Formation Markers Bone Alkaline Phosphatase Research Articles

The tardigrade-derived mitochondrial abundant heat soluble protein improves adipose-derived stem cell survival against representative stressors

Human adipose-derived stem cell (ASC) grafts have emerged as a powerful tool in regenerative medicine. However, ASC therapeutic potential is hindered by stressors throughout their use. Here we demonstrate the transgenic expression of the tardigrade-derived mitochondrial abundant heat soluble (MAHS) protein for improved ASC resistance to metabolic, mitochondrial, and injection shear stress. In vitro, MAHS-expressing ASCs demonstrate up to 61% increased cell survival following 72 h of incubation in phosphate buffered saline containing 20% media. Following up to 3.5% DMSO exposure for up to 72 h, a 14–49% increase in MAHS-expressing ASC survival was observed. Further, MAHS expression in ASCs is associated with up to 39% improved cell viability following injection through clinically relevant 27-, 32-, and 34-gauge needles. Our results reveal that MAHS expression in ASCs supports survival in response to a variety of common stressors associated with regenerative therapies, thereby motivating further investigation into MAHS as an agent for stem cell stress resistance. However, differentiation capacity in MAHS-expressing ASCs appears to be skewed in favor of osteogenesis over adipogenesis. Specifically, activity of the early bone formation marker alkaline phosphatase is increased by 74% in MAHS-expressing ASCs following 14 days in osteogenic media. Conversely, positive area of the neutral lipid droplet marker BODIPY is decreased by up to 10% in MAHS-transgenic ASCs following 14 days in adipogenic media. Interestingly, media supplementation with up to 40 mM glucose is sufficient to restore adipogenic differentiation within 14 days, prompting further analysis of mechanisms underlying interference between MAHS and differentiation processes.

Clostridium butyricum Can Promote Bone Development by Regulating Lymphocyte Function in Layer Pullets.

Bone health problems are a serious threat to laying hens; microbiome-based therapies, which are harmless and inexpensive, may be an effective solution for bone health problems. Here, we examined the impacts of supplementation with Clostridium butyricum (CB) on bone and immune homeostasis in pullets. The results of in vivo experiments showed that feeding the pullets CB was beneficial to the development of the tibia and upregulated the levels of the bone formation marker alkaline phosphatase and the marker gene runt-related transcription factor 2 (RUNX2). For the immune system, CB treatment significantly upregulated IL-10 expression and significantly increased the proportion of T regulatory (Treg) cells in the spleen and peripheral blood lymphocytes. In the in vitro test, adding CB culture supernatant or butyrate to the osteoblast culture system showed no significant effects on osteoblast bone formation, while adding lymphocyte culture supernatant significantly promoted bone formation. In addition, culture supernatants supplemented with treated lymphocytes (pretreated with CB culture supernatants) stimulated higher levels of bone formation. In sum, the addition of CB improved bone health by modulating cytokine expression and the ratio of Treg cells in the immune systems of layer pullets. Additionally, in vitro CB could promote the bone formation of laying hen osteoblasts through the mediation of lymphocytes.

Nox4 expression in osteo-progenitors controls bone development in mice during early life

Tightly regulated and cell-specific NADPH-oxidases (Nox) represent one of the major sources of reactive oxygen species (ROS) signaling molecules that are involved in tissue development and stem cell self-renewal. We have characterized the role of Nox4 in osteo-progenitors during postnatal bone development. Nox4 expression in bone and ROS generation were increased during early osteoblast differentiation and bone development. Stromal osteoblastic cell self-renewal, proliferation and ROS production were significantly lower in samples from whole-body Nox4 knockout mice (Nox4-/-) and conditional knockout (CKO) mice with depletion of Nox4 in the limb bud mesenchyme compared with those from control mice (Nox4fl/fl), but they were reversed after 9 passages. In both sexes, bone volume, trabecular number and bone mineral density were significantly lower in 3-week old CKO and Nox4-/- mice compared with Nox4fl/fl controls. This was reflected in serum levels of bone formation markers alkaline phosphatase (ALP) and procollagen 1 intact N-terminal propeptide (P1NP). However, under-developed bone formation in 3-week old CKO and Nox4-/- mice quickly caught up to levels of control mice by 6-week of age, remained no different at 13-week of age, and was reversed in 32-week old male mice. Osteoclastogenesis showed no differences among groups, however, CTX1 reflecting osteoclast activity was significantly higher in 3-week old male CKO and Nox4-/- mice compared with control mice, and significantly lower in 32-week old Nox4-/- mice compared with control mice. These data suggest that Nox4 expression and ROS signaling in bone and osteoblastic cells coordinately play an important role in osteoblast differentiation, proliferation and maturation.

