Osteocalcin Synthesis Research Articles

In Vitro Screening Studies on Eight Commercial Essential Oils-Derived Compounds to Identify Promising Natural Agents for the Prevention of Osteoporosis.

Over the years, essential oils (EOs) and their compounds have gained growing interest due to their anti-inflammatory, antimicrobial, antioxidant, and immunomodulatory properties. The aim of this study was to evaluate the effect of eight commercially available EO-derived compounds ((R)-(+)-limonene, (S)-(-)-limonene, sabinene, carvacrol, thymol, alpha-pinene (α-pinene), beta-pinene (β-pinene), and cinnamaldehyde) on the bone formation process in vitro to select the most promising natural agents that could potentially be used in the prevention or treatment of osteoporosis. Within this study, evaluation of cytotoxicity, cell proliferation, and osteogenic differentiation was performed with the use of mouse primary calvarial preosteoblasts (MC3T3-E1). Moreover, extracellular matrix (ECM) mineralization was determined using MC3T3-E1 cells and dog adipose tissue-derived mesenchymal stem cells (ADSCs). The two highest non-toxic concentrations of each of the compounds were selected and used for testing other activities. The conducted study showed that cinnamaldehyde, thymol, and (R)-(+)-limonene significantly stimulated cell proliferation. In the case of cinnamaldehyde, the doubling time (DT) for MC3T3-E1 cells was significantly shortened to approx. 27 h compared to the control cells (DT = 38 h). In turn, cinnamaldehyde, carvacrol, (R)-(+)-limonene, (S)-(-)-limonene, sabinene, and α-pinene exhibited positive effects on either the synthesis of bone ECM or/and mineral deposition in ECM of the cells. Based on the conducted research, it can be assumed that cinnamaldehyde and (R)-(+)-limonene are the most promising among all tested EO-derived compounds and can be selected for further detailed research in order to confirm their biomedical potential in the chemoprevention or treatment of osteoporosis since they not only accelerated the proliferation of preosteoblasts, but also significantly enhanced osteocalcin (OC) synthesis by preosteoblasts (the OC level was approx. 1100-1200 ng/mg compared to approx. 650 ng/mg in control cells) and ECM calcification of both preosteoblasts and mesenchymal stem cells. Importantly, cinnamaldehyde treatment led to a three-fold increase in the mineral deposition in ADSCs, whereas (R)-(+)-limonene caused a two-fold increase in the ECM mineralization of both MC3T3-E1 cells and ADSCs.

Rat Serum Osteocalcin Concentration Is Decreased by Restriction of Energy Intake

We studied the effects of energy restriction on serum osteocalcin concentration and bone formation rate in rats. The experiment was designed to achieve energy restriction by reducing the carbohydrate intake while providing identical quantities of protein, fat, vitamins and minerals. Energy intakes of three groups of post-weaning male rats were restricted by 20, 40 and 60% for 4 wk. Serum calclum, phosphorus, transthyretin, triiodothyronine (T3), thyroxine (T4), 1,25-dihydroxycholecalciferol, 25-hydroxycholecalciferol and immunoreactive parathyroid hormone (iPTH) concentrations were determined. Energy restriction (20, 40 and 60%) produced a significant and gradual drop of serum osteocalcin concentrations, although the serum concentrations of its key regulators, i.e., 1,25-dihydroxycholecalciferol and iPTH, were not significantly affected. On the contrary, serum concentrations of calcium, phosphorus, transthyretin, T3 and T4 were significantly lower in the energy-restricted groups. However, our results do not support their implication in the regulation of serum osteocalcin synthesis by energy intake. Serum osteocalcin concentration was positively correlated with bone mineral apposition (r = 0.50, P < 0.05) and bone mineralization (r = 0.50, P < 0.05) rates suggesting that its decrease resulted from a reduction of bone formation, and not from abnormal mineralization, because osteoid seam thickness was not modified. Energy intake seems to be an important determinant of serum osteocalcin concentration and bone formation; however, the exact mechanism underlying this regulation remains to be determined.

