Tag Archives: in vitro

Resveratrol and SIRT1 Reduces Sclerostin Expression In-Vitro


Low sirtuin 1 levels in human osteoarthritis subchondral osteoblasts lead to abnormal sclerostin expression which decreases Wnt/β-catenin activity.

Wnt/β-catenin (cWnt) signaling plays a key role in osteogenesis by promoting the differentiation and mineralization of osteoblasts, activities altered in human osteoarthritic subchondral osteoblast (OA Ob). Sclerostin (SOST) has been shown to alter cWnt signaling. Sirtuin 1 (SIRT1) acts as a novel bone regulator and represses SOST levels in Ob. However the role of SIRT1 and SOST in OA Ob remains unknown. Herein, we explored the role played by SIRT1 and SOST on the abnormal mineralization and cWnt signaling in OA Ob.
Primary human normal and OA Ob were prepared from tibial plateaus. SOST levels were evaluated by immunohistochemistry, the expression and production of genes by qRT-PCR and WB analysis. Their inhibitions were performed using siRNA. cWnt signaling was measured by the TOPflash TCF/lef luciferase reporter assay. Mineralization was determined by alizarin red staining.
SOST levels were significantly increased in OA Ob compared to normal and were linked with elevated TGF-β1 levels in these cells. SIRT1 expression was significantly reduced in OA Ob compared to normal yet not modified by TGF-β1. Specific inhibition of SIRT1 increased TGF-β1 and SOST expressions in OA Ob, while stimulating SIRT1 activity with β-Nicotinamide mononucleotide reduced the expression of TGF-β1 and SOST, and increased mineralization in OA Ob. Resveratrol also reduced SOST expression in OA Ob. Reduced cWnt signaling, β-catenin levels, and mineralization in OA Ob were all corrected via reducing SOST expression.
These data indicate that high level of SOST is responsible, in part, for the reduced cWnt and mineralization of human OA Ob, which in turn is linked with abnormal SIRT1 levels in these pathological cells.

Abed É, Couchourel D, Delalandre A, Duval N…
Bone Feb 2014
PMID: 24184155

Oxytocin is Lower in Osteoporosis


Oxytocin controls differentiation of human mesenchymal stem cells and reverses osteoporosis.

Osteoporosis constitutes a major worldwide public health burden characterized by enhanced skeletal fragility. Bone metabolism is the combination of bone resorption by osteoclasts and bone formation by osteoblasts. Whereas increase in bone resorption is considered as the main contributor of bone loss that may lead to osteoporosis, this loss is accompanied by increased bone marrow adiposity. Osteoblasts and adipocytes share the same precursor cell and an inverse relationship exists between the two lineages. Therefore, identifying signaling pathways that stimulate mesenchymal stem cells osteogenesis at the expense of adipogenesis is of major importance for developing new therapeutic treatments. For this purpose, we identified by transcriptomic analysis the oxytocin receptor pathway as a potential regulator of the osteoblast/adipocyte balance of human multipotent adipose-derived stem (hMADS) cells. Both oxytocin (OT) and carbetocin (a stable OT analogue) negatively modulate adipogenesis while promoting osteogenesis in both hMADS cells and human bone marrow mesenchymal stromal cells. Consistent with these observations, ovariectomized (OVX) mice and rats, which become osteoporotic and exhibit disequilibrium of this balance, have significant decreased OT levels compared to sham-operated controls. Subcutaneous OT injection reverses bone loss in OVX mice and reduces marrow adiposity. Clinically, plasma OT levels are significantly lower in postmenopausal women developing osteoporosis than in their healthy counterparts. Taken together, these results suggest that plasma OT levels represent a novel diagnostic marker for osteoporosis and that OT administration holds promise as a potential therapy for this disease.

Elabd C, Basillais A, Beaupied H, Breuil V…
Stem Cells Sep 2008
PMID: 18583541 | Free Full Text

Review: Resveratrol, Inositol, Vitamin D and K for Bone and Cardiovascular Risk


Resveratrol, inositol, vitamin D and K in the prevention of cardiovascular and osteoporotic risk: a novel approach in peri- and postmenopause.

