Category Archives: Supplements

Silibinin Promotes Osteoblasts In Vitro

Abstract

Silibinin promotes osteoblast differentiation of human bone marrow stromal cells via bone morphogenetic protein signaling.

Silibinin is the major active constituent of the natural compound silymarin; several studies suggest that silibinin possesses antihepatotoxic properties and anticancer effects against carcinoma cells. However, no study has yet investigated the effect of silibinin on osteogenic differentiation of human bone marrow stem cells (hBMSCs). The aim of this study was to evaluate the effect of silibinin on osteogenic differentiation of hBMSCs. In this study, the hBMSCs were cultured in an osteogenic medium with 0, 1, 10 or 20 μmol/l silibinin respectively. hBMSCs viability was analyzed by cell number quantification assay and cells osteogenic differentiation was evaluated by alkaline phosphatas (ALP) activity assay, Von Kossa staining and real time-polymerase chain reaction (RT-PCR). We found that silibinin promoted ALP activity in hBMSCs without affecting their proliferation. The mineralization of hBMSCs was enhanced by treatment with silibinin. Silibinin also increased the mRNA expressions of Collagen type I (COL-I), ALP, Osteocalcin (OCN), Osterix, bone morphogenetic protein-2 (BMP-2) and Runt-related transcription factor 2 (RUNX2). The BMP antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated silibinin-promoted ALP activity. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by silibinin treatment. These results indicate that silibinin enhances osteoblast differentiation probably by inducing the expressions of BMPs and activating BMP and RUNX2 pathways. Thus, silibinin may play an important therapeutic role in osteoporosis patients by improving osteogenic differentiation of BMSCs.

Ying X, Sun L, Chen X, Xu H…
Eur. J. Pharmacol. Dec 2013
PMID: 24076187

Calcium Threonate in Ester-C Enhances Vitamin C’s Bone Mineralization In Vitro

Abstract

Enhanced production of mineralized nodules and collagenous proteins in vitro by calcium ascorbate supplemented with vitamin C metabolites.

Vitamin C or ascorbate is important in wound healing due to its essential role in collagen synthesis. To study wound healing in the periodontium, cells adherent to expanded polytetrafluoroethylene (ePTFE) augmentation membranes, recovered from edentulous ridge augmentation procedures, have been established in culture in our laboratories. The objective of this study was to determine whether treatment of these cells with a calcium ascorbate, which contains vitamin C metabolites (metabolite-supplemented ascorbate), would increase the production of collagenous protein and mineralized tissue in vitro, as compared to unsupplemented calcium ascorbate (ascorbate).
Cells derived from ePTFE membranes were cultured with beta-glycerophosphate and the test agents for 2 to 5 weeks, and the surface areas of the cell cultures occupied by mineralized nodules were measured using computerized image analysis. One experiment tested the effects of calcium threonate, one of the vitamin C metabolites in metabolite-supplemented ascorbate. Incorporation of radioactive proline and glycine was used as a measure of total protein (radioactivity precipitated by trichloracetic acid) and collagenase-digestible protein (radioactivity released by collagenase digestion.) Co-localization of collagen and fibronectin was examined by immunofluorescence.
In vitro treatment of these cells with metabolite-supplemented ascorbate increased the area of the cell cultures occupied by mineralized nodules after 5 weeks. Cell cultures treated with metabolite-supplemented ascorbate also exhibited significant increases in total protein. The increase in collagenous proteins in these cultures accounted for 85% of the increase in total protein. The greatest difference between treatment groups was observed in the cell-associated fraction containing the extracellular matrix. The additional collagen exhibited normal co-distribution with fibronectin. In cultures treated with ascorbate spiked with calcium threonate, the area of mineralized tissue was significantly greater than in ascorbate-treated cultures, but was less than that observed in cultures treated with metabolite-supplemented ascorbate.
In vitro treatment with ascorbate containing vitamin C metabolites enhanced the formation of mineralized nodules and collagenous proteins. Calcium threonate may be one of the metabolites influencing the mineralization process. Identifying factors which facilitate the formation of mineralized tissue has significant clinical ramifications in terms of wound healing and bone regeneration.

