Tag Archives: animal

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

Review: Flaxseed Oil, but not Flax Lignans, may Help Bones

Abstract

Implications of dietary α-linolenic acid in bone health.

Recent evidence implies the benefit of ω-3 polyunsaturated fatty acids in bone health. Although eicosapentaenoic acid and docosahexaenoic acid, present in fish oil, have been extensively researched, much less is known about the influence of α-linolenic acid (ALA; present in flaxseeds), a metabolic precursor of eicosapentaenoic acid and docosahexaenoic acid, on bone. Our objective was to evaluate the published literature and distinguish between the individual effects of flaxseed oil and flax lignans on bone to elucidate the exact role of ALA in skeletal biology. The search was conducted in several databases resulting in 129 articles of which 30 were eligible for inclusion in this review. The studies showed that consumption of whole flaxseeds did not lead to a marked improvement of osteoporotic bones in humans and animals. However, when combined with estrogen therapy, flaxseed supplementation offered an extra benefit to bone in animal models. Similar results were found in studies conducted with flaxseed oil (predominantly ALA), but the favorable role of flaxseed oil was more obvious in various pathologic conditions (kidney disease, obesity with insulin resistance), resulting in improved bone properties. In contrast, despite a marginal estrogenic effect, the consumption of flax lignans resulted in little benefit to bone and the effect was limited to early life of females only in animal models. Based on the available studies, it could be concluded that supplementation with flaxseeds may contribute to some improvement in osteoporotic bone properties but the bone-protective effect may be attributed to ALA, not to the lignan fraction of flaxseeds.

Kim Y, Ilich JZ
Nutrition
PMID: 21726979

Fish Oil or Borage Oil Improve Bone in Mice

Abstract

Borage and fish oils lifelong supplementation decreases inflammation and improves bone health in a murine model of senile osteoporosis.

Fats are prevalent in western diets; they have known deleterious effects on muscle insulin resistance and may contribute to bone loss. However, relationships between fatty acids and locomotor system dysfunctions in elderly population remain controversial. The aim of this study was to analyze the impact of fatty acid quality on the age related evolution of the locomotor system and to understand which aging mechanisms are involved. In order to analyze age related complications, the SAMP8 mouse strain was chosen as a progeria model as compared to the SAMR1 control strain. Then, two months old mice were divided in different groups and subjected to the following diets : (1) standard “growth” diet – (2) “sunflower” diet (high ω6/ω3 ratio) – (3) “borage” diet (high γ-linolenic acid) – (4) “fish” diet (high in long chain ω3). Mice were fed ad libitum through the whole protocol. At 12 months old, the mice were sacrificed and tissues were harvested for bone studies, fat and muscle mass measures, inflammation parameters and bone cell marker expression. We demonstrated for the first time that borage and fish diets restored inflammation and bone parameters using an original model of senile osteoporosis that mimics clinical features of aging in humans. Therefore, our study strongly encourages nutritional approaches as relevant and promising strategies for preventing aged-related locomotor dysfunctions.

Wauquier F, Barquissau V, Léotoing L, Davicco MJ…
Bone Feb 2012
PMID: 21664309

High Omega-6 to Omega-3 Ratio is Associated with Lower Bone Density

Abstract

Ratio of n-6 to n-3 fatty acids and bone mineral density in older adults: the Rancho Bernardo Study.

Several lines of evidence suggest that n-3 fatty acids reduce the risk of some chronic diseases, including heart disease, diabetes, and cancer. Other research, mainly in animals, also suggests a role in bone health.
We aimed to investigate the association between the ratio of dietary n-6 to n-3 fatty acids and bone mineral density (BMD) in 1532 community-dwelling men and women aged 45-90 y.
Between 1988 and 1992, dietary data were obtained through self-administered food-frequency questionnaires, and BMD was measured at the hip and spine with the use of dual-energy X-ray absorptiometry. A medical history was obtained and current medication use was validated. Age- and multiple-adjusted linear regression analyses were performed.
There was a significant inverse association between the ratio of dietary linoleic acid to alpha-linolenic acid and BMD at the hip in 642 men, 564 women not using hormone therapy, and 326 women using hormone therapy; these results were independent of age, body mass index, and lifestyle factors. An increasing ratio of total dietary n-6 to n-3 fatty acids was also significantly and independently associated with lower BMD at the hip in all women and at the spine in women not using hormone therapy.
A higher ratio of n-6 to n-3 fatty acids is associated with lower BMD at the hip in both sexes. These findings suggest that the relative amounts of dietary polyunsaturated fatty acids may play a vital role in preserving skeletal integrity in older age.