The prevention of cardiovascular and osteoporotic risk is a topic of great importance in the peri- and postmenopausal periods. This paper reviews the role of resveratrol, inositol, vitamin D and K in the prevention of cardiovascular and osteoporotic risk in peri- and post-. The phytoestrogen-like activity of resveratrol has potential clinical implications in the gynecological practice. In particular transresveratrol inhibits low-density lipoprotein oxidation, which is a recognized risk factor for cardiovascular diseases. Resveratrol has also a documented antiplatelet effect and may prevent cardiovascular diseases inhibiting the cardiac fibroblasts proliferation. With regard to bone health, in in vitro studies resveratrol has shown activities in osteoblastic MC3T3-E1 cells. Resveratrol also interacts with vitamin D in promoting bone health. Resveratrol is considered a caloric restriction mimetic and potentially effects factors involved in the metabolic syndrome. Myo-inositol has documented in clinical studies its effectiveness in improving the metabolic syndrome in post menopausal women. Thus the supplementation with inositol and resveratrol may be useful in the prevention of insulin resistance and consequently metabolic syndrome and cardiovascular diseases risk. Finally vitamin K2 effects calcium metabolisms and subjects with higher levels of calcium in the bones tend to have a lower frequency of vascular calcifications and a lower cardiovascular risk. Vitamin K2 also has a key role in the bone homeostasis. A supplement including resveratrol, inositol, vitamin K and vitamin D offers a novel opportunity to the woman in peri- and postmenopause.

Parazzini F
Minerva Ginecol Oct 2014
PMID: 25245999

Review: Resveratrol Pre-Clinical Evidence


Resveratrol Supplementation Affects Bone Acquisition and Osteoporosis: Pre-Clinical Evidence Towards Translational Diet Therapy.

Osteoporosis is a major public health issue that is expected to rise as the global population ages. Resveratrol (RES) is a plant polyphenol with various anti-aging properties. RES treatment of bone cells results in protective effects, but dose translation from in vitro studies to clinically relevant doses is limited since bioavailability is not taken into account. The aims of this review is to evaluate in vivo evidence for a role of RES supplementation in promoting bone health to reduced osteoporosis risk and potential mechanisms of action. Due to multiple actions on both osteoblasts and osteoclasts, RES has potential to attenuate bone loss resulting from different etiologies and pathologies. Several animal models have investigated the bone protective effects of RES supplementation. Ovariectomized rodent models of rapid bone loss due to estrogen-deficiency reported that RES supplementation improved bone mass and trabecular bone without stimulating other estrogen-sensitive tissues. RES supplementation prior to age-related bone loss was beneficial. The hindlimb unloaded rat model used to investigate bone loss due to mechanical unloading showed RES supplementation attenuated bone loss in old rats, but had inconsistent bone effects in mature rats. In growing rodents, RES increased longitudinal bone growth, but had no other effects on bone. In the absence of human clinical trials, evidence for a role of RES on bone heath relies on evidence generated by animal studies. A better understanding of efficacy, safety, and molecular mechanisms of RES on bone will contribute to the determination of dietary recommendations and therapies to reduce osteoporosis. This article is part of a Special Issue entitled: Resveratol: Challenges in translating pre-clincial findigns to iproved patient outcomes.

Tou JC
Biochim. Biophys. Acta Oct 2014
PMID: 25315301

Orthosilicic Acid Stimulates Collagen and Osteoblasts In Vitro


Orthosilicic acid stimulates collagen type 1 synthesis and osteoblastic differentiation in human osteoblast-like cells in vitro.

Silicon deficiency in animals leads to bone defects. This element may therefore play an important role in bone metabolism. Silicon is absorbed from the diet as orthosilicic acid and concentrations in plasma are 5-20 microM. The in vitro effects of orthosilicic acid (0-50 microM) on collagen type 1 synthesis was investigated using the human osteosarcoma cell line (MG-63), primary osteoblast-like cells derived from human bone marrow stromal cells, and an immortalized human early osteoblastic cell line (HCC1). Collagen type 1 mRNA expression and prolyl hydroxylase activity were also determined in the MG-63 cells. Alkaline phosphatase and osteocalcin (osteoblastic differentiation) were assessed both at the protein and the mRNA level in MG-63 cells treated with orthosilicic acid. Collagen type 1 synthesis increased in all treated cells at orthosilicic acid concentrations of 10 and 20 microM, although the effects were more marked in the clonal cell lines (MG-63, HCCl 1.75- and 1.8-fold, respectively, P < 0.001, compared to 1.45-fold in the primary cell lines). Treatment at 50 microM resulted in a smaller increase in collagen type 1 synthesis (MG-63 1.45-fold, P = 0.004). The effect of orthosilicic acid was abolished in the presence of prolyl hydroxylase inhibitors. No change in collagen type 1 mRNA level was seen in treated MG-63 cells. Alkaline phosphatase activity and osteocalcin were significantly increased (1.5, 1.2-fold at concentrations of 10 and 20 microM, respectively, P < 0.05). Gene expression of alkaline phosphatase and osteocalcin also increased significantly following treatment. In conclusion, orthosilicic acid at physiological concentrations stimulates collagen type 1 synthesis in human osteoblast-like cells and enhances osteoblastic differentiation.

