Tag Archives: in vitro

Quercetin Protects Human Osteoblasts Cells Exposed to Cigarette Smoke

Abstract

Quercetin protects primary human osteoblasts exposed to cigarette smoke through activation of the antioxidative enzymes HO-1 and SOD-1.

Smokers frequently suffer from impaired fracture healing often due to poor bone quality and stability. Cigarette smoking harms bone cells and their homeostasis by increased formation of reactive oxygen species (ROS). The aim of this study was to investigate whether Quercetin, a naturally occurring antioxidant, can protect osteoblasts from the toxic effects of smoking. Human osteoblasts exposed to cigarette smoke medium (CSM) rapidly produced ROS and their viability decreased concentration- and time-dependently. Co-, pre- and postincubation with Quercetin dose-dependently improved their viability. Quercetin increased the expression of the anti-oxidative enzymes heme-oxygenase- (HO-) 1 and superoxide-dismutase- (SOD-) 1. Inhibiting HO-1 activity abolished the protective effect of Quercetin. Our results demonstrate that CSM damages human osteoblasts by accumulation of ROS. Quercetin can diminish this damage by scavenging the radicals and by upregulating the expression of HO-1 and SOD-1. Thus, a dietary supplementation with Quercetin could improve bone matter, stability and even fracture healing in smokers.

Braun KF, Ehnert S, Freude T, Egaña JT…
ScientificWorldJournal 2011
PMID: 22203790 | Free Full Text

Almonds Increase Bone Mass and Inhibit Osteoclasts

Abstract

Postprandial effects of almond consumption on human osteoclast precursors–an ex vivo study.

Consumption of almonds has been associated with increased bone mineral density, but the direct effects of almonds on bone cells are not known. We determined whether serum obtained following the consumption of a meal containing 60 g of almonds affects human osteoclast formation, function, and gene expression in vitro. Human osteoclast precursors were cultured in medium containing 10% serum obtained from 14 healthy subjects at baseline and 4 hours following the consumption of 3 test meals containing almonds, potatoes, and rice and balanced for macronutrient composition. Osteoclast formation was determined by the number of tartrate-resistant acid phosphatase (TRAP)(+) multinucleated cells, and osteoclast function was assessed by measuring TRAP activity in the culture medium and calcium released from OsteoAssay (Lonza Walkersville, Walkersville, MD, USA) plates. The expression of cathepsin K, receptor activator of nuclear factor kB, and matrix metalloproteinase-9 genes was measured by real-time reverse transcriptase-polymerase chain reaction. Compared with serum obtained at baseline, serum obtained 4 hours following the consumption of the almond meal reduced osteoclast formation by approximately 20%, TRAP activity by approximately 15%, calcium release by approximately 65%, and the expression of cathepsin K, receptor activator of nuclear factor kB, and matrix metalloproteinase-9 by 13% to 23%. No effects were observed with serum obtained from the other test meals. Serum obtained 4 hours following the consumption of an almond meal inhibits osteoclast formation, function, and gene expression in cultured human osteoclast precursors, and provides evidence for a positive effect of almonds on bone health.

Platt ID, Josse AR, Kendall CW, Jenkins DJ…
Metab. Clin. Exp. Jul 2011
PMID: 20947104

Low Dose Aspirin May Increase Bone Resorption in Diabetic Mice

Abstract

Low dose aspirin therapy decreases blood glucose levels but does not prevent type i diabetes-induced bone loss.

Diabetes is strongly associated with increased fracture risk. During T1-diabetes onset, levels of blood glucose and pro-inflammatory cytokines (including TNFα) are increased. At the same time, levels of osteoblast markers are rapidly decreased and stay decreased 40 days later at which point bone loss is clearly evident. Inflammation is known to suppress bone formation and induce bone loss. Previous co-culture studies indicate that diabetic bone is inflamed and diabetic bone marrow is capable of enhancing osteoblast death in vitro. Here we investigate a commonly used non-steroidal anti-inflammatory drug, aspirin, to prevent T1-diabetic bone loss in vivo.
We induced diabetes in 16-week-old male C57BL/6 mice and administered aspirin in the drinking water.
Our results demonstrate that aspirin therapy reduced diabetic mouse non-fasting blood glucose levels to less than 400 mg/dl, but did not prevent trabecular and cortical bone loss. In control mice, aspirin treatment increased bone formation markers but did not affect markers of bone resorption or bone density/volume. In diabetic mice, bone formation markers and bone density/volume are decreased and unaltered by aspirin treatment. Bone resorption markers, however, are increased and 2-way ANOVA analysis demonstrates an interaction between aspirin treatment and diabetes (p<0.007). Aspirin treatment did not prevent the previously reported diabetes-induced marrow adiposity.
Taken together, our results suggest that low dose aspirin therapy does not negatively impact bone density in control and diabetic mice, but could potentially increase bone resorption in T1-diabetic mice.

