Impact of vitamin D supplementation on markers of bone turnover: Systematic review and meta-analysis of randomised controlled trials.

AIM: The effects of vitamin D administration on bone turnover markers (BTMs) in adults are controversial. Thus, we carried out a meta-analysis of available randomised controlled trials (RCTs) to examine the impact of vitamin D supplementation on BTMs. METHODS: To identify relevant RCTs, we searched the PubMed/MEDLINE, Web of Science, Scopus, Cochrane Library and Embase databases for manuscripts published up to July 2022. The present study was conducted in agreement with the PRISMA guidelines. Weighed mean difference (WMD) and 95% confidence intervals (CI) were used to calculate the magnitude of the effect of the intervention. RESULTS: A total of 42 RCTs were included in the meta-analysis. The age of the participants enrolled in the RCTs ranged from 19.4 to 84 years. The pooled results depicted a decrease in deoxypyridinoline (DPD) concentrations (WMD: -1.58 nmol/mmol, 95% CI: -2.55, -.61, p = .001) following vitamin D supplementation. In addition, subgroup analyses demonstrated that vitamin D administration notably reduced procollagen type I N-terminal propeptide (PINP) levels in individuals aged >50 years and led to a pronounced decrease in alkaline phosphatase (ALP) values when the intervention lasted >12 weeks. No significant effect was observed on other BTMs, for example, collagen type 1 cross-linked C-telopeptide (CTX) and osteocalcin (OC) levels. CONCLUSION: Vitamin D administration decreases DPD, PINP and ALP levels, indicating a reduced bone turnover following the intervention. Other BTMs, for example, CTX or OC values, were not affected by vitamin D prescription. Vitamin D supplementation may exert a positive effect on some important BTMs.

Ginkgolide B facilitates muscle regeneration via rejuvenating osteocalcin-mediated bone-to-muscle modulation in aged mice.

BACKGROUND: The progressive deterioration of tissue-tissue crosstalk with aging causes a striking impairment of tissue homeostasis and functionality, particularly in the musculoskeletal system. Rejuvenation of the systemic and local milieu via interventions such as heterochronic parabiosis and exercise has been reported to improve musculoskeletal homeostasis in aged organisms. We have shown that Ginkgolide B (GB), a small molecule from Ginkgo biloba, improves bone homeostasis in aged mice by restoring local and systemic communication, implying a potential for maintaining skeletal muscle homeostasis and enhancing regeneration. In this study, we investigated the therapeutic efficacy of GB on skeletal muscle regeneration in aged mice. METHODS: Muscle injury models were established by barium chloride induction into the hind limb of 20-month-old mice (aged mice) and into C2C12-derived myotubes. Therapeutic efficacy of daily administrated GB (12 mg/kg body weight) and osteocalcin (50 µg/kg body weight) on muscle regeneration was assessed by histochemical staining, gene expression, flow cytometry, ex vivo muscle function test and rotarod test. RNA sequencing was used to explore the mechanism of GB on muscle regeneration, with subsequent in vitro and in vivo experiments validating these findings. RESULTS: GB administration in aged mice improved muscle regeneration (muscle mass, P = 0.0374; myofiber number/field, P = 0.0001; centre nucleus, embryonic myosin heavy chain-positive myofiber area, P = 0.0144), facilitated the recovery of muscle contractile properties (tetanic force, P = 0.0002; twitch force, P = 0.0005) and exercise performance (rotarod performance, P = 0.002), and reduced muscular fibrosis (collagen deposition, P < 0.0001) and inflammation (macrophage infiltration, P = 0.03). GB reversed the aging-related decrease in the expression of osteocalcin (P < 0.0001), an osteoblast-specific hormone, to promote muscle regeneration. Exogenous osteocalcin supplementation was sufficient to improve muscle regeneration (muscle mass, P = 0.0029; myofiber number/field, P < 0.0001), functional recovery (tetanic force, P = 0.0059; twitch force, P = 0.07; rotarod performance, P < 0.0001) and fibrosis (collagen deposition, P = 0.0316) in aged mice, without an increased risk of heterotopic ossification. CONCLUSIONS: GB treatment restored the bone-to-muscle endocrine axis to reverse aging-related declines in muscle regeneration and thus represents an innovative and practicable approach to managing muscle injuries. Our results revealed the critical and novel role of osteocalcin-GPRC6A-mediated bone-to-muscle communication in muscle regeneration, which provides a promising therapeutic avenue in functional muscle regeneration.

Meta-Analysis of Effects of Nutritional Intervention Combined with Calcium Carbonate D3 Tablets on Bone Mineral Density, Bone Metabolism, and Curative Effect in Patients with Osteoporosis.