The Effect of BMI, Age, Gender, and Pubertal Stage on Bone Turnover Markers in Chinese Children and Adolescents

ObjectivesTo ascertain the associations of serum bone turnover markers (BTMs) levels with body mass index (BMI) in Chinese children and adolescents, and whether the influence of BMI, age, pubertal stage on BTMs varied by gender.MethodsA total of 500 students (180 controls and 320 children and adolescents with overweight/obesity) aged 9–14 years were randomly selected from the Chinese National Survey on Students Constitution and Health Cohort. Serum levels of BTMs, including bone formation marker bone alkaline phosphatase (BAP), collagen type 1 C-terminal propeptide (CICP), and bone resorption markers C-terminal telopeptide of type-I collagen (CTX) were determined by commercial enzyme-linked immunosorbent assay kits. The associations among BMI, age, gender, pubertal stage, and BTMs were analyzed.ResultsSerum levels of CICP and CTX in overweight/obese children and adolescents were lower than those in controls (p<0.05). Moreover, after subgroup analysis stratified by gender, the decreased serum CICP and CTX levels in overweight/obese children and adolescents were observed only in boys (p<0.05). After adjustment of age and pubertal stage, there was a negative correlation between serum BAP and BMI in both boys and girls (p<0.05). However, the correlations between serum CICP, CTX levels, and BMI were significant in boys but not in girls. Serum BAP and CICP levels were independently correlated with BMI, age, gender, and pubertal stage, while CTX levels were independently correlated with BMI, age, and gender (p<0.05). BAP, CICP, and CTX levels showed a clear age, gender, and pubertal stage dependence with significantly higher values in boys (p<0.05).ConclusionsOur findings support the associations between serum BTMs levels and BMI in Chinese children and adolescents, and suggest age, gender, and pubertal stage differences in this relationship that warrant future studies.

Role of teriparatide (rh PTH) in fracture healing of osteoporotic patient

Introduction: Osteoporosis a disease where decreased bone strength increases the risk of a fracture. It is the most common reason for fracture among the elderly. Osteoporosis, an imbalance between bone resorption and bone formation. The diagnosis of osteoporosis can be made using conventional radiography and by measuring the bone mineral density. Osteoporosis is diagnosed when the bone mineral density is less than or equal to 2.5 standard deviations below that of a young reference. Teriparatide, a recombinant form of parathyroid hormone (identical to a portion of human parathyroid hormone (PTH)), intermittent use activates osteoblasts more than osteoclasts, which leads to an overall increase in bone turnover. Teriparatide is the only anabolic agent (i.e., bone growing) indicated for use in postmenopausal women with osteoporosis at a high risk for fracture or with a history of osteoporotic fracture, patients with multiple risk factors for fracture. It has been FDA-approved since 2002. Materials and Methods: Over duration of october 2016 to march 2018, 60 patients who have fracture with osteoporosis admitted in S.N. Medical College, Agra. Patient suspected to have osteoporosis based on conventional radiography. Selected patients in the study undergone confirmation of osteoporosis by dual energy absorptiometry and those who are below 2.5 standard deviation are kept in study. Patients fitting into inclusion criteria would form the study group. Data collected by interviews, observation of clinical and radiological findings. 60 patients divided in two groups as cases and controls. Cases are subjected to teriparatide therapy and controls given placebo. Results: Bone formation marker alkaline phosphatase were 150% above baseline after 8 weeks in the teriparatide-treated patient. At 8 weeks, approximately 91.037% in the teriparatide group showed improved healing of osteoporotic fracture compared to 57.14% in the placebo group. Conclusion: Our findings suggest that teriparatide provide selective advantages to fracture healing or functional recovery in the management of osteoporotic fractures. Teriparatide effective in accelerating and increasing the rate of fracture healing. However, more randomized controlled trials are needed to evaluate with certainty the impacts of Teriparatideosteoanabolic role in fracture healing to decide on incorporate this drug as a standard option for conservative management of osteoporotic fracture.

