Tocotrienol-rich fraction from annatto ameliorates expression of lysyl oxidase in human osteoblastic MG-63 cells.

Lysyl oxidase (LOX) is required for the formation of bone collagen cross-links. Inactivation of the LOX gene in osteoblasts by DNA methylation and JAK signaling has been reported to cause loss of cross-links and an increased risk of fractures. Tocotrienols (T3s) have proven benefits on bone strength, but their potential effects on LOX remain largely unknown. Thus, the present study investigates the in vitro effects of T3s on LOX expression in human osteoblastic MG-63 cells. Results indicated that Tocotrienol-Rich Fraction (TRF), the δ-T3 rich oil extracted from Annatto was the most effective and significantly increased LOX expression. TRF treatment decreased de-novo methyltransferases (DNMTs), DNMT3A and DNMT3B levels. In addition, TRF significantly inhibited JAK2 activation and decreased expression of Fli1, a transcription factor of DNMTs. We conclude that TRF induced an increase in LOX expression via inhibition of de-novo methylation and reduction of Fli1 expression by the inactivation of JAK2.Abbreviations: CpG: cytosine-guanine dinucleotide; DNMT: DNA methyltransferase; Fli1: friend leukemia virus integration 1; JAK: janus kinase; LOX: lysyl oxidase; PCR: polymerase chain reaction; STAT: signal transducers and activators of transcription; T3s: tocotrienols; TPs: tocopherols; TRF: Tocotrienol-Rich Fraction.

Bergamot Polyphenol Fraction Exerts Effects on Bone Biology by Activating ERK 1/2 and Wnt/ß-Catenin Pathway and Regulating Bone Biomarkers in Bone Cell Cultures.

Epidemiological studies show that fruit consumption may modulate bone mineral density. However, data regarding the effect of the Citrus bergamia Risso (Bergamot orange), a citrus fruit containing a high concentration of flavonoids, on bone health are still lacking. In this study, we investigated the effects of Bergamot polyphenols on the Wnt/ß-catenin pathway in two distinct bone cell types (Saos-2 and MG63). Findings showed that exposure to 0.01 and 0.1 mg/mL doses upregulate ß-catenin expression (p = 0.001), osteoblast differentiation markers (e.g., RUNX2 and COL1A), and downregulate RANKL (p = 0.028), as compared to the control. Our results highlight, for the first time, that Bergamot polyphenols act on bone cells through the ß-catenin pathway. In vivo studies are necessary to fully understand Bergamot's role against bone resorption.

Polyphenols (S3) Isolated from Cone Scales of Pinus koraiensis Alleviate Decreased Bone Formation in Rat under Simulated Microgravity.

In order to screen out an effective bone loss protectant from natural plant polyphenol and to elucidate the mechanism of the plant polyphenols that alleviate bone loss under simulated microgravity, the proliferation activities of 9 total polyphenol extracts from natural product (TPENP) on osteoblasts were measured. Polyphenols (S3) was isolated from total polyphenols of cone scales from pinus koraiensis (Korean pine). ALP activity in osteoblasts and MDA level in femur were measured. Mechanical properties and microstructure of the distal cancellous region of the femur in rat were tested. Various bone metabolism markers, enzymes activity and genes expression were also analyzed. The results showed that S3 has the highest activity of osteoblast proliferation. S3 promoted ALP activity in osteoblasts, enhanced mechanical properties and microstructure of the distal cancellous region of femur in rat, decreased MDA level, elevated the serum concentration of BALP, PINP and activities of SOD, CAT, GSH-Px in femur under simulated microgravity. In addition, S3 enhanced the expression of NRF-2, ß-catenin, p-GSK3-ß, OSX, RUNX2, Osteonectin, Osteocalcin, ALP and collagen I. These results indicated that S3 can alleviated bone loss induced by simulated microgravity through abate the inhibition of the oxidative stress on Wnt/ß-catenin signaling pathway.

Osteoblast AMP-activated protein kinase regulates glucose metabolism and bone mass in adult mice.