Modulation of Osteogenic Gene Expression by Human Osteoblasts Cultured in the Presence of Bisphenols BPF, BPS, or BPAF

Bone effects attributed to bisphenols (BPs) include the inhibition of growth and differentiation. This study analyzes the effect of BPA analogs (BPS, BPF, and BPAF) on the gene expression of the osteogenic markers RUNX2, osterix (OSX), bone morphogenetic protein-2 (BMP-2), BMP-7, alkaline phosphatase (ALP), collagen-1 (COL-1), and osteocalcin (OSC). Human osteoblasts were obtained by primary culture from bone chips harvested during routine dental work in healthy volunteers and were treated with BPF, BPS, or BPAF for 24 h at doses of 10-5, 10-6, and 10-7 M. Untreated cells were used as controls. Real-time PCR was used to determine the expression of the osteogenic marker genes RUNX2, OSX, BMP-2, BMP-7, ALP, COL-1, and OSC. The expression of all studied markers was inhibited in the presence of each analog; some markers (COL-1; OSC, BMP2) were inhibited at all three doses and others only at the highest doses (10-5 and 10-6 M). Results obtained for the gene expression of osteogenic markers reveal an adverse effect of BPA analogs (BPF, BPS, and BPAF) on the physiology of human osteoblasts. The impact on ALP, COL-1, and OSC synthesis and therefore on bone matrix formation and mineralization is similar to that observed after exposure to BPA. Further research is warranted to determine the possible contribution of BP exposure to the development of bone diseases such as osteoporosis.

Biocompatibility study of tobacco mosaic virus nanoparticles on human alveolar bone cells

One of the most important factors in a dental implant's success is an adequate quantity of supporting bone. However, there are still some limitations for the bone substitution material. Previous studies found that tobacco mosaic virus (TMV) had the potential for bone formation induction. The aim of this study was to evaluate the biocompatibility of TMV with primary human alveolar bone cells. Primary human alveolar bone cells were cultured on TMV coated substrates. Cell viability, alkaline phosphatase activity, calcium matrix mineralization forming ability, immunofluorescence staining for osteocalcin synthesis and cell morphology were assessed. The results showed that primary human alveolar bone cells cultured on the TMV coated substrates had a higher metabolic rate than the non-TMV coated control group at days 1, 3, 7 and 14. Moreover, the calcium deposition was positive and the alkaline phosphatase activity assay was found significantly greater than the control group at day 14 (p < 0.05). The osteocalcin protein synthesis was found in both the TMV coated substrates and the control group. The immunofluorescence study revealed that in the TMV coated substrates group, the cell morphology changed into a polygonal shape and aggregated more quickly than the control group. The present findings conclude that TMV is biocompatible with primary human alveolar bone cells and also shows osteoinduction potential.

Calcined Hydroxyapatite with Collagen I Foam Promotes Human MSC Osteogenic Differentiation

Collagen I-based foams were modified with calcined or noncalcined hydroxyapatite or calcium phosphates with various particle sizes and pores to monitor their effect on cell interactions. The resulting scaffolds thus differed in grain size, changing from nanoscale to microscopic, and possessed diverse morphological characteristics and resorbability. The materials’ biological action was shown on human bone marrow MSCs. Scaffold morphology was identified by SEM. Using viability test, qPCR, and immunohistochemical staining, we evaluated the biological activity of all of the materials. This study revealed that the most suitable scaffold composition for osteogenesis induction is collagen I foam with calcined hydroxyapatite with a pore size of 360 ± 130 µm and mean particle size of 0.130 µm. The expression of osteogenic markers RunX2 and ColI mRNA was promoted, and a strong synthesis of extracellular protein osteocalcin was observed. ColI/calcined HAP scaffold showed significant osteogenic potential, and can be easily manipulated and tailored to the defect size, which gives it great potential for bone tissue engineering applications.

Impact of cigarette smoke on osteogenic and osteoclast signaling in middle palatal suture.