To investigate the changes in bone mineral density, bone metabolism, and efficacy of nutritional intervention combined with calcium carbonate D3 tablets in patients with osteoporosis, a RevMan 5.2 software meta-analysis was conducted in this study. According to the therapeutic direction of nutritional intervention combined with calcium carbonate D3 tablets for osteoporosis patients, relevant literature were searched in Wanfang Medical, CNKI, VIP, and PubMed literature databases at home and abroad. Keywords included bone mineral density, bone metabolism, blood calcium (Ca), blood phosphorus (P), osteocalcin (OC), bone mineral density (BMD), serum alkaline phosphatase (ALP), efficacy, osteoporosis, and nutritional intervention. Literature that met the criteria were deleted, and meta-analysis was performed using RevMan 5.2 software. The results indicate that a total of 10 Chinese literature were included. Compared with the monotherapy group, the clinical efficacy, osteocalcin, BMD, alkaline phosphatase, calcium, and phosphorus were significantly higher in the combination group (P < 0.05). Based on calcium carbonate D3, treatment combined with nutritional intervention can enhance the clinical efficacy, bone metabolism, and bone mineral density of patients with osteoporosis, and nutritional intervention combined with calcium carbonate D3 tablets is a feasible program to promote the recovery of patients with osteoporosis.

Effects of Bisphosphonate on Osteocyte Proliferation and Bone Formation in Patients with Diabetic Osteoporosis.

Background: Bisphosphonate is currently considered one of the drugs for the first-line treatment of osteoporosis because of its ability to inhibit bone resorption, but the molecular mechanism of its effect on osteocyte proliferation and bone formation of diabetic osteoporosis is still unclear. Objective: To confirm the potential effect on of bisphosphonate on osteocyte proliferation and bone formation in patients having diabetic osteoporosis (DO). Methods: Sixty DO patients admitted to our hospital from February 2019 to April 2021 were randomly selected and divided into the bisphosphonate group and the control group. The total incidence, incidence of hip fracture, efficacy, bone mineral density, osteocalcin, pain score, osteocyte proliferation, bone formation index, serum calcium, and phosphorus contents were compared between two groups. Results: The curative effect of bisphosphonic acid group was better than that of control group, and the difference was statistically significant (P < 0.05). Compared with the control group, the bone mineral density and osteocalcin in the bisphosphonic acid group were significantly improved after treatment, and the pain score in the bisphosphonic acid group was significantly lower than that in the control group (P < 0.05). After intervention treatment, the OD and PINP values in the bisphosphonate group were significantly different from those in the control group (P < 0.05). After treatment, the contents of serum calcium and phosphorus in the bisphosphonic acid group were significantly higher than those in the control group (P < 0.05). The incidence of hip fracture, spinal fracture, and other fractures in the bisphosphonic acid group was significantly lower than that in the control group (P < 0.05). Conclusion: The treatment of DO with bisphosphonate is capability of effectively improving bone cell proliferation and bone formation, further alleviating clinical symptoms and promoting the improvement of the disease.

Ponicidin Treatment Improved the Cell Proliferation, Differentiation, and Calcium Mineralization on the Osteoblast-Like MG-63 Cells.

Osteoporosis is a general bone-related ailment characterized by reduced bone density and quality, elevated bone fragility, and fractures. It was reported that both aged men and women has an increased risks of osteoporosis. The current research work focused to unveil the beneficial roles of ponicidin treatment in the proliferation and calcium deposition on the osteoblast-like MG-63 cells. The effect of 5 and 10 µg/ml of ponicidin on the cell proliferation was assessed. The viability of ponicidin-supplemented MG-63 cells was inspected by MTT test. The contents of osteocalcin, collagen, and ALP activity in the ponicidin administered cells were assessed by kits. The level of calcium mineralization was examined by ARS staining technique. The ponicidin treatment remarkably improved the proliferation of MG-63 cells. The ponicidin did not affect the MG-63 cells viability but promoted its viability 24- and 48-h treatment. The contents of osteocalcin, collagen, and ALP activity in the 5 and 10 µg/ml of ponicidin-supplemented MG-63 cells were found increased than the control cells. The ponicidin also increased the level of calcium deposition in MG-63 cells, which is assessed by ARS staining. In conclusion, it was clear that ponicidin improved the proliferation and calcium mineralization in a MG-63 cells. Therefore, it was clear that ponicidin has helpful roles on the new bone development as a hopeful therapeutic candidate to treat the bone-related disease like osteoporosis.

Modulatory activity of a bovine hydrolyzed collagen-hydroxyapatite food complex on human primary osteoblasts after simulating its gastrointestinal digestion and absorption.