Coe LM, Denison JD, McCabe LR
Cell. Physiol. Biochem. 2011
PMID: 22178944 | Free Full Text

Review: NSAIDs May Inhibit Bone Healing

Abstract

Do nonsteroidal anti-inflammatory drugs affect bone healing? A critical analysis.

Nonsteroidal anti-inflammatory drugs (NSAIDs) play an essential part in our approach to control pain in the posttraumatic setting. Over the last decades, several studies suggested that NSAIDs interfere with bone healing while others contradict these findings. Although their analgesic potency is well proven, clinicians remain puzzled over the potential safety issues. We have systematically reviewed the available literature, analyzing and presenting the available in vitro animal and clinical studies on this field. Our comprehensive review reveals the great diversity of the presented data in all groups of studies. Animal and in vitro studies present so conflicting data that even studies with identical parameters have opposing results. Basic science research defining the exact mechanism with which NSAIDs could interfere with bone cells and also the conduction of well-randomized prospective clinical trials are warranted. In the absence of robust clinical or scientific evidence, clinicians should treat NSAIDs as a risk factor for bone healing impairment, and their administration should be avoided in high-risk patients.

Pountos I, Georgouli T, Calori GM, Giannoudis PV
ScientificWorldJournal 2012
PMID: 22272177 | Free Full Text

Beta Blocker Suppresses Resorption in Rats

Abstract

Low dose of propranolol down-modulates bone resorption by inhibiting inflammation and osteoclast differentiation.

Bones are widely innervated, suggesting an important role for the sympathetic regulation of bone metabolism, although there are controversial studies. We investigated the effects of propranolol in a model of experimental periodontal disease.
Rats were assigned as follows: animals without ligature; ligated animals receiving vehicle and ligated animals receiving 0.1, 5 or 20 mg·kg(-1) propranolol. After 30 days, haemodynamic parameters were measured by cardiac catheterization. Gingival tissues were removed and assessed for IL-1β, TNF-α and cross-linked carboxyterminal telopeptides of type I collagen (CTX) by elisa, or intercellular adhesion molecule 1 (ICAM-1), receptor activator of NF-κ B ligand (RANKL) and osteoprotegerin (OPG) by Western blot analysis. Sections from the mandibles were evaluated for bone resorption. Also, we analysed the ability of propranolol to inhibit osteoclastogenesis in vitro.
Propranolol at 0.1 and 5 mg·kg(-1) reduced the bone resorption as well as ICAM-1 and RANKL expression. However, only 0.1 mg·kg(-1) reduced IL-1β, TNF-α and CTX levels as well as increased the expression of OPG, but did not alter any of the haemodynamic parameters. Propranolol also suppressed in vitro osteoclast differentiation and resorptive activity by inhibiting the nuclear factor of activated T cells (NFATc)1 pathway and the expression of tartrate-resistant acid phosphatase (TRAP), cathepsin K and MMP-9.
Low doses of propranolol suppress bone resorption by inhibiting RANKL-mediated osteoclastogenesis as well as inflammatory markers without affecting haemodynamic parameters.

Rodrigues WF, Madeira MF, da Silva TA, Clemente-Napimoga JT…
Br. J. Pharmacol. Apr 2012
PMID: 21950592 | Free Full Text

Berberine, Icariin, and Curculigoside from Er-Xian Inhibit Resorption

Abstract

Effects and interaction of icariin, curculigoside, and berberine in er-xian decoction, a traditional chinese medicinal formula, on osteoclastic bone resorption.