Reference values for bone metabolism in a Japanese cohort survey randomly sampled from a basic elderly resident registry

The aim of this study was to provide definitive reference values for bone mineral density (BMD) and bone turnover markers in the general elderly population. Registered citizens of 50 to 89 years old were targeted for this survey. After random sampling from the resident registry of Obuse town, we established eight groups based on age (50 s, 60 s, 70 s, and 80 s) and gender. A total of 411 people were enrolled. We used a dual-energy x-ray absorptiometry device to measure and evaluate BMD. The bone formation marker bone alkaline phosphatase (BAP) was measured as a bone turnover marker. Bone quality marker pentosidine, and bone resorption markers including urinary total deoxypyridinoline (DPD), tartrate-resistant acid phosphatase 5b (TRACP-5b), 25-hydroxyvitamin D (25[OH]D), and whole parathyroid hormone (PTH) were also measured as bone turnover markers. Sixty-three people (15.3%) were diagnosed as osteoporosis. BMD decreased with age in the femoral neck and total hip. On the other hand, there was no characteristic change with age in the lumber spine. As for bone markers, pentosidine and DPD increased with aging, although 25(OH)D, whole PTH, and BAP showed no characteristic associations with gender and aging. In terms of the relationship between low BMD and bone markers, there was a significant independent association between low BMD and TRACP-5b in females. In conclusions, hip BMD decreased with aging in men and women. However, there was no characteristic decline with aging in the lumbar spine. All bone markers showed no significant independent characteristics associated with age or gender in a multivariate analysis model, except for a significant association between low BMD and TRACP-5b in females. TRACP-5b was a potentially useful marker for the detection of low BMD.

Relationship between fluoride exposure, orthopedic injuries and bone formation markers in patients with coal-burning fluorosis

Chronic exposure to fluoride is a public health problem worldwide. We explored the relationship between fluoride exposure, orthopedic injuries and bone formation markers alkaline phosphatase (ALP), bone Gla protein (BGP) in participants with coal-burning fluorosis in Hehua Village (coal-burning fluorosis endemic area) in Zhijin County of Guizhou Province and Zhangguan Village (non-fluoride contaminated area) in Anshun City of Guizhou Province. Environmental samples were collected and fluoride contents were examined using a fluoride ion-selective electrode. Dental fluorosis and skeletal fluorosis of 295 participants from Hehua Village and 85 participants from Zhangguan Village were diagnosed with informed consent. Urinary samples and peripheral blood samples were collected from the participants to determine urinary fluoride (UF), ALP acti-vity, and BGP content. The results showed that fluoride contents in rice, pepper, corn, drinking water, clay, vegetable-grown soil, coal and indoor and outdoor air were significantly higher than those in the control area, but lower than the previously reported values. With the increases of UF concentration, the ALP activity and BGP content significantly increased, the severity of skeletal fluorosis was greater, but with no significant changes in dental fluorosis. There was positive correlation between the severity of skeletal fluorosis and ALP activity, BGP content. These results indicated that low fluoride exposure could cause orthopedic injuries. ALP and BGP could be used to eva-luate the bone turnover in patients with skeletal fluorosis, which would be useful in the auxiliary diagnosis and therapeutic evaluation of skeletal fluorosis.