Introduction: Introduction: osteoporosis is the most prevalent bone disease and one of the main causes of chronic disability in middle and advanced ages. Conventional pharmacological treatments are still limited, and their prolonged use can cause adverse effects that motivate poor adherence to treatment. Nutritional strategies are traditionally based on supplementing the diet with calcium and vitamin D. Recent studies confirm that the results of this supplementation are significantly improved if it is accompanied by the intake of oral hydrolyzed collagen. Objective: to evaluate the possible in vitro osteogenic activity of a peptide-mineral complex formed by bovine hydrolyzed collagen and bovine hydroxyapatite (Phoscollagen®, PHC®). Methods: the digestion and absorption of PHC® were simulated using the dynamic gastrointestinal digester of AINIA and Caco-2 cell model, respectively. Primary cultures of human osteoblasts were treated with the resulting fraction of PHC® and changes were evaluated in the proliferation of preosteoblasts and in the mRNA expression of osteogenic biomarkers at different stages of osteoblast maturation: Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteocalcin (OC) and type I collagen (ColA1). Results: an increase in preosteoblastic proliferation was observed (p ≤ 0,05). No changes were detected in the biomarkers of osteoblasts with 5 days of differentiation, but were with 14 days, registering an increase in Runx2 (p = 0.0008), ColA1 (p = 0.035), OC (p = 0.027) and ALP (without significance). Conclusion: these results show that the PHC® peptide-mineral complex stimulates the activity of mature osteoblasts, being capable of promoting bone formation.

Introducción: Introducción: la osteoporosis es la enfermedad ósea más prevalente y una de las principales causas de discapacidad crónica en las edades medias y avanzadas. Los tratamientos farmacológicos convencionales aún son limitados y su uso prolongado puede provocar efectos adversos que motiven baja adherencia al tratamiento. Las estrategias nutricionales se basan tradicionalmente en suplementar la dieta con calcio y vitamina D. Estudios recientes confirman que los resultados de esta suplementación mejoran significativamente si se acompaña de la ingesta de colágeno hidrolizado oral. Objetivo: evaluar la posible actividad osteogénica in vitro de un complejo péptido-mineral formado por colágeno hidrolizado e hidroxiapatita bovinos (Phoscollagen®, PHC®). Métodos: se simuló la digestión y absorción de PHC® utilizando el digestor dinámico gastrointestinal de AINIA y el modelo celular Caco-2, respectivamente. Cultivos primarios de osteoblastos humanos se trataron con la fracción resultante de PHC® y se evaluaron los cambios en la proliferación de los preosteoblastos y en la expresión del ARNm de los biomarcadores osteogénicos en diferentes etapas de maduración de los osteoblastos: factor de transcripción 2 relacionado con Runt (Runx2), fosfatasa alcalina (ALP), osteocalcina (OC) y colágeno tipo I (ColA1). Resultados: se observó un incremento de la proliferación preosteoblástica (p ≤ 0,05). No se detectaron cambios en los biomarcadores de osteoblastos con 5 días de diferenciación, pero sí con 14 días, registrándose un aumento de Runx2 (p = 0,0008), ColA1 (p = 0,035), OC (p = 0,027) y ALP (sin significancia). Conclusión: estos resultados muestran que el complejo péptido-mineral PHC® estimula la actividad de los osteoblastos maduros, siendo susceptible de promover la formación ósea.

The Effects of Milk Supplementation on Bone Health Indices in Adults: A Meta-Analysis of Randomized Controlled Trials.

Milk contains a number of bone-beneficial nutrients. However, milk, due to the D-galactose content, might have unfavorable effects on bone health. A meta-analysis of randomized controlled trials (RCTs) was performed to clarify the effects of milk supplementation on bone mineral density (BMD), bone turnover markers [N-terminal telopeptide of type I collagen (NTx), C-terminal telopeptide of type 1 collagen (CTx), osteocalcin, bone alkaline phosphatase (BALP), and procollagen type 1 N-propeptide (P1NP)], and hormonal indices related to bone metabolism [parathyroid hormone (PTH), 25-hydroxyvitamin D [25(OH)D], and insulin-like growth factor 1 (IGF-1)] in adults. The PubMed and Web of Science databases were searched. A random-effects model was used to estimate the pooled effect sizes. A total of 20 RCTs were included. The trial duration ranged from 1 mo to 36 mo. Milk supplementation resulted in a small but significant increase in BMD at the hip (+0.004 g/cm2; n = 9 RCTs) and lumbar spine (+0.025 g/cm2; n = 7), but did not significantly affect whole-body BMD (n = 3) and femoral neck BMD (n = 7). Milk supplementation reduced the concentrations of P1NP (-5.20 ng/mL; n = 9), CTx (-0.16 ng/mL; n = 9), and NTx (-8.66 nmol bone collagen equivalents/mmol creatinine; n = 3). The concentrations of osteocalcin (n = 9) and BALP (n = 3) were not affected by milk supplementation. Reduced parathyroid hormone PTH (-1.01 pg/mL; n = 13) concentrations and increased IGF-1 (+1.79 nmol/l; n = 4) concentrations were observed with milk supplementation. 25(OH)D (+3.73 ng/mL; n = 11) concentrations were increased with vitamin-D fortified milk supplementation. The addition of milk to the diet may potentially increase the likelihood of preventing bone loss by restoring bone homeostasis through the modulation of the calcium-vitamin D-PTH axis, bone remodeling rate, and growth hormone/IGF-1 axis.