Rowe DJ, Ko S, Tom XM, Silverstein SJ…
J. Periodontol. Sep 1999
PMID: 10505801

This study is on Ester-C. Ester-C, PureWay-C, and AlphaSorb-C are Vitamin C products that contain Calcium-L-Threonate. Biocalth is Calcium product which is all Calcium-L-Threonate without Vitamin C.

 

L-Threonate Inhibits Resorption In Vitro

Abstract

[Effects of L-threonate on bone resorption by osteoclasts in vitro].

To clarify if calcium L-threonate and sodium L-threonate have inhibitory effects on the bone resorption of rabbit’s osteoclasts in vitro.
This study contained a total of 16 culture groups, including one group as control and 5 groups treated by 5 drugs (calcium D-threonate, sodium L-threonate, alendronate, 17beta-estradiol and calcium gluconate) each at the final concentrations of 10(-9) mol/L, 10(-7) mol/L, 10(-5) mol/L respectively. After 7 days, eight bone slices of every group were stained with toluidine blue and the areas of resorptive pits were analyzed under light microscope; the concentrations of C-telopeptide of type I collagen (CTx or Crosslaps) in culture supernatants were measured by ELISA.
(1) The resorption area and the CTx concentration of the Calcium L-threonate groups were reduced significantly as compared with those of control and of Calcium gluconate groups respectively. The resorption area and CTx level of the Sodium L-threonate groups were significantly reduced when compared with those of the control, but the effects of Calcium gluconate groups were not so. (2) The reduction in the resorption area and CTx concentration of Calcium L-threonate group was more than that of Sodium L-threonate group. (3) The reductive effect of the high concentration (10(-5)) group of Calcium L-threonate on the area and CTx level was corresponding to that of 17beta-estradiol at a concentration between 10(-7) and 10(-9). (4) The resorption area was related to the CTx concentration (r=0.876). (5) The CTX level was much more sensitive, precise and stable than the concentration.
L-threonate, especially calcium L-threonate could inhibit the bone resorption of osteoclasts in vitro, and its effect might be related to the radical of L-threonic acid. The CTx concentration in culture supernatants might be an effective marker quantitatively reflecting the bone resorption by osteoclasts in vitro.

He JH, Tong NW, Li HQ, Wu J
Sichuan Da Xue Xue Bao Yi Xue Ban Mar 2005
PMID: 15807273

Silibinin Increases Osteoblasts and Inhibits Osteoclasts in Mouse Cells

Abstract

Osteoblastogenesis and osteoprotection enhanced by flavonolignan silibinin in osteoblasts and osteoclasts.

Bone-remodeling imbalance induced by decreased osteoblastogenesis and increased bone resorption is known to cause skeletal diseases such as osteoporosis. Silibinin is the major active constituent of silymarin, the mixture of flavonolignans extracted from blessed milk thistle (Silybum marianum). Numerous studies suggest that silibinin is a powerful antioxidant and has anti-hepatotoxic properties and anti-cancer effects against carcinoma cells. This study investigated that silibinin had bone-forming and osteoprotective effects in in vitro cell systems of murine osteoblastic MC3T3-E1 cells and RAW 264.7 murine macrophages. MC3T3-E1 cells were incubated in osteogenic media in the presence of 1-20 µM silibinin up to 15 days. Silibinin accelerated cell proliferation and promoted matrix mineralization by enhancing bone nodule formation by calcium deposits. In addition, silibinin furthered the induction of osteoblastogenic biomarkers of alkaline phosphatase, collagen type 1, connective tissue growth factor, and bone morphogenetic protein-2. Differentiated MC3T3-E1 cells enhanced secretion of receptor activator of nuclear factor-κB ligand (RANKL) essential for osteoclastogenesis, which was reversed by silibinin. On the other hand, RAW 264.7 cells were pre-incubated with 1-20 µM silibinin for 5 days in the presence of RANKL. Non-toxic silibinin markedly attenuated RANK transcription and intracellular adhesion molecule-1 expression elevated by RANKL, thereby suppressing the differentiation of macrophages to multi-nucleated osteoclasts. It was also found that silibinin retarded tartrate-resistant acid phosphatase and cathepsin K induction and matrix metalloproteinase-9 activity elevated by RANKL through disturbing TRAF6-c-Src signaling pathways. These results demonstrate that silibinin was a potential therapeutic agent promoting bone-forming osteoblastogenesis and encumbering osteoclastic bone resorption.