Er-Xian decoction (EXD), a traditional Chinese medicine, has been reported to have a protective effect against bone loss in ovariectomized osteoporotic rats, and the inclusion of icariin (I), curculigoside (C), and berberine (B) in EXD displays inhibitory effects on osteoclastic bone resorption. In the present paper, we investigated the interaction and effects of I, C, B, and their combination on bone resorption activity in vitro on osteoclasts derived from rat bone marrow cells. ICB synergistically decreased the formation of bone resorption pits, the number of multinucleated osteoclasts, and the activity of tartrate-resistant acid phosphatase (TRAP) and showed antagonistic or additive effects on cathepsin K activity in the coculture system of osteoblasts and bone marrow cells in the presence of 1, 25-dihydroxyvitamin D(3) and dexamethasone. The combination of ICB also enhanced the inhibitory effects on the formation of F-actin ring, a cytoskeleton structure of osteoclasts induced from bone marrow cells with macrophage colony stimulation factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). In addition, ICB synergistically improved the ratio of protein expression of osteoprotegerin (OPG) and RANKL in osteoblasts and interfered with the mitogen-activated protein kinases (MAPKs) pathway in osteoclast. These results clearly show that I, C, B, and their combination in EXD exert effects of mutual reinforcement. However, IBC does not show an intensified adverse effect in the ovariectomized murine model, as revealed by change in body and uterine weight, confirming the safety of EXD. These observations are in agreement with the rationality of the formula used in this paper.

Xue L, Jiao L, Wang Y, Nie Y…
Evid Based Complement Alternat Med 2012
PMID: 23243450 | Free Full Text

Guduchi Increases Osteoblasts and Mineralization In Vitro

Abstract

Effects of Tinospora cordifolia (Menispermaceae) on the proliferation, osteogenic differentiation and mineralization of osteoblast model systems in vitro.

Ancient Indian ayurvedic literature prescribes Tinospora cordifolia as a remedy to rheumatoid arthritis, inflammatory and allied diseases of musculo skeletal system. To investigate the effects of the alcoholic extract of Tinospora cordifolia (TC) on the proliferation, differentiation and mineralization of bone like matrix on osteoblast model systems in vitro and hence its possible use as a potential anti-osteoporotic agent.
Two in vitro osteoblast model systems were used in the study viz., human osteoblast-like cells MG-63 and primary osteoblast cells isolated from femur of rats. Cell growth and viability was assessed by standard colorimetric assays like MTT assay. The cell differentiation into osteoblastic lineage was evaluated by the activities of bone marker alkaline phosphatase. The effect of the extract on matrix mineralization was assessed by alizarin red-s staining and Von kossa staining. Cell morphology was studied by phase contrast microscopy and light microscopy (Giemsa/crystal violet staining).
Results indicate that the alcoholic extract of TC at a dosage of 25μg/ml stimulated the growth of osteoblasts, increased the differentiation of cells into osteoblastic lineage and increased the mineralization of bone like matrix on both the osteoblast model systems used in the study. Cell morphology studies clearly indicated the increase in cell numbers and absence of adverse change in the cell morphology on treatment with the extract.
TC extract has a potential influence on osteogenesis and hence its use could be explored as a potential anti-osteoporotic agent.

Abiramasundari G, Sumalatha KR, Sreepriya M
J Ethnopharmacol May 2012
PMID: 22449439

Coptisine Inhibits Osteoclasts In Mouse Cells

Abstract

Coptisine inhibits RANKL-induced NF-κB phosphorylation in osteoclast precursors and suppresses function through the regulation of RANKL and OPG gene expression in osteoblastic cells.

Excessive receptor activator of NF-κB ligand (RANKL) signaling causes enhanced osteoclast formation and bone resorption. The downregulation of RANKL expression and its downstream signals may be an effective therapeutic approach to the treatment of bone loss diseases such as osteoporosis. Here, we found that coptisine, one of the isoquinoline alkaloids from Coptidis Rhizoma, exhibited inhibitory effects on osteoclastogenesis in vitro. Although coptisine has been studied for its antipyretic, antiphotooxidative, dampness dispelling, antidote, antinociceptive, and anti-inflammatory activities in vitro and in vivo, its effects on osteoclastogenesis have not been investigated. Therefore, we evaluated the effects of coptisine on osteoblastic cells as well as osteoclast precursors for osteoclastogenesis in vitro. The addition of coptisine to cocultures of mouse bone marrow cells and primary osteoblastic cells with 10(-8) M 1α,25(OH)(2)D(3) caused significant inhibition of osteoclast formation in a dose-dependent manner. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed that coptisine inhibited RANKL gene expression and stimulated the osteoprotegerin gene expression induced by 1α,25(OH)(2)D(3) in osteoblastic cells. Coptisine strongly inhibited RANKL-induced osteoclast formation when added during the early stage of bone marrow macrophage (BMM) cultures, suggesting that it acts on osteoclast precursors to inhibit RANKL/RANK signaling. Among the RANK signaling pathways, coptisine inhibited NF-κB p65 phosphorylations, which are regulated in response to RANKL in BMMs. Coptisine also inhibited the RANKL-induced expression of NFATc1, which is a key transcription factor. In addition, 10 μM coptisine significantly inhibited both the survival of mature osteoclasts and their pit-forming activity in cocultures. Thus, coptisine has potential for the treatment or prevention of several bone diseases characterized by excessive bone destruction.