The combination effect of vitamin K and vitamin D on human bone quality: a meta-analysis of randomized controlled trials.

BACKGROUND: Previous studies did not draw a consistent conclusion about the effects of vitamin K combined with vitamin D on human skeletal quality. METHOD AND FINDINGS: A comprehensive search on Web of Science, PubMed, Embase and the Cochrane Library (from 1950 to February 2020) and bibliographies of relevant articles was undertaken, with the meta-analysis of eight randomized controlled trials (RCTs) including a total of 971 subjects. Vitamin K combined with vitamin D significantly increased the total bone mineral density (BMD): the pooled effect size was 0.316 [95% CI (confidence interval), 0.031 to 0.601]. A significant decrease in undercarboxylated osteocalcin (-0.945, -1.113 to -0.778) can be observed with the combination of vitamin K and D. Simultaneously, subgroup analysis showed that K2 or vitamin K (not specified) supplement was less than 500 µg d-1, which when combined with vitamin D can significantly increase the total BMD compared with the control group fed a normal diet or the group with no treatment (0.479, 0.101 to 0.858 and 0.570, 0.196 to 0.945). CONCLUSIONS: The combination of vitamin K and D can significantly increase the total BMD and significantly decrease undercarboxylated osteocalcin, and a more favorable effect is expected when vitamin K2 is used.

Fermented Oyster Extract Promotes Osteoblast Differentiation by Activating the Wnt/ß-Catenin Signaling Pathway, Leading to Bone Formation.

The Pacific oyster, Crassostrea gigas, is well-known as a nutritious food. Recently, we revealed that fermented extract of C. gigas (FO) inhibited ovariectomy-induced osteoporosis, resulting from suppression of osteoclastogenesis. However, since the beneficial effect of FO on osteogenesis is poorly understood, it was examined in mouse preosteoblast MC3T3-E1 cells, human osteosarcoma MG-63 osteoblast-like cells, and zebrafish larvae in this study. We found that FO increased mitochondrial activity from days 1 to 7; however, total cell number of MC3T3-E1 cells gradually decreased without any change in cell viability, which suggests that FO stimulates the differentiation of MC3T3-E1 cells. FO also promoted the expression of osteoblast marker genes, including runt-related transcription factor 2 (mRUNX2), alkaline phosphatase (mALP), collagen type I α1 (mCol1α1), osteocalcin (mOCN), osterix (mOSX), bone morphogenetic protein 2 (mBMP2), and mBMP4 in MC3T3-E1 cells accompanied by a significant increase in ALP activity. FO also increased nuclear translocation of RUNX2 and OSX transcription factors, ALP activity, and calcification in vitro along with the upregulated expression of osteoblast-specific marker proteins such as RUNX2, ALP, Col1α1, OCN, OSX, and BMP4. Additionally, FO enhanced bone mineralization (calcein intensity) in zebrafish larvae at 9 days post-fertilization comparable to that in the ß-glycerophosphate (GP)-treated group. All the tested osteoblast marker genes, including zRUNX2a, zRUNX2b, zALP, zCol1a1, zOCN, zBMP2, and zBMP4, were also remarkably upregulated in the zebrafish larvae in response to FO. It also promoted tail fin regeneration in adult zebrafish as same as the GP-treated groups. Furthermore, not only FO positively regulate ß-catenin expression and Wnt/ß-catenin luciferase activity, but pretreatment with a Wnt/ß-catenin inhibitor (FH535) also significantly decreased FO-mediated bone mineralization in zebrafish larvae, which indicates that FO-induced osteogenesis depends on the Wnt/ß-catenin pathway. Altogether, the current study suggests that the supplemental intake of FO has a beneficial effect on osteogenesis.

Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300.