Considering that smoking is a public health problem that has been growing among adolescents, the aim of this study was to investigate the impact of cigarette smoke on osteogenic and osteoclastogenic signaling in middle palatal suture of rats. Male Wistar rats exposed (n = 30) or not to cigarette smoke (n = 30) were used. Exposure to smoke was carried out for two daily periods of 3 minutes each, with an interval of 12 hours between exposures. After the experimental periods of 3, 7, 14 and 21 days, the animals were euthanized. The collected tissues were analyzed using light microscopy and real-time RT-PCR was performed to investigate gene expression. The data obtained were compared using the Kruskal Wallis and Dunn tests (⍺ = 5%). Morphologically, there were no significant changes in the middle palatal suture of rats exposed or not to cigarette smoke during 3, 7, 14 and 21 days (p> 0.05). On the other hand, osteoclastogenic signaling was increased in animals exposed to smoke and was characterized by a higher production of RANKL at 3 and 14 days (p <0.05), with no change in the synthesis of RANK and osteoprotegerin (p> 0.05). Interestingly, in the exposed animals, an early increase in the synthesis of osteocalcin, bone sialoprotein and osteopontin was also identified at 3 days of exposure (p <0.05), not sustained over time (p> 0.05). Cigarette smoke modulates osteogenic and osteoclastogenic signaling in the middle palatal suture of young rats, although morphological changes have not been evidenced.

Metformin-Incorporated Gelatin/Nano-Hydroxyapatite Scaffolds Promotes Bone Regeneration in Critical Size Rat Alveolar Bone Defect Model.

In this study, we fabricated gelatin/nano-hydroxyapatite/metformin scaffold (GHMS) and compared its effectiveness in bone regeneration with extraction-only, Sinbone, and Bio-Oss Collagen® groups in a critical size rat alveolar bone defect model. GHMS was synthesized by co-precipitating calcium hydroxide and orthophosphoric acid within gelatin solution, incorporating metformin, and cross-linked by microbial transglutaminase. The morphology, characterization, and biocompatibility of scaffold were examined. The in vitro effects of GHMS on osteogenic gene and protein expressions were evaluated. In vivo bone formation was assessed in a critical size rat alveolar bone defect model with micro-computed tomography and histological examination by comparing GHMS with extraction-only, Sinbone, and Bio-Oss Collagen®. The synthesized GHMS had a highly interconnected porous structure with a mean pore size of 81.85 ± 13.8 µm. GHMS exhibited good biocompatibility; promoted ALPL, RUNX2, SP7, BGLAP, SPARC and Col1a1 gene expressions; and upregulated the synthesis of osteogenic proteins, including osteonectin, osteocalcin, and collagen type I. In critical size rat alveolar bone defects, GHMS showed superior bone regeneration compared to extraction-only, Sinbone, and Bio-Oss Collagen® groups as manifested by greater alveolar ridge preservation, while more bone formation with a lower percentage of connective tissue and residual scaffold at the defect sites grafted with GHMS in histological staining. The GHMS presented in this study may be used as a potential bone substitute to regenerate alveolar bone. The good biocompatibility, relatively fast degradation, interconnected pores allowing vascularization, and higher bioactivity properties of the components of the GHMS (gelatin, nHA, and metformin) may contribute to direct osteogenesis.

Highly Porous Fluorapatite/β-1,3-Glucan Composite for Bone Tissue Regeneration: Characterization and In-Vitro Assessment of Biomedical Potential.

A novel fluorapatite/glucan composite (“FAP/glucan”) was developed for the treatment of bone defects. Due to the presence of polysaccharide polymer (β-1,3-glucan), the composite is highly flexible and thus very convenient for surgery. Its physicochemical and microstructural properties were evaluated using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), mercury intrusion, mechanical testing and compared with the reference material, which was a hydroxyapatite/glucan composite (“HAP/glucan”) with hydroxyapatite granules (HAP) instead of FAP. It was found that FAP/glucan has a higher density and lower porosity than the reference material. The correlation between the Young’s modulus and the compressive strength between the materials is different in a dry and wet state. Bioactivity assessment showed a lower ability to form apatite and lower uptake of apatite-forming ions from the simulated body fluid by FAP/glucan material in comparison to the reference material. Moreover, FAP/glucan was determined to be of optimal fluoride release capacity for osteoblasts growth requirements. The results of cell culture experiments showed that fluoride-containing biomaterial was non-toxic, enhanced the synthesis of osteocalcin and stimulated the adhesion of osteogenic cells.

Rethinking Fragility Fractures in Type 2 Diabetes: The Link between Hyperinsulinaemia and Osteofragilitas.