Depletion and repletion of fruit and vegetable intake alters serum bone turnover markers: a 28-week single-arm experimental feeding intervention

This study was designed to evaluate the effects of elevated fruit and vegetable intake on bone turnover markers. In all, twenty-nine subjects (nine male and twenty female, with a mean age of 32·1 (sem 2·5) years) participated in a 28-week single-arm experimental feeding intervention trial and consumed a prescribed low-fruit and vegetable diet for 6 weeks (depletion-1), a provided high-fruit and vegetable diet for 8 weeks (fruit: 360-560 g; vegetables: 450-705 g), another prescribed low-fruit and vegetable diet for 6 weeks (depletion-2) and then their usual diets for 8 weeks (repletion). Serum bone-related biomarkers were analysed with commercial ELISA kits. Plasma carotenoid levels decreased as a result of the depletion phase and increased with the high-fruit and vegetable diet. Compared with the baseline, depletion-1 resulted in higher serum bone resorption marker C-terminal telopeptide of type 1 collagen (CTX) and lower bone formation marker alkaline phosphatase (BAP) (CTX, 0·68 (sem 0·05) v. 0·97 (sem 0·08) ng/ml and BAP, 10·7 (sem 0·7) v. 9·5 (sem 0·8) µg/l for the baseline and the depletion-1, respectively, P<0·05). High intake of fruit and vegetables decreased serum CTX (P<0·05) to 0·60 (sem 0·04) ng/ml and increased serum BAP to 11·3 (sem 0·7) µg/l (P<0·05), compared with the depletion-1 phase. Serum concentrations of CTX were inversely correlated and those of BAP were positively correlated with blood lycopene. These data show that increased fruit and vegetable consumption at or above federal dietary guidance may be beneficial to bone health.

Therapeutic Effect of Strontium Ranelate on Bone in Chemotherapy-Induced Osteopenic Rats via Increased Bone Volume and Reduced Bone Loss.

The purpose of this study was to determine whether treatment with strontium ranelate (SrR) can restore bone mass and strength at a skeletal site with established osteopenia in chemotherapy-induced rats. Forty-five Sprague-Dawley male rats were randomly assigned to three study groups (n = 15 rats per group): normal rats (control group), osteopenic rats (chemo group), and osteopenic rats with SrR (chemo-SrR group). We chose a male rat model that mimicked a clinical setting by weekly intravenous injection of cyclophosphamide at 20mg/kg. The control and chemo groups were maintained without treatment, and the chemo-SrR group was intragastrically administered strontium ranelate at a dosage of 900mg/kg/day. All animals were fed a standard laboratory diet, and blood samples were collected for biochemical analysis. After 12weeks of treatment, micro-CT, biomechanical testing, and histomorphometry were examined. In addition, bone samples were obtained to evaluate the content of mineral substances in bones. SrR treatment of chemo rats significantly increased tibial trabecular bone volume, trabecular thickness, and BV/TV. Serum levels of the bone formation marker alkaline phosphatase (ALP) in the SrR group were significantly higher than those in the chemo animals, which was accompanied by an increase in the bone mineral content, bone calcium and phosphate, as well as reduced serum Ca and P concentrations. The serum level of tartrate-resistant acid phosphatase (TRAP) in the SrR treatment group showed no obvious changes. Histomorphological analyses revealed that chemotherapy resulted in decreased osteoclast number, which may be due to the inhibition of bone turnover. However, SrR treatment enhanced the number of osteoblasts while restoring bone mass and improving bone strength in chemo rats. Therefore, the results of this study indicate that SrR treatment has a positive effect on bone in chemotherapy-induced osteoporosis.

Serum bone alkaline phosphatase is a useful marker to evaluate lumbar bone mineral density in Japanese postmenopausal osteoporotic women during denosumab treatment.