Previous studies have shown that AMP-activated protein kinase (AMPK), a crucial regulator of energy homeostasis, plays important roles in osteoblast differentiation and mineralization. However, little is known about in vivo roles of osteoblastic AMPK in glucose metabolism and bone mass regulation in adult mice. Here, we used the inducible Cre system to control the onset of Ampk disruption after birth by removing doxycycline supplementation. We conditionally inactivated Ampk in osterix (Osx)-expressing cells in 3-week-old Ampk-/- mice. After 6 months of Ampk inactivation, the Ampk-/- mice displayed lower serum osteocalcin levels as well as glucose intolerance and insulin resistance, as indicated by glucose tolerance and insulin tolerance tests, respectively, when compared with wild-type mice. After 18 months of Ampk inactivation, micro computed tomography showed significant reductions in trabecular bone volume and cortical bone thickness in the femur of Ampk-/- mice when compared with wild-type mice. Moreover, bone stiffness was significantly lower in Ampk-/- mice than in wild-type mice. This is the first study to show that osteoblast AMPK plays an important roles in glucose metabolism and in maintaining trabecular bone volume, cortical thickness, and bone strength in adult mice.

Ophiopogonin D improves osteointegration of titanium alloy implants under diabetic conditions by inhibition of ROS overproduction via Wnt/ß-catenin signaling pathway.

A high failure rate of titanium implants in diabetic patients has been indicated in clinical evidences. Excessive oxidative stress at the bone-implant interface plays an important role in the impaired osteointegration under diabetic conditions. While the underlying mechanisms remain unknown and the targeted treatments are urgently needed. Ophiopogonin D (OP-D), isolated from Chinese herbal Radix Ophiopogon japonicus, is generally reported to be a potent antioxidant agent. In the present study, we hypothesized that OP-D exerted promotive effects on osteointegration against oxidative stress, and investigated the underlying mechanisms associated with alteration of Wnt/ß-catenin signaling pathway. Rabbit osteoblasts incubated on titanium alloy implant were co-cultured with normal serum (NS), diabetic serum (DS), DS + OP-D, DS + NAC (a potent ROS inhibitor) and DS + OP-D + Dkk1 (a Wnt inhibitor) for examinations of osteoblast behaviors. For in vivo study, titanium alloy implants were implanted into the femoral condyle defects on diabetic rabbits. Results demonstrated that diabetes-induced oxidative stress resulted in osteoblast dysfunctions and apoptotic injury at the bone-implant interface, concomitant with the inactivation of Wnt/ß-catenin signaling. Importantly, OP-D administration attenuated oxidative stress, directly reactivating Wnt/ß-catenin signaling. Osteoblast dysfunctions were thus reversed as evidenced by improved osteoblast adhesion, proliferation and differentiation, and ameliorated apoptotic injury, exerting similar effects to NAC treatment. In addition, the positive effects afforded by OP-D were confirmed by improved osteointegration and oetogenesis within the titanium alloy implants in vivo by Micro-CT and histological analyses. Furthermore, the pro-osteogenic effects of OP-D were almost completely abolished by the Wnt inhibitor Dkk1. These results demonstrated, for the first time, OP-D administration alleviated the damaged osteointegration of titanium alloy implants under diabetic conditions by means of inhibiting oxidative stress via a Wnt/ß-catenin-dependent mechanism. The OP-D administration would become a reliable treatment strategy for implant failure therapy in diabetics due to the optimal anti-oxidative and pro-osteogenic properties.

Effect of buffalo casein-derived novel bioactive peptides on osteoblast differentiation.

PURPOSE: Epidemiological and intervention studies show that milk consumption in childhood and during adolescence is related to higher bone mineral density. Milk and milk products prevent the bone loss in pre- and postmenopausal women. Apart from calcium, there are other biologically active compounds in milk such as bioactive peptides which may play a role in promoting bone health. Casein is the major protein in milk which has also been reported to have numerous biological active peptides within it. The hypothesis of the present study was to identify the key peptides behind osteoanabolic nature of the milk protein, which further can be used to prepare functional foods to alleviate bone diseases like osteoporosis. Hence, this study was carried out to investigate osteogenic nature of four novel bioactive peptides [PEP1 (EDVPSER), PEP2 (NAVPITPTL), PEP3 (VLPVPQK) and PEP4 (HPHPHLSF)] derived from buffalo casein by in vitro osteoblast differentiation model. METHODS: Calvaria cells were isolated from 3-day-old rat pups, cultured under in vitro conditions till confluence and further used for experiments. Calvarial osteoblast cells were cultured in the presence or absence of peptides including positive controls up to 21 days. Effect of peptides was checked at regular intervals by quantifying osteoblast differentiation marker genes (ALP, OCN and COL-1) expression, alkaline phosphatase activity, osteocalcin level in culture supernatants, mineral deposition by alizarin red staining and caspase-3 and 9 assays. RESULTS: The osteoblast differentiation marker genes (ALP, OCN and COL-1) expression was significantly [(p < 0.01) (p < 0.001)] up-regulated in the presence of these peptides. The peptides also significantly induced alkaline phosphatase activity, osteocalcin level and mineral deposition in comparison with the control. It was also observed that all the four peptides did not show any cytotoxic effect during 21-day treatment period. CONCLUSION: All peptides enhanced osteoblast differentiation along with the positive controls. These results hold an immense scope to use peptides as preventive measure for reducing incidence of osteoporosis. These peptides can also be used as drugs and can be utilized as functional ingredients in functional foods preparation for osteoporosis therapy, but in vivo studies are required for further confirmation.