The uncarboxylated form of osteocalcin (GluOC) regulates glucose and lipid metabolism in mice. We previously showed that low-dose (≤10 ng/ml) GluOC induces the expression of adiponectin and peroxisome proliferator-activated receptor γ (PPARγ) via a cAMP-PKA-ERK-CREB signaling pathway in 3T3-L1 adipocytes. We also noticed that high-dose (≥20 ng/ml) GluOC inhibits the expression of adiponectin and PPARγ in these cells. We have here explored the mechanism underlying these effects of high-dose GluOC. High-dose GluOC triggered morphological changes in 3T3-L1 adipocytes suggestive of the induction of cell death. It activated the putative GluOC receptor GPRC6A and thereby induced the production of cAMP and activation of protein kinase A (PKA), similar to signaling by low-dose GluOC with the exception that the catalytic subunit of PKA also entered the nucleus. Cytosolic PKA induced phosphorylation of cAMP response element-binding protein (CREB) at serine-133 via extracellular signal-regulated kinase (ERK). Nuclear PKA appeared to mediate the inhibitory phosphorylation of salt-inducible kinase 2 (SIK2) at serine-358 and thereby to alleviate the inhibitory phosphorylation of the CREB co-activator p300 at serine-89. The activation of CREB and p300 resulted in increased expression of the transcription factor FoxO1 and consequent upregulation of Fas ligand (FasL) at the plasma membrane. The interaction of FasL with Fas on neighboring adipocytes triggered the phosphorylation at threonine-357/serine-358 and homotrimerization of mixed-lineage kinase domain-like protein (MLKL), a key regulator of necroptosis, as well as Ca2+ influx via transient receptor potential melastatin 7 (TRPM7), the generation of reactive oxygen species and lipid peroxides, and dephosphorylation of dynamin-related protein 1 (DRP1) at serine-637, resulting in mitochondrial fragmentation. Together, our results indicate that high-dose GluOC triggers necroptosis through upregulation of FasL at the plasma membrane in a manner dependent of activation of CREB-p300, followed by the activation of Fas signaling in neighboring adipocytes.

Long-term oral administration of osteocalcin induces insulin resistance in male mice fed a high-fat, high-sucrose diet.

Uncarboxylated osteocalcin (GluOC), a bone-derived hormone, regulates energy metabolism by stimulating insulin secretion, pancreatic ß-cell proliferation, and adiponectin expression in adipocytes. Previously, we showed that long-term intermittent or daily oral administration of GluOC reduced the fasting blood glucose level, improved glucose tolerance, and increased the fasting serum insulin concentration as well as pancreatic ß-cell area in female mice fed a normal or high-fat, high-sucrose diet. We have now performed similar experiments with male mice and found that such GluOC administration induced glucose intolerance, insulin resistance, and adipocyte hypertrophy in those fed a high-fat, high-sucrose diet. In addition, GluOC increased the circulating concentration of testosterone and reduced that of adiponectin in such mice. These phenotypes were not observed in male mice fed a high-fat, high-sucrose diet after orchidectomy, but they were apparent in orchidectomized male mice or intact female mice that were fed such a diet and subjected to continuous testosterone supplementation. Our results thus reveal a sex difference in the effects of GluOC on glucose homeostasis. Given that oral administration of GluOC has been considered a potentially safe and convenient option for the treatment or prevention of metabolic disorders, this sex difference will need to be taken into account in further investigations.

Osteocalcin attenuates high fat diet-induced impairment of endothelium-dependent relaxation through Akt/eNOS-dependent pathway.

BACKGROUND: Recent studies have demonstrated a protective effect of osteocalcin (OCN) on glucose homeostasis and metabolic syndrome. However, its role in vascular function remains unknown. This study investigated the contribution of OCN to the pathogenesis of endothelial dysfunction in the thoracic aorta of apolipoprotein E-deficient (ApoE-KO) mice. METHODS: Eight-week-old ApoE-KO mice were given chow or high fat diet (HFD) for 12 weeks with or without daily intraperitoneal injection of OCN. Intraperitoneal glucose tolerance test (IPGTT), insulin tolerance test (ITT),measurement of serum lipid profiles and blood pressure were carried out. Endothelium-dependent relaxation (EDR) was measured by wire myography. Human umbilical vein endothelial cells (HUVECs) were used to study the role of OCN on eNOS levels in vitro. PI3K inhibitor (LY294002) and Akt inhibitor V were used ex-vivo to determine whether PI3K/Akt/eNOS contributes to the beneficial effect of OCN for the vascular or not. RESULTS: Daily injections of OCN can significantly improve lipid metabolism, glucose tolerance and insulin sensitivity in ApoE-KO mice. In ApoE-KO mice fed with HFD, the OCN-treated mice displayed an improved acetylcholine-stimulated EDR compared to the vehicle-treated group. In addition, compared to vehicle-treated HUVECs, OCN-treated HUVECs displayed increased activation of the Akt-eNOS signaling pathway, as evidenced by significantly higher levels of phosphorylated Akt and eNOS. Furthermore, a similar beneficial effect of OCN on thoracic aorta was observed using ex vivo organ culture of isolated mouse aortic segment. However, this effect was attenuated upon co-incubation with PI3K inhibitor or Akt inhibitor V. CONCLUSIONS: Our study demonstrates that OCN has an endothelial-protective effect in atherosclerosis through mediating the PI3K/Akt/eNOS signaling pathway.