Reffitt DM, Ogston N, Jugdaohsingh R, Cheung HF…
Bone Feb 2003
PMID: 12633784

Soy Isoflavones + Vitamin D3 Improve Bone Density, Stimulate Osteoblasts, and Inhibit Osteoclasts in Ovariectomized Rats


Combined effect of soy isoflavones and vitamin D3 on bone loss in ovariectomized rats.

Several studies have shown that soy isoflavones have estrogen-like activities and might constitute an alternative to hormone replacement treatment. The present study investigated the effects of soy isoflavones alone and combined with vitamin D3 on prevention of bone loss.
Sprague-Dawley rats were sham-operated (n = 8) or ovariectomized (OVX; n = 40), and then the OVX rats were randomly assigned to five groups that were untreated or treated for 14 wk with vitamin D3, 17β-estradiol, soy isoflavone extract (SIE), or vitamin D3 plus SIE. The effects of the isoflavones and 1α,25(OH)(2)D(3) on cultured osteoblasts and osteoclasts also were investigated.
In OVX rats, the bone mineral density and trabecular bone volume loss were improved by 17β-estradiol, SIE, or SIE plus vitamin D3 treatment. SIE treatment was more effective than vitamin D3 or 17β-estradiol in inhibiting increases in serum tumor necrosis factor-α levels and osteoblast osteoprotegerin expression. SIE plus vitamin D3 was more effective in increasing osterix expression than each alone. Bone cell cultures showed that the isoflavones induced preosteoblasts to differentiate into osteoblasts and increased osteoblast mineralization. Isoflavones inhibited preosteoclasts and osteoclast proliferation and decreased osteoclast resorption. The combination of isoflavones plus 1α,25(OH)(2)D(3) showed additive effects on the increase in cell proliferation of cultured preosteoblasts.
Treatment with soy isoflavones might be an alternative to hormone replacement therapy in decreasing bone loss from postmenopausal estrogen deficiency. In addition, there are further effects on increasing transcription factor osterix expression and preosteoblast proliferation when these were combined with vitamin D3.

Chang KL, Hu YC, Hsieh BS, Cheng HL…
Nutrition Jan 2013
PMID: 22858193

Blueberry May Prevent Collagen and Bone Loss in Rat Cells


Blueberry consumption prevents loss of collagen in bone matrix and inhibits senescence pathways in osteoblastic cells.

Ovariectomy (OVX)-induced bone loss has been linked to increased bone turnover and higher bone matrix collagen degradation as the result of osteoclast activation. However, the role of degraded collagen matrix in the fate of resident bone-forming cells is unclear. In this report, we show that OVX-induced bone loss is associated with profound decreases in collagen 1 and Sirt1. This was accompanied by increases in expression and activity of the senescence marker collagenase and expression of p16/p21 in bone. Feeding a diet supplemented with blueberries (BB) to pre-pubertal rats throughout development or only prior to puberty [postnatal day 21 (PND21) to PND34] prevents OVX-induced effects on expression of these molecules at PND68. In order to provide more evidence and gain a better understanding on the association between bone collagen matrix and resident bone cell fate, in vitro studies on the cellular senescence pathway using primary calvarial cells and three cell lines (ST2 cells, OB6, and MLO-Y4) were conducted. We found that senescence was inhibited by collagen in a dose-response manner. Treatment of cells with serum from OVX rats accelerated osteoblastic cell senescence pathways, but serum from BB-fed OVX rats had no effect. In the presence of low collagen or treatment with OVX rat serum, ST2 cells exhibited higher potential to differentiate into adipocytes. Finally, we demonstrated that bone cell senescence is associated with decreased Sirt1 expression and activated p53, p16, and p21. These results suggest that (1) a significant prevention of OVX-induced bone cell senescence from adult rats can occur after only 14 days consumption of a BB-containing diet immediately prior to puberty, and (2) the molecular mechanisms underlying this effect involves, at least in part, prevention of collagen degradation.

