteogenic Markers ◽  
Polymerase Chain ◽  
Dose Dependent

Background: 25-hydroxyvitamin D3 (25[OH]VD3) has recently been found to be an active hormone. Its biological actions are also demonstrated in various cell types. However, the precise influences of vitamin D3 (VD3) and its metabolites (25[OH]VD3, 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2VD3]) on the osteoblast differentiation remain largely unknown. In this work, we investigated the effects of VD3 and its metabolites in different concentrations on the early and later osteoblast differentiation and biomineralization. Methods: We first used quantitative real-time polymerase chain reaction (RT-qPCR) to evaluate the responsiveness of osteoblasts to VD3, 25(OH)VD3 or 1α,25-(OH)2VD3. We also evaluated the proliferation, differentiation and biomineralization of osteoblast at different time points via cell counting kit-8 assay and the analysis of osteogenic markers. Results: The experimental results confirmed that osteoblasts could be responsive to 25(OH)VD3 and 1α,25-(OH)2VD3 but could not directly metabolize VD3 and 25(OH)VD3. Only 200 nmol/L VD3 significantly promoted osteoblast proliferation, while 25(OH)VD3 and 1α,25-(OH)2VD3 did not show obvious actions. Moreover, the early osteogenic markers were increased by 25(OH)VD3 and 1α,25-(OH)2VD3 in a dose-dependent manner. More importantly, only 25(OH)VD3 had accelerated the gene and protein expressions of osteocalcin and the biomineralization level of osteoblasts. Conclusions: Our findings provide reliable evidence that 25(OH)VD3 at 100–200 nmol/L can induce the early and later osteoblast differentiation and biomineralization for clinical bone tissue engineering.

scholarly journals RETRACTED ARTICLE: Titanium particles damage osteocytes and inhibit osteoblast differentiation

2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Li Chen ◽  
Ziyue Wang ◽  
Wei Xu ◽  
Qirong Dong
Keyword(s):  
Cell Viability ◽  
Osteoblast Differentiation ◽  
Cell Counting ◽  
Cell Contact ◽  
Dependent Manner ◽  
Retracted Article ◽  
Ti Particles ◽  
Dose Dependent Decrease ◽  
Dose Dependent

Abstract Purposes to study the effect of titanium particles on MLO-Y4 and the effects of osteocytes alterations on osteoblasts. Methods cultured MLO-Y4 osteocytes were exposed to different concentrations of titanium (Ti) particles, cell viability was measured using the Cell Counting Kit-8 (CCK-8) assay, apoptosis of MLO-Y4 cells was evaluated by flow cytometry, Real-time PCR quantification of mRNA expression of SOST, at the same time with Western Blot detection sclerosteosis protein expression levels.MC3T3-E1 cells culture with MLO-Y4 cells exposed to different concentrations of titanium (Ti) particles in vitro, in order to detection of osteoblast osteogenetic activity. Results Our results showed that Ti particles inhibited cell viability of MLO-Y4 osteocytes in a dose-dependent manner. Incubation with Ti particles caused apoptosis of MLO-Y4cells.Treatment with Ti particles significantly increased expression of the osteocytic marker SOST/sclerostin. Furthermore, treatment of MLO-Y4 cells with Ti particles produced a dose-dependent decrease in ALP activity and decreased mineralization of MC3T3-E1 cells through direct cell-cell contact. Conclusions Titanium particles damage osteocytes and inhibit osteoblast differentiation.

Hemostatic Properties of Infusible Trehalose-Stabilized Lyophilized Platelet Derivatives.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 834-834
Author(s):  
Keith A. Moskowitz ◽  
Josh Dee ◽  
Jason Barnidge ◽  
Ruth Sum ◽  
David Ho ◽  
...  
Keyword(s):  
Fluid Phase ◽  
Fold Increase ◽  
Submicron Particles ◽  
Cell Counting ◽  
Attractive Alternative ◽  
Dependent Manner ◽  
Clot Retraction ◽  
Drug Induced ◽  
Dose Dependent