Effect of magnesium on the osteogenesis of normal human osteoblasts.

Biomaterials containing magnesium are used for implants and bone regeneration. However, mechanisms underlying the biologic effects of magnesium are still largely unknown and have not been examined on normal human osteoblasts. This study was designed to test the effect of supplemented Mg2+ concentrations between 0.5 mM and 16 mM on the osteogenic behaviors of normal human primary osteoblasts. Human primary osteoblasts were cultured in the groups with various concentrations of supplemented magnesium for various time intervals. Cell proliferation was measured using crystal violet staining. Degree of Alkaline Phosphatase (ALP) activity was measured by fluorometric assay. Expression of osteocalcin was measured by immunosorbent assay. Mineralization of cultures was determined by Alizarin Red S staining. Results showed that initial cell attachment efficiency was not affected by supplemented Mg2+ (P > 0.05). At 21 days, proliferation rates increased in groups containing 0.5 mM-4 mM supplemented Mg2+ and decreased in groups of supplemented 8 mM and 16 mM Mg2+. ALP activity and osteocalcin expression were upregulated in groups of supplemented Mg2+ between 0.5 mM-2.0 mM (P < 0.05), but downregulated in groups with supplemented Mg2+ concentrations of 4mM and above (P < 0.05). Cultures with 1 mM and 2 mM supplemented Mg2+ showed upregulated mineralization activity compared to the control (P < 0.05), but downregulated in groups with supplemented Mg2+ concentrations of 4 mM and above (P < 0.05). The present study based on an experimental design demonstrated the impact of 2 mM supplemented Mg2+ on induced-proliferation and differentiation of normal human osteoblasts.

FGF18 inhibits MC3T3­E1 cell osteogenic differentiation via the ERK signaling pathway.

Fibroblast growth factor (FGF) 18 is a member of the FGF family and serves a key role in skeletal growth and development. The present study investigated the effect of FGF18 on pre­osteoblast MC3T3-E1 cells and the signaling pathways involved by performing an alkaline phosphatase (ALP) assay and reverse transcription­quantitative polymerase chain reaction. MC3T3­E1 cells incubated in a culture medium supplemented with FGF18 exhibited increased viability when compared with the untreated control cells. In addition, ALP activity was decreased in MC3T3­E1 cells treated with FGF18 plus an osteogenic medium (OM) for 7 and 14 days when compared with untreated and OM­treated controls. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) results demonstrated that the expression of osteoblastic­associated genes was significantly repressed in FGF18 plus OM­treated MC3T3­E1 cells, including ALP, collagen type I, osteocalcin, bone sialo protein and osterix. These results suggested that the expression levels of genes associated with osteogenesis were mainly repressed. In addition, combined treatment of MC3T3­E1 cells with OM and FGF18 led to a significant reduction in mineral deposition when compared with the OM­only treated group. Furthermore, FGF18 activated the extracellular signal­regulated kinase pathway in MC3T3­E1 cells, which may have been responsible for the observed decrease in the expression of osteoblastic­associated genes. In conclusion, the results suggest that FGF18 may be involved in MC3T3­E1 cell proliferation and osteoblastic differentiation.

Dried Plum Ingestion Increases the Osteoblastogenic Capacity of Human Serum.