Lee JW, Iwahashi A, Hasegawa S, Yonezawa T…
J Nat Med Jan 2012
PMID: 21656335

Berberine Decreases Bone Loss in Rat Cells

Abstract

Effects of berberine on differentiation and bone resorption of osteoclasts derived from rat bone marrow cells.

To observe the effects of berberine on osteoclastic differentiation and bone resorption action in vitro, and to investigate the cellular mechanism of its inhibitory effects on bone resorption.
The multinucleated osteoclasts (MNCs) were derived by 1,25-dihydroxyvitamin D3 and dexamethasone from bone marrow cells in the coculture system with primary osteoblastic cells. The tartrate-resistant acid phosphatase (TRAP) staining and image analysis of bone resorption pit on dental slices were used to identify osteoclast. The activity of TRAP was measured by p-nitrophenyl sodium phosphate assay. The bone resorption pit area on the bone slices formed by osteoclasts was measured by computer image processing.
At the concentrations of 0.1, 1 and 10 micromol/L, berberine dose-dependently suppressed the formation of TRAP-positive multinucleated cells, the TRAP activity and the osteoclastic bone resorption. The strongest inhibitory effect was exhibited at the concentration of 10 micromol/L, with the inhibiting rate of 60.45%, 42.12% and 72.69% respectively.
Berberine can decrease bone loss through inhibition of osteoclast formation, differentiation and bone resorption.

Wei P, Jiao L, Qin LP, Yan F…
Zhong Xi Yi Jie He Xue Bao Apr 2009
PMID: 19361364 | Free Full Text

Palmatine Inhibits Resorption in Mouse Cells

Abstract

Palmatine attenuates osteoclast differentiation and function through inhibition of receptor activator of nuclear factor-κb ligand expression in osteoblast cells.

Osteoclasts are the only cell type capable of resorbing mineralized bone, and they act under the control of numerous cytokines produced by supporting cells such as osteoblasts and stromal cells. Among cytokines, receptor activator of nuclear factor-κB ligand (RANKL) was found to be a key osteoclastogenetic molecule that directly binds to its cognate receptor, RANK, on osteoclast precursor cells. In turn, RANKL, which is an essential factor for differentiation and activation of osteoclasts, is one of the major targets of anti-resorptive agents. In this study, we found that palmatine, an isoquinoline alkaloid originally isolated from Coptis chinensis, had an inhibitory effect on osteoclast differentiation and function in vitro. Palmatine inhibited osteoclast formation in the co-culture system with mouse bone marrow cells (BMC) and osteoblasts in the presence of 10 nM 1α,25-(OH)(2)D(3). Palmatine did not affect osteoclast formation induced by RANKL in the BMC cultures. Reverse-transcription polymerase chain reaction (RT-PCR) analysis showed that palmatine significantly inhibited the expression of 1α,25-(OH)(2)D(3)-induced expression of RANKL mRNAs in stromal cells without loss of cell viability. Moreover, palmatine suppressed resorption pit formation by mature osteoclasts on dentin slices and induced disruption of actin ring formation in mature osteoclasts with an impact on cell viability. Taken together, these results suggest that palmatine attenuates osteoclast differentiation through inhibition of RANKL expression in osteoblast cells, and its inhibitory effect on bone resorption is due to its disruptive effect on actin rings in mature osteoclasts. Therefore, palmatine might be an ideal candidate as an anti-resorptive agent for the prevention and treatment of bone disorders such as osteoporosis.

Lee JW, Mase N, Yonezawa T, Seo HJ…
Biol. Pharm. Bull. 2010
PMID: 20930384 | Free Full Text