Abstract Availability of platelet concentrates for treatment of bleeding associated with thrombocytopenia, trauma, or drug-induced coagulopathies is problematic due to the short 5 day platelet storage time and because platelets require controlled shaking at ambient temperature in order to remain viable, a condition which augments bacterial growth. To address the platelet availability problem we expanded upon trehalose cryo-preservation technology to create a lyophilized hemostatic platelet derivative. Washed platelets were stabilized by accumulation of 5–10 mM intracellular trehalose via fluid phase endocytosis then formulated with excipients and lyophilized. Lyophilized platelets were instantaneously rehydrated with > 90% recovery and were stable for at least 3–6 months at ambient temperatures. Rehydrated (RH) platelets responded quantitatively to α-and γ-thrombin and ristocetin by transmittance aggregometry and were partially agglutinated by collagen as judged by aggregometry and single cell counting using the Platelet Works® system. RH platelets co-aggregated in a dose dependent manner when mixed with fresh autologous platelets during collagen-induced activation. Aggregation response to low-dose thrombin and collagen was inhibited by the GPIIb/IIIa antagonist RGDS and by EGTA. RH platelets were quantitatively incorporated into fibrin clots and elicited platelet-dependent fibrin-clot retraction ~ 60% as well as fresh platelets. RH platelets were similar in size to fresh and had less than 25% submicron particles as judged by electronic particle counting and flow cytometry scatter profiles. RH platelets were partially activated upon rehydration as judged by anti P-selectin and anti-LAMP-3 binding, yet GPIIb/IIIa remained in a resting conformation, as judged by a lack of PAC-1 binding. GPIIb/IIIa receptors were present as judged by the binding of complex-dependent (clone 5B12) and function-blocking (clone P2) antibodies. RH platelets also contained intact GPIbα as judged by binding of the function-blocking MoAb AN51. Function of GPIIb/IIIa and collagen receptors on RH platelets was further demonstrated as RH platelets adhered to immobilized fibrinogen and collagen in the absence of added agonists and in a dose-dependent manner. Moreover, RH platelets exhibited a two-fold increase in platelet procoagulant activity in the presence of thrombin receptor agonist peptide SFLLRN as judged by Annexin-V binding. Procoagulant and hemostatic activity was further demonstrated as RH platelets accelerated the clotting of recalcified whole thrombocytopenic blood in a dose-dependent manner similarly to fresh platelets. Lastly, RH platelets corrected the coagulopathy induced by contact pathway inhibition with aprotinin during the recalcification of citrated whole blood. The technology has been scaled to single donor platelet aphaeresis units, equivalent to a standard transfusion dose. Preclinical animal models of safety, efficacy, and circulation persistence are currently being evaluated. In summary, trehalose- stabilized lyophilized platelet derivatives contain numerous in vitro hemostatic properties and may offer an attractive alternative to fresh platelet transfusions when the latter are indicated yet unavailable.

scholarly journals Lipoxygenase metabolites of arachidonic acid modulate hematopoiesis

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1675-1679 ◽  
Author(s):  
DS Snyder ◽  
JF Desforges
Keyword(s):  
Arachidonic Acid ◽  
Mononuclear Cells ◽  
Nordihydroguaiaretic Acid ◽  
Cell Types ◽  
Dependent Manner ◽  
Myristate Acetate ◽  
Human Erythropoietin ◽  
Dose Dependent