A diarylheptanoid phytoestrogen from Curcuma comosa, 1,7-diphenyl-4,6-heptadien-3-ol, accelerates human osteoblast proliferation and differentiation.

Curcuma comosa Roxb. is ginger-family plant used to relieve menopausal symptoms. Previous work showed that C. comosa extracts protect mice from ovariectomy-induced osteopenia with minimal effects on reproductive organs, and identified the diarylheptanoid (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (DPHD) as the major active component of C. comosa rhizomes. At 1-10µM, DPHD increased differentiation in transformed mouse osteoblasts, but the effect of DPHD on normal bone cells was unknown. We examined the concentration dependency and mechanism of action of DPHD relative to 17ß-estradiol in nontransformed human osteoblasts (h-OB). The h-OB were 10-100 fold more sensitive to DPHD than transformed osteoblasts: DPHD increased h-OB proliferation at 10nM and, at 100nM, activated MAP kinase signaling within 30 min. In long-term differentiation assays, responses of h-OB to DPHD were significant at 10nM, and optimal response in most cases was at 100 nM. At 7-21 days, DPHD accelerated osteoblast differentiation, indicated by alkaline phosphatase activity and osteoblast-specific mRNA production. Effects of DPHD were eliminated by the estrogen receptor antagonist ICI182780. During differentiation, DPHD promoted early expression of osteoblast transcription factors, RUNX2 and osterix. Subsequently, DPHD accelerated production of bone structural genes, including COL1A1 and osteocalcin comparably to 17ß-estradiol. In h-OB, DPHD increased the osteoprotegerin to RANKL ratio and supported mineralization more efficiently than 10nM 17ß-estradiol. We conclude that DPHD promotes human osteoblast function in vitro effectively at nanomolar concentrations, making it a promising compound to protect bone in menopausal women.

The role of vitamin K in soft-tissue calcification.

Seventeen vitamin K-dependent proteins have been identified to date of which several are involved in regulating soft-tissue calcification. Osteocalcin, matrix Gla protein (MGP), and possibly Gla-rich protein are all inhibitors of soft-tissue calcification and need vitamin K-dependent carboxylation for activity. A common characteristic is their low molecular weight, and it has been postulated that their small size is essential for calcification inhibition within tissues. MGP is synthesized by vascular smooth muscle cells and is the most important inhibitor of arterial mineralization currently known. Remarkably, the extrahepatic Gla proteins mentioned are only partly carboxylated in the healthy adult population, suggesting vitamin K insufficiency. Because carboxylation of the most essential Gla proteins is localized in the liver and that of the less essential Gla proteins in the extrahepatic tissues, a transport system has evolved ensuring preferential distribution of dietary vitamin K to the liver when vitamin K is limiting. This is why the first signs of vitamin K insufficiency are seen as undercarboxylation of the extrahepatic Gla proteins. New conformation-specific assays for circulating uncarboxylated MGP were developed; an assay for desphospho-uncarboxylated matrix Gla protein and another assay for total uncarboxylated matrix Gla protein. Circulating desphospho-uncarboxylated matrix Gla protein was found to be predictive of cardiovascular risk and mortality, whereas circulating total uncarboxylated matrix Gla protein was associated with the extent of prevalent arterial calcification. Vitamin K intervention studies have shown that MGP carboxylation can be increased dose dependently, but thus far only 1 study with clinical endpoints has been completed. This study showed maintenance of vascular elasticity during a 3-y supplementation period, with a parallel 12% loss of elasticity in the placebo group. More studies, both in healthy subjects and in patients at risk of vascular calcification, are required before conclusions can be drawn.

Protective effect of green tea polyphenols on bone loss in middle-aged female rats.

UNLABELLED: Recent studies have suggested that green tea polyphenols (GTP) are promising agents for preventing bone loss in women. Findings that GTP supplementation resulted in increased urinary GTP concentrations and bone mass via an increase of antioxidant capacity and/or a decrease of oxidative stress damage suggest a significant role of GTP in bone health of women. INTRODUCTION: Recent studies suggested that green tea polyphenols (GTP) are promising agents for preventing bone loss in women. However, the mechanism related to the possible protective role of GTP in bone loss is not well understood. METHODS: This study evaluated bioavailability, mechanisms, bone mass, and safety of GTP in preventing bone loss in middle-aged rats without (sham, SH) and with ovariectomy (OVX). A 16-week study of 2 (SH vs. OVX) x 3 (no GTP, 0.1% GTP, and 0.5% GTP in drinking water) factorial design using 14-month-old female rats (n = 10/group) was performed. An additional 10 rats in baseline group were euthanized at the beginning of study to provide baseline parameters. RESULTS: There was no difference in femur bone mineral density between baseline and the SH+0.5% GTP group. Ovariectomy resulted in lower values for liver glutathione peroxidase activity, serum estradiol, and bone mineral density. GTP supplementation resulted in increased urinary epigallocatechin and epicatechin concentrations, liver glutathione peroxidase activity and femur bone mineral density, decreased urinary 8-hydroxy-2'-deoxyguanosine and urinary calcium levels, but no effect on serum estradiol and blood chemistry levels. CONCLUSION: We conclude that a bone-protective role of GTP may contribute to an increase of antioxidant capacity and/or a decrease of oxidative stress damage.