Patients with type 2 diabetes mellitus (T2DM) and/or cardiovascular disease (CVD), conditions of hyperinsulinaemia, have lower levels of osteocalcin and bone remodelling, and increased rates of fragility fractures. Unlike osteoporosis with lower bone mineral density (BMD), T2DM bone fragility “hyperinsulinaemia-osteofragilitas” phenotype presents with normal to increased BMD. Hyperinsulinaemia and insulin resistance positively associate with increased BMD and fragility fractures. Hyperinsulinaemia enforces glucose fuelling, which decreases NAD+-dependent antioxidant activity. This increases reactive oxygen species and mitochondrial fission, and decreases oxidative phosphorylation high-energy production capacity, required for osteoblasto/cytogenesis. Osteocytes directly mineralise and resorb bone, and inhibit mineralisation of their lacunocanalicular space via pyrophosphate. Hyperinsulinaemia decreases vitamin D availability via adipocyte sequestration, reducing dendrite connectivity, and compromising osteocyte viability. Decreased bone remodelling and micropetrosis ensues. Trapped/entombed magnesium within micropetrosis fossilisation spaces propagates magnesium deficiency (MgD), potentiating hyperinsulinaemia and decreases vitamin D transport. Vitamin D deficiency reduces osteocalcin synthesis and favours osteocyte apoptosis. Carbohydrate restriction/fasting/ketosis increases beta-oxidation, ketolysis, NAD+-dependent antioxidant activity, osteocyte viability and osteocalcin, and decreases excess insulin exposure. Osteocalcin is required for hydroxyapatite alignment, conferring bone structural integrity, decreasing fracture risk and improving metabolic/endocrine homeodynamics. Patients presenting with fracture and normal BMD should be investigated for T2DM and hyperinsulinaemia.

Leptin Mediated Pathways Stabilize Posttraumatic Insulin and Osteocalcin Patterns after Long Bone Fracture and Concomitant Traumatic Brain Injury and Thus Influence Fracture Healing in a Combined Murine Trauma Model.

Recent studies on insulin, leptin, osteocalcin (OCN), and bone remodeling have evoked interest in the interdependence of bone formation and energy household. Accordingly, this study attempts to investigate trauma specific hormone changes in a murine trauma model and its influence on fracture healing. Thereunto 120 female wild type (WT) and leptin-deficient mice underwent either long bone fracture (Fx), traumatic brain injury (TBI), combined trauma (Combined), or neither of it and therefore served as controls (C). Blood samples were taken weekly after trauma and analyzed for insulin and OCN concentrations. Here, WT-mice with Fx and, moreover, with combined trauma showed a greater change in posttraumatic insulin and OCN levels than mice with TBI alone. In the case of leptin-deficiency, insulin changes were still increased after bony lesion, but the posttraumatic OCN was no longer trauma specific. Four weeks after trauma, hormone levels recovered to normal/basal line level in both mouse strains. Thus, WT- and leptin-deficient mice show a trauma specific hyperinsulinaemic stress reaction leading to a reduction in OCN synthesis and release. In WT-mice, this causes a disinhibition and acceleration of fracture healing after combined trauma. In leptin-deficiency, posttraumatic OCN changes are no longer specific and fracture healing is impaired regardless of the preceding trauma.

Functional State of Mesenchymal Stem Cells upon Exposure to Bioactive Coatings on Titanium Alloys.

Bioactive coatings on implants affect osteogenic differentiation of mesenchymal stem cells (MSC). We studied the morphofunctional state of bone marrow MSC cultured on the surface of calcium phosphate coatings on titanium formed by plasma electrolytic oxidation (PEO). The biocompatible properties of the coatings manifested in the absence of the cytotoxic effect on cells. High expression of receptors (CD90, CD29, and CD106), enhanced synthesis of osteocalcin and osteopontin, and changes in surface architectonics of MSC adherent to the samples confirmed osteoinductive properties of the calcium phosphate PEO coating.