Abstract Lipoxygenase (LPO) metabolites of arachidonic acid participate in the activation and/or proliferation of a variety of cell types. In this study, we examined the role of LPO metabolites in controlling myelopoiesis and erythropoiesis in vitro. Monocyte depleted cells (MDC) prepared from human whole blood or whole mononuclear cells from human bone marrow were cultured in methylcellulose in the presence of various growth factors. Conditioned media containing human colony stimulating factors (CSF) or the tumor-promoting phorbol ester, phorbol myristate acetate (PMA), were added to induce myelopoiesis. Semipurified human erythropoietin (EPO) was added along with an endogenous source of burst- promoting activity (BPA) to induce erythropoiesis. The LPO inhibitor BW755C blocked all types of colony formation in a dose-dependent manner, with ID50 of 20 and 5 micrograms/mL for myeloid and erythroid colonies, respectively. MDC depleted of T cells were similarly inhibited by BW755C. Similar results were seen with two other LPO inhibitors, 1-phenyl-3-pyrazolidone and butylated hydroxyanisole. A fourth LPO inhibitor, nordihydroguaiaretic acid, inhibited at higher concentrations. Indomethacin, at concentrations that inhibit cyclooxygenase, had no significant effect, either alone or in combination with the LPO inhibitors. These results suggest that certain LPO products may be important mediators of both CSF- and PMA-induced myelopoiesis, and of BPA/EPO-induced erythropoiesis.

scholarly journals Lipoxygenase metabolites of arachidonic acid modulate hematopoiesis

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1675-1679
Author(s):  
DS Snyder ◽  
JF Desforges
Keyword(s):  
Arachidonic Acid ◽  
Mononuclear Cells ◽  
Nordihydroguaiaretic Acid ◽  
Cell Types ◽  
Dependent Manner ◽  
Myristate Acetate ◽  
Human Erythropoietin ◽  
Dose Dependent

Lipoxygenase (LPO) metabolites of arachidonic acid participate in the activation and/or proliferation of a variety of cell types. In this study, we examined the role of LPO metabolites in controlling myelopoiesis and erythropoiesis in vitro. Monocyte depleted cells (MDC) prepared from human whole blood or whole mononuclear cells from human bone marrow were cultured in methylcellulose in the presence of various growth factors. Conditioned media containing human colony stimulating factors (CSF) or the tumor-promoting phorbol ester, phorbol myristate acetate (PMA), were added to induce myelopoiesis. Semipurified human erythropoietin (EPO) was added along with an endogenous source of burst- promoting activity (BPA) to induce erythropoiesis. The LPO inhibitor BW755C blocked all types of colony formation in a dose-dependent manner, with ID50 of 20 and 5 micrograms/mL for myeloid and erythroid colonies, respectively. MDC depleted of T cells were similarly inhibited by BW755C. Similar results were seen with two other LPO inhibitors, 1-phenyl-3-pyrazolidone and butylated hydroxyanisole. A fourth LPO inhibitor, nordihydroguaiaretic acid, inhibited at higher concentrations. Indomethacin, at concentrations that inhibit cyclooxygenase, had no significant effect, either alone or in combination with the LPO inhibitors. These results suggest that certain LPO products may be important mediators of both CSF- and PMA-induced myelopoiesis, and of BPA/EPO-induced erythropoiesis.

scholarly journals Strontium Promotes the Proliferation and Osteogenic Differentiation of Human Placental Decidual Basalis- and Bone Marrow-Derived MSCs in a Dose-Dependent Manner

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Yi-Zhou Huang ◽  
Cheng-Guang Wu ◽  
Hui-Qi Xie ◽  
Zhao-Yang Li ◽  
Antonietta Silini ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Early Stage ◽  
Cell Types ◽  
Osteogenic Potential ◽  
Dependent Manner ◽  
Gene Expressions ◽  
Dose Dependent ◽  
Osteogenic Gene

The osteogenic potential of mesenchymal stromal cells (MSCs) varies among different tissue sources. Strontium enhances the osteogenic differentiation of bone marrow-derived MSCs (BM-MSCs), but whether it exerts similar effects on placental decidual basalis-derived MSCs (PDB-MSCs) remains unknown. Here, we compared the influence of strontium on the proliferation and osteogenic differentiation of human PDB- and BM-MSCs in vitro. We found that 1 mM and 10 mM strontium, but not 0.1 mM strontium, evidently promoted the proliferation of human PDB- and BM-MSCs. These doses of strontium showed a comparable alkaline phosphatase activity in both cell types, but their osteogenic gene expressions were promoted in a dose-dependent manner. Strontium at doses of 0.1 mM and 1 mM elevated several osteogenic gene expressions of PDB-MSCs, but not those of BM-MSCs at an early stage. Nevertheless, they failed to enhance the mineralization of either cell type. By contrast, 10 mM strontium facilitated the osteogenic gene expression as well as the mineralization of human PDB- and BM-MSCs. Collectively, this study demonstrated that human PDB- and BM-MSCs shared a great similarity in response to strontium, which promoted their proliferation and osteogenic differentiation in a dose-dependent manner.