A worldwide health concern, osteoporosis (OP), increases the risk of bone fracture and results in morbidity. This study examined whether the representative bone absorption marker serum tartrate-resistant acid phosphatase 5b (TRACP-5b) or bone formation marker bone alkaline phosphatase (BAP) could estimate primary OP status and denosumab efficacy in a real-world setting. We retrospectively enrolled 114 female postmenopausal primary OP patients in Japan. Values and percent changes in TRACP-5b, BAP, lumbar 1–4 bone mineral density (L-BMD), and total hip BMD (H-BMD) were assessed before treatment and at 4, 8, and 12 months of therapy to identify the correlations between the percent changes in bone metabolic markers and BMD. We also established two sets of subgroups based on the upper limits of reference values in Japan for serum: TRACP-5b (<420 mU/dL) and (≥420 mU/dL) and BAP (<14.5 µg/L) and (≥14.5 µg/L). Negative correlations were observed for the percent changes of TRACP-5b and H-BMD at 4 months (r=−0.3476) and 8 months (r=−0.3880), for the percent changes of BAP and H-BMD at 8 months (r=−0.3354), and for the percent changes of BAP and L-BMD at 12 months (r=−0.3186). We observed a significant difference between the subgroups for the percent changes of L-BMD at 8 months (p=0.013) and 12 months (p=0.004) in BAP values. These results suggest that TRACP-5b and BAP had negative correlations with BMD, and that BAP represented a useful serum marker to evaluate L-BMD during denosumab therapy for OP.

Therapeutic Effect of Cistanoside A on Bone Metabolism of Ovariectomized Mice.

Cistanoside A (Cis A), an active phenylethanoid glycoside isolated from Cistanche deserticola Y. C. Ma, has received our attention because of its possible role in the treatment of osteoporosis. In the present study, we evaluated the effects of Cis A on an ovariectomized (OVX) mice model and investigated its underlying molecular mechanisms of action. After 12 weeks of orally-administrated intervention, Cis A (20, 40 and 80 mg/kg body weight/day) exhibited significant antiosteoporotic effects on OVX mice, evidenced by enhanced bone strength, bone mineral density and improved trabecular bone microarchitecture. Meanwhile, the activities of bone resorption markers, including tartrate-resistant acid phosphatase (TRAP), deoxypyridinoline (DPD) and cathepsin K, were decreased, and the bioactivity of bone formation marker alkaline phosphatase (ALP) was increased. Mechanistically, Cis A inhibited the expression of TNF-receptor associated factor 6 (TRAF6), an upstream molecule that is shared by both nuclear factor kappa-light chain enhancer of activated B cells (NF-κB) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways and subsequently suppressed the levels of receptor activators of nuclear factor kappaB ligand (RANKL), downregulated the expression of NF-κB and upregulated osteoprotegerin (OPG), PI3K and Akt, which means Cis A possessed antiosteoporotic activity in ovariectomized mice via TRAF6-mediated NF-kappaB inactivation and PI3K/Akt activation. Put together, we present novel findings that Cis A, by downregulating TRAF6, coordinates the inhibition of NF-κB and stimulation of PI3K/Akt pathways to promote bone formation and prevent bone resorption. These data demonstrated the potential of Cis A as a promising agent for the treatment of osteoporosis disease.

Long-Term Oral Toxicity and Anti-osteoporotic Effect of Sintered Dicalcium Pyrophosphate in Rat Model of Postmenopausal Osteoporosis

Sintered dicalcium pyrophosphate (SDCP), a synthetic pyrophosphate analog, has shown potential for the management of osteoporosis. The long-term oral toxicity and anti-osteoporotic effect of SDCP in a postmenopausal osteoporosis rat model were evaluated in this study. SDCP was orally administered to bilateral ovariectomized (OVX) Wistar rats at a dose of 0.75 mg/kg daily for 24 weeks following by 2 weeks of observation. There were no abnormal findings in clinical signs of toxicity, food consumption, body weight, blood examination, necropsy, and histological inspection attributable to the ingestion of SDCP. The serum level of type I collagen fragments, a bone resorption marker, decreased in SDCP-treated rats, and the bone formation markers alkaline phosphatase, osteocalcin, and osteopontin significantly decreased. These findings indicate that the bone turnover rate decreased in SDCP-treated animals. Relative to OVX rats, the increase in serum tartrate-resistant acid phosphatase 5b level represents an increase in bony tissues in the SDCP-treated rats. Histological examinations of distal femoral metaphyses further revealed that the ingestion of SDCP improved the trabecular bone architecture and decreased bone porosity. Analysis of limb bone ashes showed a significant increase in bone mineral content. Our results show that SDCP inhibits bone resorption to restore bone mass in OVX rats without deleterious effects, and therefore that SDCP has potential in the management of osteoporosis.