Weiss LA, Barrett-Connor E, von Mühlen D
Am. J. Clin. Nutr. Apr 2005
PMID: 15817874 | Free Full Text

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 K2 Reversed Bone Loss in Obese Mice

Abstract

Vitamin K1 (phylloquinone) and K2 (menaquinone-4) supplementation improves bone formation in a high-fat diet-induced obese mice.

Several reports suggest that obesity is a risk factor for osteoporosis. Vitamin K plays an important role in improving bone metabolism. This study examined the effects of vitamin K1 and vitamin K2 supplementation on the biochemical markers of bone turnover and morphological microstructure of the bones by using an obese mouse model. Four-week-old C57BL/6J male mice were fed a 10% fat normal diet group or a 45% kcal high-fat diet group, with or without 200 mg/1000 g vitamin K1 (Normal diet + K1, high-fat diet + K1) and 200 mg/1000 g vitamin K2 (Normal diet + K2, high-fat diet + K2) for 12 weeks. Serum levels of osteocalcin were higher in the high-fat diet + K2 group than in the high-fat diet group. Serum OPG level of the high-fat diet group, high-fat diet + K1 group, and high-fat diet + K2 group was 2.31 ± 0.31 ng/ml, 2.35 ± 0.12 ng/ml, and 2.90 ± 0.11 ng/ml, respectively. Serum level of RANKL in the high-fat diet group was significantly higher than that in the high-fat diet + K1 group and high-fat diet + K2 group (p<0.05). Vitamin K supplementation seems to tend to prevent bone loss in high-fat diet induced obese state. These findings suggest that vitamin K supplementation reversed the high fat diet induced bone deterioration by modulating osteoblast and osteoclast activities and prevent bone loss in a high-fat diet-induced obese mice.

Kim M, Na W, Sohn C
J Clin Biochem Nutr Sep 2013
PMID: 24062608 | Free Full Text

Vitamin K is related to blood coagulation, assisting the promotion of OC carboxylation of γ-glutamic acid, which is produced by osteoblasts, and aiding in bone formation by coupling carboxylated OC with phosphine.(15) Many studies have demonstrated that low intake of vitamin K decreases bone density, and that this is a factor that increases osteoporosis and bone fracture.(16) In the study by Booth et al.,(17) low intake of vitamin K1 led to low bone density, and was a factor for increased risk of bone fracture. When vitamin K1 was administered to human bone marrow culture, osteoclast formation was inhibited.(13) After administering vitamin K2 to osteoblasts, real-time gene expression analysis found that the OC, OPG, and RANKL genes were expressed, demonstrating that vitamin K2 has an influence on osteoblasts and osteoclasts.(18) In addition, vitamin K2 supplementation in patients with osteoporosis necessitated by the administration of glucocorticoids inhibited OPG decrease, and had effects of bone loss prevention.(19) Vitamin K2 supplementation in patients with rheumatoid arthritis accompanied with osteoporosis decreased RANKL levels and inhibited osteoclast activation.(20) Therefore, vitamin K affects bone condition both in healthy adults and in patients with specific diseases.

[…]

The results of the bone density analysis revealed an increase with the vitamin K1 and K2 supplementation in high-fat diets. Studies on the relationship between bone density and vitamin K generally have used dual-energy x-ray absorptiometry or ultrasonic densitometry,(32) but this study used high-resolution 3D micro-CT to analyze the morphologic microstructure of trabecular bone. In the study by Fujikawa et al.,(24) the Tb.N increased when vitamin K2 and calcium were fed to ovariectomized mice, and the Tb.Sp decreased. Yamaguchi et al.,(33) also fed vitamin K2 to ovariectomized rats, and reported that it prevented bone loss. These two studies used osteoporosis-induced animals, and the methods differed from those in this study, in which obesity-induced mice were fed vitamin K supplements. In this study, even though there was no significantly statistical difference in the microstructure analysis between the groups, but BV, Tb.N, and Tb.Sp were seemed to be better in the vitamin K2-supplemented group than those in the HF group, indicating that vitamin K2 may play a role in protecting the structures of trabecular bone.