scholarly journals Comparison of Antiviral Activity of Gemcitabine with 2′-Fluoro-2′-Deoxycytidine and Combination Therapy with Remdesivir against SARS-CoV-2

2021 ◽  
Vol 22 (4) ◽  
pp. 1581
Author(s):  
Yejin Jang ◽  
Jin Soo Shin ◽  
Myoung Kyu Lee ◽  
Eunhye Jung ◽  
Timothy An ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Human Lung ◽  
Lung Epithelial Cells ◽  
Dependent Manner ◽  
Fluorescent Image ◽  
Lung Epithelial ◽  
Infected Cells ◽  
Polymerase Chain ◽  
Dose Dependent

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. The virus still spreads globally through human-to-human transmission. Nevertheless, there are no specific treatments clinically approved. This study aimed to compare antiviral activity of gemcitabine and its analogue 2′-fluoro-2′-deoxycytidine (2FdC) against SARS-CoV-2 as well as cytotoxicity in vitro. Fluorescent image-based antiviral assays revealed that gemcitabine was highly potent, with a 50% effective concentration (EC50) of 1.2 μM, more active than the well-known nucleoside monophosphate remdesivir (EC50 = 35.4 μM). In contrast, 2FdC was marginally active (EC50 = 175.2 μM). For all three compounds, the 50% cytotoxic concentration (CC50) values were over 300 μM toward Vero CCL-81 cells. Western blot and quantitative reverse-transcription polymerase chain reaction analyses verified that gemcitabine blocked viral protein expression in virus-infected cells, not only Vero CCL-81 cells but also Calu-3 human lung epithelial cells in a dose-dependent manner. It was found that gemcitabine has a synergistic effect when combined with remdesivir. This report suggests that the difluoro group of gemcitabine is critical for the antiviral activity and that its combination with other evaluated antiviral drugs, such as remdesivir, could be a desirable option to treat SARS-CoV-2 infection.

Metabolism of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3 and its sensitivity to ketoconazole in 1α,25-dihydroxyvitamin D3-differentiated HL60 cells

1989 ◽  
Vol 3 (3) ◽  
pp. 199-205 ◽  
Author(s):  
M. E. Hayes ◽  
D. Bayley ◽  
E. B. Mawer
Keyword(s):  
High Performance ◽  
Cell Number ◽  
Dependent Manner ◽  
Macrophage Phenotype ◽  
Dihydroxyvitamin D3 ◽  
Hydroxyvitamin D ◽  
Dihydroxyvitamin D ◽  
25 Hydroxyvitamin D ◽  
Dose Dependent

ABSTRACT Regulation of the metabolism of [3H]25-hydroxyvitamin D3 ([3H]25-(OH)D3) in vitro to material with the characteristics of [3H]24,25-dihydroxyvitamin D3 ([3H]24,25-(OH)2D3) has been studied in the human promyelocytic cell line HL60. Synthesis of 24,25-(OH)2D3 was induced in a dose-dependent manner in cells pretreated with 0·1–100 nm 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3) for 4 days. This treatment also inhibited cell proliferation and stimulated differentiation to a macrophage phenotype that was characterized by staining for non-specific esterase (NSE) activity. The ability to synthesize [3H]24,25-(OH)2D3 from [3H]25-(OH)D3 and the expression of NSE activity both responded to changes in concentration of 1α,25-(OH)2D3 in the culture medium in a parallel manner. Synthesis of [3H]24,25-(OH)2D3 was linear when the incubation time was between 1 and 8 h and the cell number between 1 and 12×106 cells/incubation. The optimum substrate concentration for its synthesis was 125 nm, giving an apparent Michaelis constant of 360 nm. The identity of the [3H]24,25-(OH)2D3 synthesized by these cells was confirmed by co-chromatography with authentic 24,25-(OH)2D3 on normal-phase and reverse-phase high-performance liquid chromatography systems and by its reaction to sodium-m-periodate. Cells that had been exposed to 100 nm 1α,25-(OH)2D3 for 4 days synthesized 2·17±0·07 (s.e.m.) pmol 24,25-(OH)2D3/106 cells per h. This synthesis was inhibited in a dose-dependent manner over a concentration range of 0·01–1 μm by the drug ketoconazole, an antimycotic imidazole which is a known inhibitor of certain cytochrome P-450 enzyme systems, suggesting that the HL60 25-(OH)D3-24-hydroxylase is also a P-450-dependent enzyme system.