Characterisation of cryopreserved cells freshly isolated from human bone marrow.

Osteoblast progenitor cells (OBPCs) isolated from bone marrow have the ability to differentiate into osteoblasts and thus potential therapeutic use to tissue-engineer bone. In order for OBPCs to be available for clinical use a means of storing viable cells is necessary. The aim of this study was to determine whether a simple method of cryopreservation had an effect on osteogenic differentiation or growth of OBPCs isolated from fresh human bone marrow. Stro-1 was used to identify the isolated OBPCs. The osteoblastic potential of the marrow cells was confirmed as culture with osteogenic supplements (OS) significantly increased osteoblastic protein production (alkaline phosphatase (ALP), osteopontin and osteocalcin) compared with standard conditions (P less than 0.05). Ten further marrow aspirates were harvested; each was halved for either cryopreservation or control culture. Primary cultures from both populations formed colonies with recognised OBPC morphology. OS stimulated both cryopreserved and control populations to produce significantly more osteoblastic proteins (P less than 0.05) and there was no significant difference between the increase in osteogenic proteins when cultured with OS (P great than 0.2). The proliferation rate after 5 days in culture was not significantly affected by cryopreservation (P greater than 0.05). It has been suggested that OBPCs are immuno-privileged; so allogenic cells could be implanted into patients for tissue engineering bone without causing a hypersensitivity reaction. Our study demonstrates a method of storage, which allows OBPCs to be available for use without affecting osteoblastic potential or viability.

Short-term reduction in bone markers with high-dose simvastatin.

The effect of statins on bone mass and fracture rates is uncertain. Therefore, we investigated whether statin therapy acutely altered bone turnover as measured by changes in bone serum markers (bone-specific alkaline phosphatase, osteocalcin, and type I collagen N-telopeptide cross-links). Fasting blood samples were obtained from 55 (M/F 39/16) healthy nonsmoking adults (mean +/- standard deviation: age, 50.4+/-7.5 years; body mass index, 27.8+/-4.9 kg/m(2)) with low-density lipoprotein cholesterol concentrations between 3.38-4.90 mmol/l. Subjects were randomized to four possible 8-week treatment regimens: placebo (n =14), pravastatin 40 mg/daily (n =12), simvastatin 20 mg/daily (n =14) or simvastatin 80 mg/daily (n =15). High-dose simvastatin (80 mg/daily) produced a significant reduction in bone-specific alkaline phosphatase as compared with other treatment regimens (p =0.009). However, there were no changes in urinary N-telopeptide cross-links, a sensitive marker of bone resorption. Short-term use of high-dose simvastatin lowers the level of the serum bone marker bone-specific alkaline phosphatase, which suggests the possibility of reduced bone turnover.

Bone tissue composition, dimensions and strength in female rats given an increased dietary level of vitamin A or exposed to 3,3',4, 4',5-pentachlorobiphenyl (PCB126) alone or in combination with vitamin C.

In previous studies we have described structural and functional changes in rat bone tissue caused by 3,3',4,4',5-pentachlorobiphenyl (PCB126). Some of the effects caused by PCB126 resemble those found in vitamin C-deficient rats, as well as those found in rats with a high dietary intake of vitamin A. The present investigation was designed to determine if these PCB126-induced changes could be inhibited by addition of vitamin C to the drinking water and if they could be evoked by vitamin A administration. Five groups of female rats were used in this study, which lasted for 12 weeks. Three of the groups were exposed to PCB126 (total dose 320 microgram/kg, bw), either alone or in combination with vitamin C added to the drinking water (1 and 10 g/l, respectively). One group was given feed with increased level of vitamin A (600000 U/kg pellet) and the fifth group served as controls. Using peripheral quantitative computed tomography (pQCT), it was found that PCB126 increased trabecular density and cortical thickness, but reduced the trabecular area. Furthermore, maximum torque and stiffness of the humerus during torsional testing and serum osteocalcin levels were reduced by PCB126. Of the PCB126 induced effects observed, addition of vitamin C only inhibited the reduction of serum osteocalcin. Like PCB126 vitamin A supplementation increased the inorganic content and the bone density and also reduced the trabecular area and polar moment of inertia but did not increase the cortical thickness or reduce maximum torque, stiffness or serum osteocalcin level. Apparently, the effects induced by PCB126 are not mediated either via decreased vitamin C level or increased vitamin A level.