[…]

The effects of vitamin K1 and K2 supplementation in normal diet on bone metabolism were not statistically significant. However, vitamin K1 and K2 supplementation in a high-fat diet could prevent a decrease in bone density, and vitamin K2 had a greater effect on this parameter. Therefore, vitamin K2 increases OPG, a marker related to bone density and the metabolism of osteoclasts and osteoblasts, and it decreases RANKL, and thus has an influence on bone metabolism. This study has showed the effects of vitamin K on bone density and metabolism in animals, but further studies are needed to determine whether the same holds true for obese humans. Future studies would need to perform bone measurement and biochemical examinations on the bone microstructures and metabolism in humans.

 

Citric Acid Increases Bone Density and Strength in Chicks

Abstract

Effect of dietary citric acid on the performance and mineral metabolism of broiler.

The objective of this study was to investigate the effect of dietary citric acid (CA) on the performance and mineral metabolism of broiler chicks. A total of 1720 Ross PM3 broiler chicks (days old) were randomly assigned to four groups (430 in each) and reared for a period of 35 days. The diets of groups 1, 2, 3 and 4 were supplemented with 0%, 0.25%, 0.75% or 1.25% CA by weight respectively. Feed and faeces samples were collected weekly and analysed for acid insoluble ash, calcium (Ca), phosphorus (P) and magnesium (Mg). The pH was measured in feed and faeces. At the age of 28 days, 10 birds from each group were slaughtered; tibiae were collected from each bird for the determination of bone mineral density, total ash, Ca, P, Mg and bone-breaking strength, and blood was collected for the measurement of osteocalcin, serum CrossLaps(®), Ca, P, Mg and 1,25(OH)(2)Vit-D in serum. After finishing the trial on day 37, all chicks were slaughtered by using the approved procedure. Birds that were fed CA diets were heavier (average body weights of 2030, 2079 and 2086 g in the 0.25%, 0.75% and 1.25% CA groups, respectively, relative to the control birds (1986 g). Feed conversion efficiency (weight gain in g per kg of feed intake) was also higher in birds of the CA-fed groups (582, 595 and 587 g/kg feed intake for 0.25%, 0.75% and 1.25% CA respectively), relative to the control birds (565 g/kg feed intake). The digestibility of Ca, P and Mg increased in the CA-fed groups, especially for the diets supplemented with 0.25% and 0.75% CA. Support for finding was also indicated in the results of the analysis of the tibia. At slaughter, the birds had higher carcass weights and higher graded carcasses in the groups that were fed the CA diets. The estimated profit margin was highest for birds fed the diet containing 0.25% CA. Birds of the 0.75% CA group were found to have the second highest estimated profit margin. Addition of CA up to a level of 1.25% of the diet increased performance, feed conversion efficiency, carcass weight and carcass quality, but only in numerical terms. The addition of CA up to 0.75% significantly increased the digestibility of macro minerals, bone ash content, bone mineral density and bone strength of the broiler chicks. It may, therefore, be concluded that the addition of 0.75% CA in a standard diet is suitable for growth, carcass traits, macromineral digestibility and bone mineral density of broiler chicks.

Islam KM, Schaeublin H, Wenk C, Wanner M…
J Anim Physiol Anim Nutr (Berl) Oct 2012
PMID: 22093035

High Sodium Causes Loss of Calcium in Rats

Abstract

Calcium metabolism and bone calcium content in normal and oophorectomized rats consuming various levels of saline for 12 months.

The effect of different intakes of salt for 12 mo on bone calcium content and urinary excretion of calcium and hydroxyproline were examined in sham operated and oophorectomized (OX) rats to determine the long term effects of high sodium intake and its interaction with estrogen deficiency. Sham operated (n = 24) and OX (n = 24) rats were divided into groups of six rats in a 2 x 4 design. One group of sham and one of OX rats were given 0, 2, 6 or 18 g/L sodium chloride to drink. Urine samples were collected at 0, 2, 4, 6, 10 and 12 mo for the measurement of sodium, calcium, creatinine and hydroxyproline. At the end of 12 mo, blood was taken for measurement of calcium, albumin, alkaline phosphatase and creatinine and the left femur was removed and analyzed for calcium and phosphate. Body weights of the OX rats were higher than the sham operated controls. At the start of the experiment (10 d after OX) urinary excretions of calcium and hydroxyproline were significantly higher in OX rats. However, after 4-6 mo, they were significantly lower in OX rats. Calcium excretion and hydroxyproline excretion were increased by high salt intake, and there was a significant correlation between sodium and calcium excretion (r = 0.962). Bone calcium content of OX rats was lower than their corresponding sham-operated controls. Sodium intake also had a significant effect on bone calcium content. Multiple regression analysis showed that OX and sodium intake explained 7.6% and 1.5% of the variation in bone calcium content. We conclude that high sodium intake causes increased loss of calcium and reduces bone calcium content in sham-operated as well as OX rats.