Effect of 1,25 dihydroxyvitamin D3 on isolated islets from vitamin D3-deprived rats

1990 ◽  
Vol 258 (4) ◽  
pp. E643-E648 ◽  
Author(s):  
B. J. Billaudel ◽  
A. G. Faure ◽  
B. C. Sutter
Keyword(s):  
Insulin Release ◽  
Vitamin D3 ◽  
Insulin Response ◽  
Dihydroxyvitamin D3 ◽  
1,25 Dihydroxyvitamin D3 ◽  
Dose Dependent ◽  
Vitamin D3 Deficiency ◽  
Calcium Redistribution

Insulin release is impaired by vitamin D3 deficiency but can be restored by in vivo administration of 1,25 dihydroxyvitamin D3 [1,25(OH)2D3]. A direct influence of 1,25(OH)2D3 on the B-cell was studied in vitro with islets from 5-wk vitamin D3-deprived rats. This hormone (10(-12) to 10(-6) mol/l) added to the incubation medium had a stimulatory dose-dependent effect on insulin response to 8.3 mmol/l glucose 6 h later. Moreover, perifusion experiments performed after different times of incubation demonstrated that after 6 h 1,25(OH)2D3 increased in particular the first phase of insulin response to 16.7 mmol/l glucose. The 45Ca fluxes, followed in parallel, were never modified by 1,25(OH)2D3 in the absence of glucose but were enhanced during the glucose stimulus, whereas 86Rb fluxes were never affected by 1,25(OH)2D3. These results demonstrated that 1,25(OH)2D3 acts in vitro on B-cells, but with a 6-h delay to potentiate the glucose-induced insulin release, concomitant with intracellular calcium redistribution.

Oxygenation of 18-hydroxycorticosterone as the final reaction for aldosterone biosynthesis

1984 ◽  
Vol 107 (3) ◽  
pp. 395-400 ◽  
Author(s):  
Itaru Kojima ◽  
Etsuro Ogata ◽  
Hiroshi Inano ◽  
Bun-ichi Tamaoki
Keyword(s):  
Carbon Monoxide ◽  
Hydroxysteroid Dehydrogenase ◽  
Dependent Manner ◽  
In Vitro Production ◽  
Mitochondrial Preparation ◽  
Final Reaction ◽  
Vitro Production ◽  
Dose Dependent ◽  
Cytochrome P 450

Abstract. Incubation of 18-hydroxycorticosterone with the sonicated mitochondrial preparation of bovine adrenal glomerulosa tissue leads to the production of aldosterone, as measured by radioimmunoassay. The in vitro production of aldosterone from 18-hydroxycorticosterone requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide. Cytochrome P-450 inhibitors such as metyrapone, SU 8000. SU 10603, SKF 525A, amphenone B and spironolactone decrease the biosynthesis of aldosterone from 18-hydroxycorticosterone. These results support the conclusion that the final reaction in aldosterone synthesis from 18-hydroxycorticosterone is catalyzed by an oxygenase, but not by 18-hydroxysteroid dehydrogenase. By the same preparation, the production of [3H]aldosterone but not [3H]18-hydroxycorticosterone from [1,2-3H ]corticosterone is decreased in a dose-dependent manner by addition of non-radioactive 18-hydroxycorticosterone.

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