In cell culture studies, dried plum (Prunus domestica L.) polyphenols increased osteoblast alkaline phosphatase (ALP) activity, mineralized nodule formation, and the expression of the bone marker genes runt-related transcription factor 2 (RUNX2) and osterix. The purpose of this study was to determine whether human serum collected 1 and 2 h after dried plum ingestion influenced osteoblast cell activity and gene expression. Five healthy women ingested 100 g of dried plum, and serum samples were collected at baseline (before dried plum ingestion) and 1 and 2 h postingestion of dried plum. MC3T3-E1 osteoblast cells were treated (2% of medium) with these serum samples for 3 or 9 days. Intracellular and extracellular ALP activities were significantly increased after 3 or 9 days of treatment with serum both postingestion time points, with no effect seen in baseline samples. Also, serum obtained 1 and 2 h postingestion significantly increased the mRNA expression of bone markers RUNX2 and connexin43 (CX43) after both 3 and 9 days of incubation periods. Finally, serum obtained 1 and 2 h postingestion increased the mRNA expression of ß-catenin after 9 days of incubation. We conclude that osteoblast activity and function are increased by dried plum ingestion, which may, in part, explain its beneficial effects on bone health.

Low level laser therapy modulates viability, alkaline phosphatase and matrix metalloproteinase-2 activities of osteoblasts.

Low level laser therapy (LLLT) has been shown to stimulate bone cell metabolism but their impact on the matrix metalloproteinase (MMP) expression and activity is little explored. This study evaluated the influence of LLLT at two different wavelengths, red and infrared, on MC3T3-E1 preosteoblast viability, alkaline phosphatase (ALP) and MMP-2 and -9 activities. To accomplish this, MC3T3-E1 cells were irradiated with a punctual application of either red (660nm; InGaAIP active medium) or infrared (780nm; GaAlAs active medium) lasers both at a potency of 20mW, energy dose of 0.08 or 0.16J, and energy density of 1.9J/cm2 or 3.8J/cm2, respectively. The control group received no irradiation. Cellular viability, ALP and MMP-2 and -9 activities were assessed by MTT assay, enzymatic activity and zymography, respectively, at 24, 48 and 72h. The treatment of cells with both red and infrared lasers significantly increased the cellular viability compared to the non-irradiated control group at 24 and 48h. The ALP activity was also up modulated in infrared groups at 24 and 72h, depending on the energy densities. In addition, the irradiation with red laser at the energy density of 1.9J/cm2 promoted an enhancement of MMP-2 activity at 48 and 72h. However, no differences were observed for the MMP-9 activity. In conclusion, when used at these specific parameters, LLL modulates both preosteoblast viability and differentiation highlighted by the increased ALP and MMP-2 activities induced by irradiation.

Ethanol Extract of Cissus quadrangularis Enhances Osteoblast Differentiation and Mineralization of Murine Pre-Osteoblastic MC3T3-E1 Cells.

Traditional medicinal literature and previous studies have reported the possible role of Cissus quadrangularis (CQ) as an anti-osteoporotic agent. This study examines the effectiveness of CQ in promoting osteoblast differentiation of the murine pre-osteoblast cell line, MC3T3-E1. Ethanolic extract of CQ (CQ-E) was found to affect growth kinetics of MC3T3-E1 cells in a dosage-dependent manner. High concentrations of CQ-E (more than 10 µg/ml) have particularly adverse effects, while lower concentrations of 0.1 and 1 µg/ml were non-toxic and did not affect cell viability. Notably, cell proliferation was significantly increased at the lower concentrations of CQ-E. CQ-E treatment also augmented osteoblast differentiation, as reflected by a substantial increase in expression of the early osteoblast marker ALP activity, and at later stage, by mineralization of extracellular matrix compared to the control group. These findings suggest dose-dependent effect of CQ-E with lower concentrations exhibiting anabolic and osteogenic properties. J. Cell. Physiol. 232: 540-547, 2017. © 2016 Wiley Periodicals, Inc.

Photothermal effects of laser-activated surface plasmonic gold nanoparticles on the apoptosis and osteogenesis of osteoblast-like cells.

