Tag Archives: in vitro

Chondroitin Suppresses Osteoclasts In Vitro

Abstract

Comparison of the ability of chondroitin sulfate derived from bovine, fish and pigs to suppress human osteoclast activity in vitro.

Chondroitin sulfate (CS) compounds are commonly used to manage OA symptoms. Recent literature has indicated that abnormal subchondral bone metabolism may have a role in the pathogenesis of OA. The aim of this study was to access the effects of chondroitin sulfate obtained from bovine, fish and porcine sources on human osteoclast formation and activity in vitro. Human osteoclasts were generated from blood mononuclear cells. Cells were cultured over 17 days with the addition of macrophage colony stimulating factor (M-CSF) and then stimulated with receptor activator of nuclear factor kappa B ligand from day 7. Cells were treated with the CS commencing from day 7 onwards. To assess effects on osteoclasts, tartrate resistant acid phosphatate (TRAP) expression and resorption of whale dentine assays were used. Bovine-derived CS consistently suppressed osteoclast activity at concentrations as low as 1 μg/ml. Fish and porcine CS was less consistent in their effects varying with different donor cells. All CS compounds had little effect on TRAP activity. mRNA analysis using real-time PCR of bovine CS treated cells indicated that the inhibition of activity was not due to inhibition of the late stage NFATc1 transcription factor (p > 0.05). These results are consistent with CS inhibition of mature osteoclast activity rather than the formation of mature osteoclasts. It would appear that there are differences in activity of the different CS compounds with bovine-derived CS being the most consistently effective inhibitor of osteoclast resorption, but the results need to be confirmed.

Cantley MD, Rainsford KD, Haynes DR
Inflammopharmacology Dec 2013
PMID: 23644893

Chondroitin May Benefit Bones

Abstract

A potential role of chondroitin sulfate on bone in osteoarthritis: inhibition of prostaglandin E₂ and matrix metalloproteinases synthesis in interleukin-1β-stimulated osteoblasts.

To determine the effect of chondroitin sulfate (CS) on inflammatory mediators and proteolytic enzymes induced by interleukin-1β (IL-1β) and related to cartilage catabolism in murine osteoblasts.
Osteoblasts were obtained by enzymatic digestion of calvaria from Swiss mice and cultured for 3 weeks as a primary culture. Cells were then stimulated with IL-1β (1 or 10 ng/ml). CS-treated osteoblasts were incubated with 100 μg/ml of CS during the last week of culture w/o IL-1β for the last 24 h. Expressions of cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1), 15-PG dehydrogenase (15-PGDH), matrix metalloproteinases-3 and -13 (MMP-3 and -13), osteoprotegerin (OPG) and receptor activator of nuclear factor-kappa B ligand (RANKL) were determined by real-time polymerase chain reaction (PCR). PGE₂, MMP-3 and MMP-13 release were assessed in the medium by enzyme-linked immunosorbent assay or western-blotting.
IL-1β increased COX-2, mPGES-1, MMP-3, MMP-13, RANKL expressions, decreased 15-PGDH expression, and increased PGE₂, MMP-3 and MMP-13 release. Interestingly, 7 days of CS treatment significantly counteracted IL-1β-induced expression of COX-2 (-62%, P<0.001), mPGES-1 (-63%, P<0.001), MMP-3 (-39%, P=0.08), MMP-13 (-60%, P<0.001) and RANKL (-84%, P<0.001). Accordingly, IL-1β-induced PGE₂, MMP-3 and MMP-13 releases were inhibited by 86% (P<0.001), 58%(P<0.001) and 38% (P<0.01) respectively.
In conclusion, our data demonstrate that, in an inflammatory context, CS inhibits the production of PGE₂ and MMPs. Since CS has previously been shown to counteract the production of these mediators in chondrocytes, we speculate that the beneficial effect of CS in Osteoarthritis (OA) could not only be due to its action on cartilage but also on subchondral bone.

Pecchi E, Priam S, Mladenovic Z, Gosset M…
Osteoarthr. Cartil. Feb 2012
PMID: 22179028

MK-7 Suppresses Osteoblast Proliferation and Enhances RANKL In Vitro

Abstract

Menaquinone-7 regulates the expressions of osteocalcin, OPG, RANKL and RANK in osteoblastic MC3T3E1 cells.