Blueberry Diet Derived 3‐(3‐hydroxyphenyl) Propionic Acid (PPA) Suppresses Osteoblastic Cell Senescence to Promote Bone Accretion in Mice

A blueberry (BB) supplemented diet has been previously shown to significantly stimulate bone formation in rapidly growing male and female rodents. Phenolic acids (PAs) are metabolites derived from polyphenols found in fruits and vegetables as a result of the actions of gut bacteria, and the levels of seven individual PAs were found to be at least 10 times higher in the serum of rats fed a BB‐containing diet compared to those fed a control diet. We have characterized the effects of one such BB‐associated serum PA, 3‐(3‐hydroxyphenyl) propionic acid (PPA), on stimulating osteoblastogenesis. To better understand the mechanistic actions of PPA on bone formation, we administered four doses of PPA (0.1, 0.5, 1 and 5 mg/kg/d) to one‐month‐old female C57BL6/J mice for thirty days. We found that stimulation of osteoblast differentiation and proliferation occurred in PPA treated osteoblastic cells. Using peripheral quantitative CT scan (pQCT) and micro‐CT analyses, we demonstrated differences in bone phenotypes between control and PPA‐treated animals, i.e., bone mass was significantly higher in 1 to 5 mg/kg/d PPA‐treated animals compared to controls (p&lt;0.05). Micro‐CT showed bone volume and trabecular number in 1 mg/kg/d treated animals were 0.14±0.007 and 4.76±0.095 versus 0.12±0.02 and 4.33±0.33 in control animals (p&lt;0.05). The bone formation marker alkaline phosphatase in both serum and bone marrow plasma was dose‐dependently increased in PPA‐treated animals. After aspiration of bone marrow, femur bone protein and RNA were isolated for Western blot and real‐time PCR analysis of signaling transduction. We found that PPA was able to suppress bone senescence signaling as evaluated by measuring senescence‐associated beta‐galactosidase activity, PPARγ, p53 and p21 expression (p&lt;0.05 vs control). In conclusion, blueberry‐derived PPA is capable of altering the mesenchymal stem cell differentiation program and bone cell senescence. Thus, PPA or blueberry‐rich diets merit investigation as potential dietary alternatives to drugs for degenerative bone disorders or to promote bone growth during development. Supported in part by USDA‐ARS Project 6026‐51000‐010‐05S.Support or Funding InformationSupported in part by USDA‐ARS Project 6026‐51000‐010‐05S

Changes in serum vitamin D and PTH values using denosumab with or without bisphosphonate pre-treatment in osteoporotic patients: a short-term study.

BackgroundDenosumab is a fully human monoclonal antibody that inhibits receptor activator of nuclear factor kappa-β ligand (RANKL). Previous reports have shown that denosumab treatment of osteoporotic patients decreases bone resorption and fracture risk, but there have been no clinical studies on changes in bone turnover markers, 1,25(OH)2D3, or parathyroid hormone (PTH) in denosumab therapy with or without bisphosphonate (BP) pre-treatment in Japan.MethodsHere, we report such findings in 22 patients (11 in the denosumab alone group and 11 in the BP pre-treated group) with osteoporosis following 4 months of treatment. Bone metabolism had been inhibited by prior BP administration in the BP pre-treated group.ResultsThe bone resorption markers serum tartrate-resistant acid phosphatase type 5b and urinary type I collagen cross-linked N-telopeptide were significantly decreased from baseline values for the entire study period in both groups. The bone formation marker bone alkaline phosphatase was significantly decreased at 4 months in the denosumab alone group only, and N-terminal propeptide of type 1 procollagen was significantly decreased at 2 and 4 months in the denosumab alone group versus no remarkable change in the BP pre-treated group. In the denosumab alone group, 1,25(OH)2D3 and PTH were significantly increased at 1 week and decreased gradually thereafter, but these did not change notably in the BP pre-treated group.ConclusionsOur results suggest that treatment with denosumab causes a strong inhibitory effect on bone resorption markers and mild inhibitory effect on bone formation markers. 1,25(OH)2D3 and PTH were significantly increased by denosumab but these did not change in the BP pre-treated group.Trial registrationCurrent Controlled Trials NCT02156960. Registered 31 May 2014.