Kim JL, Kang SW, Kang MK, Gong JH…
J. Cell. Biochem. Jan 2012
PMID: 21898547

Diosgenin and Lovastatin Prevent Bone Loss in Ovariectomized Rat

Abstract

Osteoprotective effect of Monascus-fermented dioscorea in ovariectomized rat model of postmenopausal osteoporosis.

This experiment established the ovariectomized (OVX) rat model of postmenopausal osteoporosis and examined the effect of the oral administration of different dosages of dioscorea, red mold dioscorea (RMD), and soy isoflavones on bone mineral density (BMD). Three months after osteoporosis had been induced and 4 weeks after feeding had begun, the tibia and femur BMD of OVX rats administered RMD showed significant increases compared with that of all other groups of OVX rats. Closer examination using microcomputed tomography also revealed that the RMD-administered rats had denser trabecular bone volume and a higher trabecular number compared to all other rat groups. Reconstructed 3D imaging indicated increases in cancellous bone mineral content, cancellous bone mineral density, and cortical bone mineral content of the proximal tibia in OVX rats. These findings indicate that administration of monacolin K and phytoestrogen diosgenin could prevent bone loss induced by estrogen deficiency.

Chiang SS, Chang SP, Pan TM
J. Agric. Food Chem. Sep 2011
PMID: 21800902

Diosgenin Stimulates Bone Formation In Mouse Osteoblasts

Abstract

Diosgenin stimulates osteogenic activity by increasing bone matrix protein synthesis and bone-specific transcription factor Runx2 in osteoblastic MC3T3-E1 cells.

Diosgenin, a steroid saponin extracted from the root of wild yam (Dioscorea villossa) is claimed to have osteogenic property. However, detailed studies providing evidence to this claim have not been fully undertaken. In this study, we investigated the effect of diosgenin on the osteogenesis of murine MC3T3-E1 osteoblastic cells. Cells were cultured with varying levels of diosgenin (0-10 μM) within 25 days of bone formation period. Diosgenin was found to stimulate proliferation within the range of 0.01-5 μM using MTT assay. The medium and cellular levels of Type 1 collagen and alkaline phosphatase (ALP), both of which are major bone matrix proteins, increased within the low range of diosgenin concentration (>0-3 μM), and this pattern was further confirmed by collagen and ALP staining of the extracellular matrix (ECM). The cellular protein expression of ALP and collagen Type 1 was also increased at 0.1-1 μM diosgenin treatment as analyzed by Western blot. Calcium deposition within the ECM also showed the same pattern as assessed by Alizarin Red S and Von Kossa staining. Bone-specific transcription factor runt-related transcription factor 2 (Runx2) and Runx2-regulated osteopontin protein expressions were induced at low concentration (0.1-1 μM) and again decreased with high diosgenin concentrations. Based on our findings, our study suggests that diosgenin can enhance bone formation by stimulating the synthesis and secretion of Type 1 collagen and ALP and bone marker proteins Runx2 and osteopontin expression. The increased levels of these marker proteins, in turn, can increase the formation of calcium deposits within the ECM thereby increasing bone formation.

Alcantara EH, Shin MY, Sohn HY, Park YM…
J. Nutr. Biochem. Nov 2011
PMID: 21292464

Diosgenin Promotes Angiogenesis in Preosteoblast-Like Mouse Cells

Abstract

Diosgenin induces hypoxia-inducible factor-1 activation and angiogenesis through estrogen receptor-related phosphatidylinositol 3-kinase/Akt and p38 mitogen-activated protein kinase pathways in osteoblasts.