Epidemiological studies show that dietary intake of natto, which contains significant amount of vitamin K(2), reduces the risk of bone formation loss. However, many confounding factors, such as calcium and isoflavone, are found in natto, because it is made from soybeans. In this study, the direct effects of MK-7, a vitamin K(2) analogue, were assessed in osteoblasts. Osteoblastic MC3T3E1 cells were cultured with or without MK-7 for 10 days and the number of cells was calculated. The cell count was not different between MK-7 treated cells and control cells for 1, 2, and 4 days. However, it was significantly suppressed in MK-7 treated cells at 10 days, suggesting that MK-7 suppressed cell proliferation. Real-time PCR analysis showed that mRNAs of osteocalcin (OC), osteoprotegerin (OPG), and the receptor activator of the NFkappaB ligand (RANKL) were induced after MK-7 administration to the culture medium. RANK mRNA expression was also enhanced by MK-7 administration. Immunocytochemical analysis showed that MK-7 increased the protein levels of OC and RANKL. RANK protein was also enhanced, but this induction was suppressed by anti-RANK antibody administration. This suppression was recovered when anti-RANK antibody and MK-7 were administered. These observations suggest that MK-7 may directly affect MC3T3E1 cells and stimulate osteoblastic differentiation, not proliferation. Katsuyama H, Otsuki T, Tomita M, Fukunaga M… Int. J. Mol. Med. Feb 2005 PMID: 15647836

MK-7 does some bad things. This says it supresses poliferation of osteoblasts, and enhances RANKL. This is in vitro and there were positive effects as well. This is interesting, but in vivo studies would be good to look at.

Vitamin K1 and MK-4 Stimulate Osteoblasts and Inhibit Osteoclasts In Vitro

Abstract

Vitamin K stimulates osteoblastogenesis and inhibits osteoclastogenesis in human bone marrow cell culture.

Accumulating evidence indicates that menaquinone-4 (MK-4), a vitamin K(2) with four isoprene units, inhibits osteoclastogenesis in murine bone marrow culture, but the reason for this inhibition is not yet clear, especially in human bone marrow culture. To clarify the inhibitory mechanism, we investigated the differentiation of colony-forming-unit fibroblasts (CFU-Fs) and osteoclasts in human bone marrow culture, to learn whether the enhancement of the differentiation of CFU-Fs from progenitor cells might relate to inhibition of osteoclast formation. Human bone marrow cells were grown in alpha-minimal essential medium with horse serum in the presence of MK-4 until adherent cells formed colonies (CFU-Fs). Colonies that stained positive for alkaline phosphatase activity (CFU-F/ALP(+)) were considered to have osteogenic potential. MK-4 stimulated the number of CFU-F/ALP(+) colonies in the presence or absence of dexamethasone. The stimulation was also seen in vitamin K(1) treatment. These cells had the ability to mineralize in the presence of alpha-glycerophosphate. In contrast, both MK-4 and vitamin K(1) inhibited 1,25 dihydroxyvitamin D(3)-induced osteoclast formation and increased stromal cell formation in human bone marrow culture. These stromal cells expressed ALP and Cbfa1. Moreover, both types of vitamin K treatment decreased the expression of receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor (RANKL/ODF) and enhanced the expression of osteoprotegerin/osteoclast inhibitory factor (OPG/OCIF) in the stromal cells. The effective concentrations were 1.0 microM and 10 microM for the expression of RANKL/ODF and OPG/OCIF respectively. Vitamin K might stimulate osteoblastogenesis in bone marrow cells, regulating osteoclastogenesis through the expression of RANKL/ODF more than through that of OPG/OCIF.

Koshihara Y, Hoshi K, Okawara R, Ishibashi H…
J. Endocrinol. Mar 2003
PMID: 12630919 | Free Full Text

Curcumol Suppresses Osteoclast Formation In Vitro

Abstract

Curcumol suppresses RANKL-induced osteoclast formation by attenuating the JNK signaling pathway.

Osteoclasts, derived from hemopoietic progenitors of the monocyte/macrophage lineage, have a unique role in bone resorption, and are considered a potential therapeutic target in the treatment of such pathologic bone diseases as osteoporosis, rheumatoid arthritis, and periodontitis. In the present study, we demonstrate that curcumol, one of the major components of the essential oil of Rhizoma Curcumae, exhibits an inhibitory effect on receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclast differentiation with both bone marrow-derived macrophages and RAW264.7 cells in a dose-dependent manner. In addition, RANKL-induced mRNA expression of osteoclast-specific genes, such as tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K, is prominently reduced in the presence of curcumol. Furthermore, the molecular mechanism of action was investigated, and curcumol inhibited osteoclastogenesis by specifically impairing RANKL-induced c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) signaling, which was further identified in rescue studies by means of anisomycin, a JNK signaling-specific activator. Taken together, these findings suggest that curcumol suppresses RANKL-induced osteoclast differentiation through the JNK/AP-1 signaling pathway, and may be useful as a therapeutic treatment for bone resorption-associated diseases.

Yu M, Chen X, Lv C, Yi X…
Biochem. Biophys. Res. Commun. Apr 2014
PMID: 24732351

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 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