Vitamin D(3) and vitamin K(1) supplementation of Dutch postmenopausal women with normal and low bone mineral densities: effects on serum 25-hydroxyvitamin D and carboxylated osteocalcin.

OBJECTIVE: Improvement of vitamin D and K status of about 60 -y-old postmenopausal Dutch women. DESIGN: In a randomized study postmenopausal women with normal (T-score >-1; n=96) and low (T-score< or =-1; n=45) bone mineral density (BMD) of the lumbar spine, were supplemented with 350-400 IU vitamin D(3), 80 microg vitamins K(1) vitamins K(1)+D(3), or placebo for 1 y. Serum 25-hydroxyvitamin D [25(OH)D] and percentage carboxylated osteocalcin (%carbOC) were measured at baseline and after 3, 6 and 12 months. RESULTS: Baseline %carbOC of the entire study population was positively correlated with BMD of the lumbar spine and femoral neck. Correspondingly, women with low BMD had lower %carbOC at baseline than women with normal BMD but this difference disappeared after 1 y of supplementation with vitamin K(1) ((mean+/-s.d.) 68+/-11% (95% CI, 64. 5-71.2%) vs 72+/-6% (95% CI, 70.1-72.9%), respectively). One year of supplementation with vitamin D(3) showed maximum increases in 25(OH)D of 33+/-29% (95% CI, 24.8-41.8%) and 68+/-58% (95% CI, 50.1-84.6%) in women with normal and low BMD, respectively. During winter, however, a 29% decline in maximum 25(OH)D levels was not prevented in women with low BMD. CONCLUSION: Daily supplementation of Dutch postmenopausal women with >400 IU vitamin D(3) is indicated to prevent a winter decline in 25(OH)D and to control serum parathyroid hormone levels. Daily supplementation with 80 microg vitamin K(1) seems to be necessary to reach premenopausal %carbOC levels. A stimulatory effect of calcium and/or vitamin D on %carbOC cannot be excluded. European Journal of Clinical Nutrition (2000) 54, 626-631.

Msx-2/Hox 8.1: a transcriptional regulator of the rat osteocalcin promoter.

We recently defined an element (ACTAATTGG) within the rat osteocalcin (OC) promoter at -84 to -92 which provides approximately 70% of basal promoter activity in osteoblastic cell lines and binds a specific nuclear factor found in OC-producing ROS 17/2.8 osteosarcoma cells. Since this element closely resembles the recently described Msx-1 (Hox 7.1) homeodomain DNA binding cognate, we examined rodent osteoblastic cells lines for expression of Msx homeodomain-encoding messages. We have found and cloned a cDNA for rat Msx-2 (Hox 8.1) from a ROS 17/2.8 library and detect high levels of expression in various osteoblastic cell lines (ROS 17/2.8, RCT3, RCT1) as well as in culture passage 3 neonatal rat calvarial osteoblastic cells. Little to no expression was detected in phenotypically immature MC3T3E1 osteoblastic cells or in a variety of nonosteoblastic (ROS 25/1, C2C12, TRAB 11) mesenchymal cell lines. Dexamethasone (DEX) down-regulates Msx-2 message levels in both RCT3 and ROS 17/2.8 cells. Recombinant rat Msx-2 homeodomain expressed in Escherichia coli as a glutathione-S-transferase fusion protein binds to the rat OC promoter region -74 to -100 as determined by gel shift analysis. Recognition is dependent upon the intact ACTAATTGG motif at -84 to -92. In transient cotransfection assays using MC3T3E1 cells (which expresses very little or no endogenous Msx-2), Msx-2 suppresses the rat OC promoter 2- to 3-fold via the Msx-2 binding motif at -84 to -92. However, in ROS 17/2.8 cells, where a high level of endogenous Msx-2 mRNA is present, expression of exogenous Msx-2 does not suppress the rat OC promoter; surprisingly, Msx-2 further augments basal promoter activity by approximately 50-70%, again dependent upon the ACTAATTGG motif at -84 to -92. These data directly demonstrate that the Msx-2 homeodomain binds the rat OC promoter and that Msx-2 can act as a sequence-specific transcriptional regulator of the rat OC promoter in cultured osteoblastic cell lines. This activity is dependent upon the specific osteoblastic cellular context, similar to previous observations in nonosseous systems with other homeodomain transcription factors. These data suggest that Msx-2 may play a role in the transcriptional regulation of the osteoblast phenotype during development in the morphogenetic fields where it is expressed.