Repair processes of flat bones formed via intramembranous versus endochondral ossification

ObjectivesAlthough fractures occur in various bones, including long, short, and flat bones, fracture repair investigations focus on the diaphysis of the long bone. The cell composition, osteogenic capacity, and bone matrix differ among osteogenesis patterns. However, the differences in the bone repair process have not been studied. Here, we compared the bone repair processes in the parietal bone and scapula of adolescent mice. MethodsBone apertures were created in the parietal bone and scapula. Samples were collected at indicated times after surgery, and the repair process was analyzed using micro-computed tomography, histological, immunohistochemical, and mRNA expression analyses. ResultsIn both repair processes, cartilage formation was not detected on the periosteum side. The parietal bone aperture was gradually filled with newly formed bone produced from the edge of the aperture by day 14 but was not completely repaired even by day 49. In the scapula, a bony callus was detected on the periosteum at day 7, and the aperture was bridged by day 14. Subsequently, the bony callus was remodeled to the original bone architecture. Alkaline phosphatase activity and osteocalcin synthesis occurred earlier in the repair region of the scapular periosteum, compared with that in the parietal periosteum. The mRNA expression of osteogenic markers in the periosteum was markedly upregulated in the scapula versus the parietal bone. ConclusionOur study findings clarify the differences between parietal bone and scapula repair and suggest that the bone repair process differs among ossification patterns.

3D Human Periodontal Stem Cells and Endothelial Cells Promote Bone Development in Bovine Pericardium-Based Tissue Biomaterial.

Bone defects repair represents a public and urgent problem in clinical practice, in fact, every year, more than two million patients required new treatments for bone injuries. Today a complete vascularization is strategic in bone formation, representing a new frontier for clinical application. Aim of this research has been developed a three-dimensional (3D) coculture platform using a bovine pericardium collagen membrane (BioR) loaded with human periodontal ligament stem cells (hPDLSCs) and endothelial differentiated cells from hPDLSCs (E-hPDLSCs) able to undergo toward osteoangiogenesis differentiation process. First, we have characterized at confocal laser scanning microscopy (CLSM) level the E-hPDLSCs phenotype profile, through CD31 and CD34 markers expression and the ability to tube vessel formation. Real Time-Polimerase Chain Reaction (RT-PCR) and western blotting analyses revealed the upregulation of Runt-related transcription factor 2 (RUNX2), Collagen 1A1 (COL1A1), Vascular Endothelial Growth Factor-A (VEGF-A) genes and proteins in the living construct composed by hPDLSCs + E-hPDSCs/BioR. Human PDLSCs + E-hPDLSCs/BioR construct showed also an enhacement of de novo synthesis of osteocalcin. Given that, the extracellular-signal-regulated kinase (ERK)/mitogen activated protein kinase (MAPK) transduction signaling was involved in the osteogenesis and angiogenesis process, the ERK1/2 protein level at biochemical level, in our experimental model, has been investigated. Our results evidenced an upregulation of ERK1/2 proteins level born in the living construct. In conclusion, we believe that the use of the hPDLSCs and E-hPDLSCs coculture togheter with BioR as substrate, could represent an efficient model able to activate through ERK1/2 signaling pathway the osteoangiogenesis process, and then representing a new potential engineered platform for surgeons during the repair and the healing of bone defects.

A comparatively study of menaquinone-7 isolated from Cheonggukjang with vitamin K1 and menaquinone-4 on osteoblastic cells differentiation and mineralization

The effect of menaquinone-7 isolated from cheonggukjang was comparatively investigated with vitamin K1 and menaquinone-4 on cell differentiation and mineralization of the osteoblastic cell line MC3T3-E1. Results indicated that all vitamin K species significantly increased MC3T3-E1 cell proliferation, cellular alkaline phosphatase activity, osteocalcin synthesis, and calcium deposition in a dose-dependent manner. Menaquinone-4 and menaquinone-7 had more potent effects on calcium deposition than vitamin K1, and their effects were only partly reduced by warfarin (γ-carboxylation inhibitor) treatment, while warfarin abolished the induction activity of vitamin K1 on calcification. This suggests that vitamin K1 and K2 (menaquinone-4 & menaquinone-7) may have different mechanisms in stimulating osteoblast mineralization. In addition, the mRNA expression ratio of osteoprotegerin and the receptor activator of nuclear factor-kB ligand was also dramatically increased by treatment with vitamin K1 (62%), menaquinone-4 (247%), and menaquinone-7 (329%), suggesting that vitamin K may suppress the formation of osteoclast by up-regulating the ratio of osteoprotegerin/receptor activator of nuclear factor-kB ligand in osteoblasts. These results provide compelling evidence that vitamin K1, menaquinone-4, and menaquinone-7 all can promote bone health, which might be associated with elevations in the osteoprotegerin/receptor activator of nuclear factor-kB ligand ratio.