Zhang J, Lazarenko OP, Blackburn ML, Badger TM…
Age (Dordr) Jun 2013
PMID: 22555620

Gamma-Tocotrienol Inhibits Osteoclasts In Vitro


Direct inhibition of osteoclast formation and activity by the vitamin E isomer gamma-tocotrienol.

Vitamin E homologues, specifically tocotrienols, have been shown to have favorable effects on bone. They possess properties that are indicative of anti-resorptive activity, suggesting the potential for vitamin E in preventing bone loss. To investigate the anti-resorptive activity of the various vitamin E homologues, we cultured human osteoclasts from blood-derived CD14+ cells on collagen, dentin, and calcium phosphate substrates, with some samples supplemented with vitamin E homologues in their cell culture medium. These were compared to the clinically used bisphosphonate, pamidronate. Compounds were either added at the start of culture to study effects on osteoclast formation, or at the start of osteoclastic resorption to determine their effects on activity. The alpha- and gamma-tocotrienol isomers inhibited osteoclast formation without consequent reduction in total cell number. Only gamma-tocotrienol inhibited osteoclast activity without toxicity. Gamma-tocotrienol was the most potent inhibitor of both osteoclast formation and activity and requires further investigation into its anti-resorptive effects on bone.

Brooks R, Kalia P, Ireland DC, Beeton C…
Int J Vitam Nutr Res Nov 2011
PMID: 22673919

Icariin Inhibits Osteoclasts In Vitro


Icariin inhibits the osteoclast formation induced by RANKL and macrophage-colony stimulating factor in mouse bone marrow culture.

Icariin is a prenylated flavonol glycoside contained in the herb Epimedium, which has long been used to improve bone fracture healing or prevent osteoporosis because of the belief that the herb has bone-strengthening action. We have previously demonstrated that icariin enhances the osteogenic differentiation of rat bone marrow stromal cells, and partially explained the bone-strengthening mechanism of the herb. In the present study, the effect of icariin on osteoclastogenesis and bone resorption activity was investigated in mouse bone marrow culture. It was found that icariin dose-dependently inhibited the growth and differentiation of hemopoietic cells from which osteoclasts were formed. Far less TRAP+ multinuclear cells appeared in the 10 microM icariin group than in the control. The bone resorption pits formed in the 10 microM icariin group was also significantly less than that of the control. RT-PCR analysis showed that the gene expression of TRAP, RANK and CTR was obviously lower than that of the control. It can be concluded that icariin has the ability to inhibit the formation and bone resorption activity of osteoclasts, which suggests that icariin should be the effective component for the bone-strengthening action of herb Epimedium.

Chen KM, Ge BF, Liu XY, Ma PH…
Pharmazie May 2007
PMID: 17557750

Horny Goat Weed Icariin Metabolites Enhance Osteoblasts and Inhibit Osteoclasts In Vitro


Icaritin and its glycosides enhance osteoblastic, but suppress osteoclastic, differentiation and activity in vitro.

Icariin, a principal flavonoid glycoside in Herba Epimedii, is hypothesized to possess beneficial effects on bone mass. Icariin is metabolized to icariside II and then to icaritin in vivo. In the present study, we investigated the in vitro effects of icariin, icariside II and icaritin on both osteoblasts and osteoclasts. After treatment with these compounds at concentrations 10(-5)-10(-8) mol/l, osteoblasts were examined for proliferation, alkaline phosphatase activity, osteocalcin secretion and matrix mineralization, as well as expression levels of bone-related proteins. The formation of osteoclasts was assessed by counting the number of multinucleated TRAP-positive cells. The activity of isolated rat osteoclasts was evaluated by measuring pit area, actin rings and superoxide generation. Icariside II and icaritin increased the mRNA expression of ALP, OC, COL-1 and OPG, but suppressed that of RANKL. In addition, these compounds reduced the number of multinucleated TRAP-positive cells and the osteoclastic resorption area. Also decreases were observed in superoxide generation and actin ring formation that are required for osteoclast survival and bone resorption activity. These findings suggest that icaritin, which was more potent than icariin and icariside II, enhanced the differentiation and proliferation of osteoblasts, and facilitated matrix calcification; meanwhile it inhibited osteoclastic differentiation in both osteoblast-preosteoclast coculture and osteoclast progenitor cell culture, and reduced the motility and bone resorption activity of isolated osteoclasts.

Huang J, Yuan L, Wang X, Zhang TL…
Life Sci. Aug 2007
PMID: 17764702