Diosgenin, extracted from the root of wild yam (Dioscorea villosa), has been reported to demonstrate an opportunity for medical application. Vascular endothelial growth factor-A (VEGF-A) plays an important role in bone-related angiogenesis, a critical process occurring during bone formation and fracture healing. In this study, we examine whether diosgenin is able to induce VEGF-A expression and to promote angiogenesis in osteoblasts. For murine MC3T3-E1 preosteoblast-like cells, VEGF-A mRNA and protein expression seemed to be significantly elevated in response to diosgenin in a concentration-dependent fashion. Conditioned media prepared from cells treated with diosgenin induced strong angiogenic activity in either in vitro or ex vivo angiogenesis assay. Furthermore, diosgenin treatment increased the stability and activity of HIF-1alpha protein. Inhibition of HIF-1alpha activity by transfection with DN-HIF-1alpha significantly diminished diosgenin-mediated VEGF-A up-regulation. The use of pharmacological inhibitors or genetic inhibition revealed that both the phosphatidylinositol 3-kinase (PI3K)/Akt and p38 signaling pathways were potentially required for diosgenin-induced HIF-1 activation and subsequent VEGF-A up-regulation. It is noteworthy that an estrogen receptor binding assay revealed that diosgenin has the strong ability to replace [(3)H]estradiol bound to estrogen receptor (IC(50), 10 nM). In addition, the specific estrogen receptor antagonists ICI 182,780 (faslodex) and tamoxifen were noted to be able to strongly inhibit diosgenin-induced, src kinase-dependent Akt and p38 MAPK activation. Taken together, such results provide evidence that diosgenin up-regulates VEGF-A and promotes angiogenesis in preosteoblast-like cells by a hypoxia-inducible factor-1alpha-dependent mechanism involving the activation of src kinase, p38 MAPK, and Akt signaling pathways via estrogen receptor.

Yen ML, Su JL, Chien CL, Tseng KW…
Mol. Pharmacol. Oct 2005
PMID: 15998873 | Free Full Text

DHEA or Diosgenin Prevents Bone Loss in Ovariectomized Rats

Abstract

The use of estrogen, DHEA, and diosgenin in a sustained delivery setting as a novel treatment approach for osteoporosis in the ovariectomized adult rat model.

It is well established that the pattern of bone loss from the cortex in osteoporotic bone begins from the endosteal surface of the cortex, where there is enlargement of the medullary canal at the expense of the inner cortex. Bone loss does not occur at the periosteal surface. The objective of the following study was to induce osteoporosis in female rats by ovariectomy, followed by treatment with sustained delivery of Diosgenin (DG), dehydroepiandrosterone (DHEA), or estrogen (E) after clinical signs of osteoporosis. Female Sprague Dawley rats were divided randomly into five groups containing four rats/group. Rats comprising group 1 were left intact and served as a control group. Animals in groups 2-5 were ovariectomized (OVX) and, after a 14 day delay to allow for induction of osteoporosis, were implanted with TCPL capsules containing DG, DHEA, and E, respectively. The experiment was ceased after 33 days of treatment, at which time the vital and reproductive organs for each group were collected, weighed, and analyzed histomorphometrically for differences. Further analysis of the progression of osteoporosis in the experimental animals was obtained by performing x-ray analysis of each group on a semi-weekly basis. By collecting and analyzing the femurs from each animal, we were also able to obtain important information about the histologic changes associated with osteoporosis (left femur), as well as data regarding the effects of osteoporosis on the mechanical strength of bone via three point bending analysis (right femur). The data generated by this study revealed important information as to the efficacy and safety of the alternative treatments DHEA, E, and DG for osteoporosis. First, histomorphometric analysis revealed that treatment with DHEA, E, and DG reduced the endosteal perimeter and cortical area to values very similar to controls (intact). Second, results of the bending stress and modulus in OVX and treated animals were not statistically different from the intact control animals, which suggests that the material properties of the bone were unaltered. Third, there is an increase in total body weight associated with OVX that is reduced to control levels after replacement therapy. Finally, OVX also resulted in reproductive tissue atrophy, which was reversed by all three of the treatment regimens in this study. These data suggest that bone loss after OVX can be significantly reduced by supplementation with sustained levels of DHEA, E, and DG without jeopardizing other body organs.