Xenograft of Human Umbilical Mesenchymal Stem Cells from Wharton’s Jelly Differentiating into Osteocytes and Reducing Osteoclast Activity Reverses Osteoporosis in Ovariectomized Rats

We examined the effects of human umbilical cord-derived mesenchymal stem cells (HUMSCs) in Wharton’s jelly on ovariectomy (OVX)-induced osteoporosis by using in vitro and in vivo experiments. Two months after OVX, the rats gained weight and had a decreased serum estradiol level . Both micro-computed tomography (micro-CT) and histochemical analyses revealed a marked decrease in the bone volume (BV) and collagen content within the head, neck, and distal condyle of the femur, indicating that the osteoporosis animal model was successfully established 2 mo after bilateral OVX. Subsequently, 2.5 × 106 HUMSCs were injected into the bone marrow cavity of the left femurs 2 mo after OVX. The rats were divided into the following groups: normal + phosphate-buffered saline (PBS), normal + HUMSCs, OVX + PBS, and OVX + HUMSCs. Two months after transplantation, both micro-CT imaging and histochemical staining revealed that the normal + HUMSCs group had higher BV and collagen content in the epiphysis and metaphysis than did the normal + PBS group. In the OVX + HUMSCs group, a substantial increase in the rod-shaped trabecular bone and the abundant accumulation of collagen were observed around the site of HUMSC transplantation. Plenty of transplanted HUMSCs remained viable and differentiated into osteoblasts. In addition, HUMSC transplantation reduced the number of osteoclasts. Compared with HUMSCs cultured alone, HUMSCs cocultured with osteoblasts showed that the percentage of cells differentiating into osteoblasts significantly increased. Furthermore, osteoclasts cocultured with HUMSCs had significantly decreased cellular activity and differentiation capability. HUMSC transplantation into the distal femur of OVX rats could locally stimulate osteocalcin synthesis, increase the trabecular bone, and inhibit osteoclast activity.

Influence of hydrodynamic pressure on the proliferation and osteogenic differentiation of bone mesenchymal stromal cells seeded on polyurethane scaffolds.

Hydraulic pressure has recently been introduced as an effective stimulation in the field of tissue engineering. In this study, a polymer scaffold consisting of polyurethane (PU)-based 1, 4-butanediisocyanate was fabricated. A self-designed bioreactor was employed to produce perfusion and hydrodynamic pressure stimulations. The viability, proliferation and osteogenic differentiation of the rat bone mesenchymal stromal cell (rBMSC) growing in the polymer scaffold were investigated after hydrodynamic pressure stimulation. Additionally, the mechanical properties of the cell-laden constructs were also evaluated. Our findings suggested that the perfusion rate (10 mL/min) and low hydrodynamic pressure stimulation (60 mmHg, 0.5 Hz) maintained the viability of rBMSC during 2 weeks cultivation. The cell proliferation was promoted by 60 mmHg stimulation in the first week. The synthesis of alkaline phosphates and osteocalcin was enhanced after 2 weeks stimulation. Meanwhile, the equilibrium modulus of scaffold was increased by 1.85-fold using 60 mmHg hydrodynamic pressure stimulation. Additionally, type I and III procollagen produced by rBMSC was increased 4.92- and 3.02-fold, respectively. However, no encouraging results were detected in 120 mmHg hydrodynamic pressure group. Our study suggests that the 60 mmHg hydrodynamic pressure is a promising approach to enhance the functional properties of the rBMSC-laden PU-based bone scaffold. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3445-3455, 2017.

Bergenin increases osteogenic differentiation and prevents methylglyoxal-induced cytotoxicity in MC3T3-E1 osteoblasts.

Bergenin, an active component of plants in the genus Bergenia, has multiple biological activities, including anti-inflammatory and immunomodulatory properties. We investigated the effects of bergenin on MC3T3-E1 osteoblasts. Bergenin treatment significantly elevated collagen synthesis, alkaline phosphatase activity, osteocalcin synthesis, and mineralization in the cells (p<0.05). Additionally, bergenin increased the ratio of osteoprotegerin to receptor activator of nuclear factor kappa-B ligand, and cyclophilin B release. Methylglyoxal (MG), a highly reactive dicarbonyl compound, is the major precursor in the formation of advanced glycation end products. Pretreatment of MC3T3-E1 cells with bergenin prevented MG-induced cell death. Furthermore, bergenin treatment significantly reduced the induction of activating transcription factor 6 and autophagy by MG. These results indicate that bergenin may have positive effects on critical osteoblastic cell functions.