The specific properties of gold nanoparticles (AuNPs) make them a novel class of photothermal agents that can induce cancer cell damage and even death through the conversion of optical energy to thermal energy. Most relevant studies have focused on increasing the precision of cell targeting, improving the efficacy of energy transfer, and exploring additional functions. Nevertheless, most cells can uptake nanosized particles through nonspecific endocytosis; therefore, before hyperthermia via AuNPs can be applied for clinical use, it is important to understand the adverse optical-thermal effects of AuNPs on nontargeted cells. However, few studies have investigated the thermal effects induced by pulsed laser-activated AuNPs on nearby healthy cells due to nonspecific treatment. The aim of this study is to evaluate the photothermal effects induced by AuNPs plus a pulsed laser on MG63, an osteoblast-like cell line, specifically examining the effects on cell morphology, viability, death program, and differentiation. The cells were treated with media containing 50 nm AuNPs at a concentration of 5 ppm for 1 hour. Cultured cells were then exposed to irradiation at 60 mW/cm(2) and 80 mW/cm(2) by a Nd:YAG laser (532 nm wavelength). We observed that the cytoskeletons of MG63 cells treated with bare AuNPs followed by pulsed laser irradiation were damaged, and these cells had few bubbles on the cell membrane compared with those that were not treated (control) or were treated with AuNPs or the laser alone. There were no significant differences between the AuNPs plus laser treatment group and the other groups in terms of cell viability, death program analysis results, or alkaline phosphatase and calcium accumulation during culture for up to 21 days. However, the calcium deposit areas in the cells treated with AuNPs plus laser were larger than those in other groups during the early culture period.

Therapeutic potential of compound K as an IKK inhibitor with implications for osteoarthritis prevention: an in silico and in vitro study.

Ginsenosides have been used traditionally as an oriental medicine. However, the anti-osteoarthritic effect of ginsenoside compound K (hereafter referred to as CK) has not been reported. Therefore, in this study, the protective effects of CK were evaluated in silico and in vitro using H2O2-stimulated MC3T3-E1 cells by measuring the levels of proinflammatory cytokines responsible for articular cartilage degradation. In silico results demonstrated that, among the selected ginsenosides, CK is a non-toxic drug-like molecule with strong binding affinity for selected cytokine-activated kinase such as IkBα kinase (IKK). The molecular binding energy of CK with the active sites of IKK suggests anti-osteoarthritic functions. Cultured H2O2-stimulated MC3T3-E1 cells that were exposed to CK showed dramatically increased expression of osteoblast differentiation markers such as alkaline phosphatase (ALP) activity, type I collagen (Col-I) content, and mineralization. During aging, H2O2 also leads to the production of reactive oxygen species (ROS) and nitric oxide (NO), which play important roles in the development of osteoarthritis (OA). Therefore, the effect of CK on ROS and NO generation was also examined. Our results showed that CK dose-dependently inhibited H2O2-induced ROS and NO production in MC3T3-E1 cells. Moreover, qRT-PCR data showed that CK increased expression of osteogenic markers such as ALP and Col-I but decreased expression of inflammatory-related genes including IKK and interleukin 1ß (IL-1ß) in a dose-dependent manner in H2O2-stimulated MC3T3-E1 cells. The findings of this study suggest the use of CK as a novel protective and therapeutic agent in AO.

Estrogenic activity of osthole and imperatorin in MCF-7 cells and their osteoblastic effects in Saos-2 cells.

There is an increasing interest in phytoestrogens due to their potential medical usage in hormone replacement therapy (HRT). The present study was designed to investigate the in vitro effects of estrogen-like activities of two widespread coumarins, osthole and imperatorin, using the MCF-7 cell proliferation assay and their alkaline phosphatase (ALP) activities in osteoblasts Saos-2 cells. The two compounds were found to strongly stimulate the proliferation of MCF-7 cells. The estrogen receptor-regulated ERα, progesterone receptor (PR) and PS2 mRNA levels were increased by treatment with osthole and imperatorin. All these effects were significantly inhibited by the specific estrogen receptor antagonist ICI182, 780. Cell cycle analysis revealed that their proliferation stimulatory effect was associated with a marked increase in the number of MCF-7 cells in S phase, which was similar to that observed with estradiol. It was also observed that they significantly increased ALP activity, which was reversed by ICI182,780. These results suggested that osthole and imperatorin could stimulate osteoblastic activity by displaying estrogenic properties or through the ER pathway. In conclusion, osthole and imperatorin may represent new pharmacological tools for the treatment of osteoporosis.

A Nutraceutical Composition Decreases CPK Levels in Saos-2 Cells and in Patients with Elevated Serum Levels of This Enzyme.