Higdon K, Scott A, Tucci M, Benghuzzi H…
Biomed Sci Instrum 2001
PMID: 11347403

Review: Creatine May Benefit Bone and Muscle

Abstract

Creatine supplementation: can it improve quality of life in the elderly without associated resistance training?

Introduction: Ageing is associated with decreased muscle mass, strength, power and function, and reduction in bone density and mineral content, leading to reduced independence and increased risk of falls. Creatine supplementation is reported to improve muscular strength and performance with training in younger athletes, and therefore could benefit older individuals. Aims: This review critically appraises the current literature on whether creatine supplementation enhances muscular performance and function, body composition, bone mineral density and content in older adults without the addition of resistance training, and thus determines whether creatine supplementation can lead to an improved lifestyle for the sedentary elderly population. Results: There is conflicting evidence regarding the usefulness of creatine supplementation in older subjects. Generally, however, creatine supplementation, without associated resistance training, seems to enhance muscular strength, power and endurance, increase lean body mass (LBM) and improve the functional capacity of the elderly. Furthermore, it has been demonstrated that increased muscle mass due to creatine supplementation can result in increased local bone density. It appears that the effect of creatine supplementation is more beneficial in larger muscles and less effective in smaller muscles, however there are exceptions. The mechanism by which creatine supplementation works requires further research, however it is likely that the effects of creatine are related to creatine kinase activity, providing enhanced energy production for greater muscular contraction. Conclusions: These data indicate that creatine supplementation without associated training in the elderly could potentially delay atrophy of muscle mass, improve endurance and strength, and increase bone strength, and thus may be a safe therapeutic strategy to help decrease loss in functional performance of everyday tasks.

Moon A, Heywood L, Rutherford S, Cobbold C
Curr Aging Sci Dec 2013
PMID: 24304199

Olive Oil or Fish Oil, but Not Sunflower Oil, Prevent Age-Related Bone Resorption

Abstract

Diets based on virgin olive oil or fish oil but not on sunflower oil prevent age-related alveolar bone resorption by mitochondrial-related mechanisms.

Aging enhances frequency of chronic diseases like cardiovascular diseases or periodontitis. Here we reproduced an age-dependent model of the periodontium, a fully physiological approach to periodontal conditions, to evaluate the impact of dietary fat type on gingival tissue of young (6 months old) and old (24 months old) rats.
Animals were fed life-long on diets based on monounsaturated fatty acids (MUFA) as virgin olive oil, n-6 polyunsaturated fatty acids (n-6PUFA), as sunflower oil, or n-3PUFA, as fish oil. Age-related alveolar bone loss was higher in n-6PUFA fed rats, probably as a consequence of the ablation of the cell capacity to adapt to aging. Gene expression analysis suggests that MUFA or n-3PUFA allowed mitochondria to maintain an adequate turnover through induction of biogenesis, autophagy and the antioxidant systems, and avoiding mitochondrial electron transport system alterations.
The main finding is that the enhanced alveolar bone loss associated to age may be targeted by an appropriate dietary treatment. The mechanisms involved in this phenomenon are related with an ablation of the cell capacity to adapt to aging. Thus, MUFA or n-3PUFA might allow mitochondrial maintaining turnover through biogenesis or autophagy. They might also be able to induce the corresponding antioxidant systems to counteract age-related oxidative stress, and do not inhibit mitochondrial electron transport chain. From the nutritional and clinical point of view, it is noteworthy that the potential treatments to attenuate alveolar bone loss (a feature of periodontal disease) associated to age could be similar to some of the proposed for the prevention and treatment of cardiovascular diseases, a group of pathologies recently associated with age-related periodontitis.

Bullon P, Battino M, Varela-Lopez A, Perez-Lopez P…
PLoS ONE 2013
PMID: 24066124 | Free Full Text