Limonene attenuates methylglyoxal-induced dysfunction in MC3T3-E1 osteoblastic cells

ABSTRACTLimonene is a common natural terpene with powerful antioxidative properties. This study investigated the effects of limonene, a terpene found in citrus fruits, on the function of the murine pre-osteoblast cell line, MC3T3-E1 cells. The results showed that limonene treatment significantly elevated collagen synthesis, alkaline phosphatase activity, osteocalcin synthesis, and mineralization in osteoblastic cells. Methylglyoxal (MG), a highly reactive dicarbonyl metabolite, is a major precursor of advanced glycation end products, which are involved in the pathogenesis of diabetic osteopathy. We therefore investigated the effects of limonene on MG-induced cytotoxicity. Pre-treatment of MC3T3-E1 cells with limonene prevented MG-induced cell death and apoptosis. Limonene also reduced MG-triggered endoplasmic reticulum (ER) stress, as indicated by decreases in the levels of the ER-localized transmembrane signal transducers ATF-6 and IRE1. Furthermore, limonene treatment significantly reduced MG-induced autophagic activity and reactive oxygen species release. These results suggest that limonene may prevent the development of diabetic osteopathy.

Resveratrol suppresses thyroid hormone‑induced osteocalcin synthesis in osteoblasts.

Resveratrol, a polyphenolic compound that is present in grape skins, berries and red wine, may be beneficial for human health through its anti‑inflammatory and anti‑oxidant effects. It has been previously demonstrated that resveratrol exerts its biological effects primarily via sirtuin1 (SIRT1) activation. We previously reported that triiodothyronine (T3) induces osteocalcin synthesis in osteoblast‑like MC3T3‑E1 cells, and that p38 mitogen‑activated protein (MAP) kinase mediates the T3‑stimulated synthesis of osteocalcin. The present study investigated the effect of resveratrol on T3‑induced osteocalcin synthesis and its underlying mechanism in MC3T3‑E1 cells. Cultured cells were stimulated with T3, and osteocalcin release from MC3T3‑E1 cells was measured by ELISA and phosphorylation of p38 MAP kinase was analyzed by western blotting. Resveratrol significantly suppressed the release of osteocalcin stimulated by T3, and SRT1720, a SIRT1 activator, significantly reduced T3‑induced osteocalcin release. The expression level of osteocalcin mRNA stimulated by T3 was significantly attenuated by resveratrol and T3‑induced transactivation activity of the thyroid hormone‑responsive element was significantly diminished by resveratrol. However, only limited effects of resveratrol on the T3‑induced phosphorylation of p38 MAP kinase were observed. The results of the present study demonstrated that resveratrol suppresses T3‑stimulated osteocalcin synthesis at a point upstream of transcription in osteoblasts, and that the inhibitory effect of resveratrol is mediated, at least partially, through SIRT1 activation. These results indicate that there may be a novel role for the polyphenol in the modulation of bone metabolism.

Synthesis of 2-methyl-1,4-naphthoquinones with higher gamma-glutamyl carboxylase activity than MK-4 both in vitro and in vivo

Vitamin K is the collective term for compounds that share a 2-methyl-1,4-naphthoquinone ring, but differ in the side-chain at the 3-position. We synthesized novel 2-methyl-1,4-naphthoquinone derivatives with different side chain length at the 3-position. Derivatives with C-14 and C-16 tails showed the highest in vitro bioactivity resulting in 2.5 and 2-fold higher carboxylated osteocalcin synthesis in MG63 cells than menaquinone-4 (MK-4, form of vitamin K2). Longer side chain lengths resulted in lower bioactivity. The in vivo vitamin K activity of the C-14 tail derivative was further tested in WKY rats receiving a vitamin K-deficient diet that resulted in a 40% decrease of prothrombin activity. The C-14 tail derivative was able to counteract the effects on vitamin K deficiency induced by the diet and resulted in the complete restoration of prothrombin activity. Compared to naturally occurring forms of vitamin K, synthetic vitamin K derivatives may have higher bioactivity and different pharmacological characteristics that are more favorable for use as supplements or in clinical settings.