OBJECTIVES: This study aimed to investigate the effects of a nutraceutical composition on the expression of some genes involved in muscle cells and functioning in osteoblast cells. The effects of nutraceutical composition have been compared to the effects of atorvastatin, which induces muscle pain and elevated creatine phosphokinase (CPK) serum level when administered to patients. In particular, we analyzed the MyoD-1 gene, which is responsible for modulation of the CPK gene, which is a marker of muscle pain and damage. METHODS: The effects of nutraceutical composition on Saos-2 cells were compared with the effects of atorvastatin. The mRNAs were extracted and the expression levels of mitochondrial and cytoplasmic CPK genes and MyoD-1 were analyzed by real-time polymerase chain reaction (RT-PCR). Moreover, the effects on lactate dehydrogenase (LDH) activity and adenosine triphosphate (ATP) synthesis were measured in the osteoblast cell line. Furthermore, 11 patients with muscle pain or elevated CPK serum levels received a supplementation of the nutraceutical composition to test whether CPK levels could be downregulated. RESULTS: The analysis in Saos-2 cells showed that the nutraceutical composition upregulates the gene expression of MyoD-1 and downregulates the expression of the cytoplasmic isoform of CPK gene expression (p ≤ 0.05); moreover, it slightly increases ATP amount and decreases LDH activity. Conversely, atorvastatin represses the expression of MyoD-1 gene without significant changing into the expression levels of both cytoplasmic and mitochondrial CPK genes. Moreover, atorvastatin does not increase the ATP amount or increase LDH activity. Remarkable, the nutraceutical composition is able to decrease CPK levels in serum of patients and in some cases improve myalgia symptoms. CONCLUSION: The nutraceutical composition decreases CPK levels both in vitro and in vivo, suggesting that it might be useful to management of nonneurological myalgia symptoms.

Effect of NSAIDs on the aminopeptidase activity of cultured human osteoblasts.

Aminopeptidases (APs) are involved in various physiological and pathological processes. In tumor tissues the expression of APs, cyclooxygenase-2 and its metabolites are increased. The objective was to determine the effect of certain NSAIDs on the AP activity of osteoblasts. Primary cultures of osteoblast were treated with different concentrations of indomethacin, meloxicam, naproxen, nimesulide, and piroxicam. The AP activity was fluorimetrically determined using aminoacyl-ß-naphthylamides (aa-ßNAs) as substrates: Ala-ßNA, Arg-ßNA, Gly-ßNA, Leu-ßNA, Lys-ßNA, Met-ßNA, and Phe-ßNA. The five NSAIDs showed an inhibitory effect of AP activity against the study substrates depending on the dose tested. Meloxicam and piroxicam had the highest inhibitory effect on enzymatic activity, with an IC50 of around 70 µM. Our results suggest that the physiological alteration of osteoblasts in the presence of NSAIDs may be a consequence of AP inhibition, suggesting a potential clinical role for these drugs against cancer in combination with chemotherapeutic agents.

Effects on MC3T3-E1 Cells and In silico Toxicological Study of Two 6-(Propan-2-yl)-4-methyl-morpholine-2,5-diones.

Recently, we found that two cyclodidepsipeptides, 3,6-di-(propan-2-yl)-4-methyl-morpholine-2,5-dione (1) and 3-(2-methylpropyl)-6-(propan-2-yl)-4-methyl- morpholine-2,5-dione (2), are excellent inhibitors of xanthine oxidase. In order to obtain more information about the toxicological potential of compounds 1 and 2 on bone cells, the current study was designed to evaluate the effect of these compounds on viability and proliferation of MC3T3-E1 cells. Compound 1 showed neither cytotoxic nor stimulatory effect on cell viability, while compound 2 showed a slight stimulatory effect on cell viability. Both studied compounds showed slight stimulatory effects on proliferation of MC3T3-E1 cells, in a dose dependent manner. Additionally, an in silico toxicological study of compounds 1 and 2 was performed, and the results indicate that they have a good probability of safe biological intake.

Alkaline phosphatase activity-guided isolation of active compounds and new dammarane-type triterpenes from Cissus quadrangularis hexane extract.