Chan EL, Swaminathan R
J. Nutr. Mar 1998
PMID: 9482774 | Free Full Text

Fish Oil Benefits Bone in Salt-Loaded Rats

Abstract

Benefits of omega-3 fatty acid against bone changes in salt-loaded rats: possible role of kidney.

There is evidence that dietary fats are important components contributing in bone health and that bone mineral density is inversely related to sodium intake. Salt loading is also known to impose negative effects on renal function. The present study aimed to determine the effect of the polyunsaturated fatty acid omega-3 on bone changes imposed by salt loading, highlighting the role of kidney as a potential mechanism involved in this effect. Male Wistar rats were divided into three groups: control group, salt-loaded group consuming 2% NaCl solution as drinking water for 8 weeks, and omega-3-treated salt-loaded group receiving 1 g/kg/day omega-3 by gavage with consumption of 2% NaCl solution for 8 weeks. Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), and heart rate (HR) were recorded. Plasma levels of sodium, potassium, calcium, inorganic phosphorus (Pi), alkaline phosphatase (ALP), creatinine, urea, 1,25-dihydroxyvitamin D [1,25(OH)2D3], and transforming growth factor-beta1 (TGF-β1) were measured. The right tibia and kidney were removed for histologic examination and renal immunohistochemical analysis for endothelial nitric oxide synthase (eNOS) was performed. The results revealed that omega-3 reduced SBP, DBP, and MAP and plasma levels of sodium, potassium, Pi, creatinine, urea, and TGF-β1, but increased plasma levels of calcium, ALP, and 1,25(OH)2D3 as well as renal eNOS. Omega-3 increased cortical and trabecular bone thickness, decreased osteoclast number, and increased newly formed osteoid bone. Renal morphology was found preserved. In conclusion, omega-3 prevents the disturbed bone status imposed by salt loading. This osteoprotective effect is possibly mediated by attenuation of alterations in Ca(2+), Pi, and ALP, and improvement of renal function and arterial blood pressure.

Ahmed MA, Abd El Samad AA
Physiol Rep Oct 2013
PMID: 24303178 | Free Full Text

The acquisition and maintenance of bone mass and strength are influenced by environmental factors, including physical activity and nutrition (Massey and Whiting 1996). Nutrition is important to bone health, and a number of minerals and vitamins have been identified as playing a potential role in the prevention of bone diseases, particularly osteoporosis (Massey and Whiting 1996). Evidence indicates that dietary fats can influence bone health (Tartibian et al. 2010), in particular the omega-3 (n-3) polyunsaturated fatty acids (PUFAs), as they have been shown to inhibit osteoclast activity and enhance osteoblast activity (Watkins et al. 2003). Eicosapentaenoic acid (EPA) supplementation was found to increase bone mineral density in postmenopausal women (Terano 2001). Beneficial effects of n-3 PUFAs on markers of bone resorption and formation in animal (Shen et al. 2006) and human (Griel et al. 2007) studies have, also, been observed.

On the other hand, a number of studies suggested a detrimental effect of dietary salt on bone. Devine et al. (1995) showed that change in bone mineral density was inversely related to sodium intake and that both dietary calcium and urinary sodium excretion were significant determinants of the change in bone mass. High-sodium diet was found to increase urinary calcium excretion and cause loss of bone calcium (Chan and Swaminathan 1998), while reducing sodium intake complemented the beneficial skeletal effects of the Dietary Approaches to Stop Hypertension diet (Lin et al. 2003). Furthermore, an epidemiological study of men and women has shown that salt intake is associated with markers of bone resorption and appears likely to be a risk factor for osteoporosis (Jones et al. 1997). Similar effect of sodium loading has been demonstrated in animal model (Gold and Gouldin 1995).