ETHNOPHARMACOLOGICAL RELEVANCE: The stem of Cissus quadrangularis L. (CQ) is used in traditional medicine to treat bone fractures and swelling. Anti-osteoporotic activity of CQ hexane extract has been reported, but the active compounds in this extract remain unknown. Thus, we aimed to identify the active compounds in CQ hexane extract using bioassay-guided isolation. MATERIALS AND METHODS: The CQ hexane extract was fractionated sequentially with benzene, dichloromethane, ethyl acetate, and methanol. The examination of CQ extract and its fractions was guided by bioassays for alkaline phosphatase (ALP) activity during the differentiation of MC3T3-E1 osteoblastic cells. The cells were treated with or without the CQ extract and its fractions for a period of time, and then the stimulatory effect of the alkaline phosphatase enzyme, a bone differentiation marker, was investigated. The compounds obtained were structurally elucidated using spectroscopic techniques and re-evaluated for activity during bone differentiation. RESULTS: A total of 29 compounds were isolated, viz., triterpenes, fatty acid methyl esters, glycerolipids, steroids, phytols, and cerebrosides. Four new dammarane-type triterpenes were isolated for the first time from nature, and this report is the first to identify this group of compounds from the Vitaceae family. Seven compounds, viz., glycerolipids and squalene, stimulated ALP activity at a dose of 10µg/mL. Moreover, the synergistic effect of these compounds on bone formation was demonstrated. CONCLUSION: This report describes, for the first time, the isolation of active compounds from CQ hexane extract; these active compounds will be useful for the quality control of extracts from this plant used to treat osteoporosis.

Icariin attenuates hypoxia-induced oxidative stress and apoptosis in osteoblasts and preserves their osteogenic differentiation potential in vitro.

OBJECTIVES: Icariin, a prenylated flavonol glycoside isolated from traditional Chinese medicinal herb of the genus Epimedium, has been demonstrated to be a potential alternative therapy for osteoporosis, and its action mechanism so far has been mainly attributed to its phytoestrogenic property. As blood supply to bone is considerably reduced with ageing and by the menopause, we hypothesized that icariin treatment would reduce bone loss by preventing ischaemia-induced hypoxic damages to bone. MATERIALS AND METHODS: To investigate effects of icariin treatment on cultured rat calvarial osteoblasts exposed to hypoxic conditions (2% oxygen). RESULTS: Compared to normoxic control, cell viability decreased with time to 50% by 48 h in the hypoxic group, and icariin attenuated the reduction, dose dependently, with 10(-6) and 10(-5)  m concentrations showing significant protective effects. Icariin also inhibited increase of lactate dehydrogenase activity in culture media. Measurements on oxidative stress, cell cycling and cell survival indicated that icariin protected osteoblasts by reducing production of reactive oxygen species and malondialdehyde, increasing superoxide dismutase activity, arresting the cell cycle and inhibiting apoptosis. Icariin also preserved osteogenic differentiation potential of the hypoxic cells in a dose-dependent manner, compared to the hypoxia alone group, as revealed by increased levels of RUNX-2, OSX and BMP-2 gene expression, alkaline phosphatase activity, and formation of mineralized nodules. CONCLUSIONS: Our results demonstrated that icariin attenuated oxidative stress and apoptosis and preserved viability and osteogenic potential of osteoblasts exposed to hypoxia in vitro, and suggested that its anti-osteoporotic effect may be attributed to its anti-hypoxic activity and phytoestrogenic properties.

The effects of pulsed electromagnetic field on the functions of osteoblasts on implant surfaces with different topographies.

The use of pulsed electromagnetic fields (PEMFs) is a promising approach to promote osteogenesis. However, few studies have reported the effects of this technique on the osseointegration of endosseous implants, especially with regard to different implant topographies. We focused on how the initial interaction between cells and the titanium surface is enhanced by a PEMF and the possible regulatory mechanisms in this study. Rat osteoblasts were cultured on three types of titanium surfaces (Flat, Micro and Nano) under PEMF stimulation or control conditions. Protein adsorption was significantly increased by the PEMF. The number of osteoblasts attached to the surfaces in the PEMF group was substantially greater than that in the control group after 1.5h incubation. PEMF stimulation oriented the osteoblasts perpendicular to the electromagnetic field lines and increased the number of microfilaments and pseudopodia formed by the osteoblasts. The cell proliferation on the implant surfaces was significantly promoted by the PEMF. Significantly increased extracellular matrix mineralization nodules were observed under PEMF stimulation. The expression of osteogenesis-related genes, including BMP-2, OCN, Col-1,ALP, Runx2 and OSX, were up-regulated on all the surfaces by PEMF stimulation. Our findings suggest that PEMFs enhance the osteoblast compatibility on titanium surfaces but to different extents with regard to implant surface topographies. The use of PEMFs might be a potential adjuvant treatment for improving the osseointegration process.