Combination chemotherapy with cyclophosphamide, epirubicin and 5-fluorouracil causes trabecular bone loss, bone marrow cell depletion and marrow adiposity in female rats

The introduction of anthracyclines to adjuvant chemotherapy has increased survival rates among breast cancer patients. Cyclophosphamide, epirubicin and 5-fluorouracil (CEF) combination therapy is now one of the preferred regimens for treating node-positive breast cancer due to better survival with less toxicity involved. Despite the increasing use of CEF, its potential in causing adverse skeletal effects remains unclear. Using a mature female rat model mimicking the clinical setting, this study examined the effects of CEF treatment on bone and bone marrow in long bones. Following six cycles of CEF treatment (weekly intravenous injections of cyclophosphamide at 10mg/kg, epirubicin at 2.5mg/kg and 5-flurouracil at 10mg/kg), a significant reduction in trabecular bone volume was observed at the metaphysis, which was associated with a reduced serum level of bone formation marker alkaline phosphatase (ALP), increased trends of osteoclast density and osteoclast area at the metaphysis, as well as an increased size of osteoclasts being formed from the bone marrow cells ex vivo. Moreover, a severe reduction of bone marrow cellularity was observed following CEF treatment, which was accompanied by an increase in marrow adipose tissue volume. This increase in marrow adiposity was associated with an expansion in adipocyte size but not in marrow adipocyte density. Overall, this study indicates that six cycles of CEF chemotherapy may induce some bone loss and severe bone marrow damage. Mechanisms for CEF-induced bone/bone marrow pathologies and potential preventive strategies warrant further investigation.

Effects of resveratrol supplementation on bone growth in young rats and microarchitecture and remodeling in ageing rats.

Osteoporosis is a highly prevalent skeletal disorder in the elderly that causes serious bone fractures. Peak bone mass achieved at adolescence has been shown to predict bone mass and osteoporosis related risk fracture later in life. Resveratrol, a natural polyphenol compound, may have the potential to promote bone formation and reduce bone resorption. However, it is unclear whether it can aid bone growth and bone mass accumulation during rapid growth and modulate bone metabolism during ageing. Using rat models, the current study investigated the potential effects of resveratrol supplementation during the rapid postnatal growth period and in late adulthood (early ageing) on bone microarchitecture and metabolism. In the growth trial, 4-week-old male hooded Wistar rats on a normal chow diet were given resveratrol (2.5 mg/kg/day) or vehicle control for 5 weeks. In the ageing trial, 6-month-old male hooded Wistar rats were treated with resveratrol (20 mg/kg/day) or vehicle for 3 months. Treatment effects in the tibia were examined by μ-computer tomography (μ-CT) analysis, bone histomorphometric measurements and reverse transcription-polymerase chain reaction (RT-PCR) gene expression analysis. Resveratrol treatment did not affect trabecular bone volume and bone remodeling indices in the youth animal model. Resveratrol supplementation in the early ageing rats tended to decrease trabecular bone volume, Sirt1 gene expression and increased expression of adipogenesis-related genes in bone, all of which were statistically insignificant. However, it decreased osteocalcin expression (p = 0.03). Furthermore, serum levels of bone resorption marker C-terminal telopeptides type I collagen (CTX-1) were significantly elevated in the resveratrol supplementation group (p = 0.02) with no changes observed in serum levels of bone formation marker alkaline phosphatase (ALP). These results in rat models suggest that resveratrol supplementation does not significantly affect bone volume during the rapid growth phase but may potentially have negative effects on male skeleton during early ageing.

ERRγ is not required for skeletal development but is a RUNX2-dependent negative regulator of postnatal bone formation in male mice.

To assess the effects of the orphan nuclear Estrogen receptor-related receptor gamma (ERRγ) deficiency on skeletal development and bone turnover, we utilized an ERRγ global knockout mouse line. While we observed no gross morphological anomalies or difference in skeletal length in newborn mice, by 8 weeks of age ERRγ +/− males but not females exhibited increased trabecular bone, which was further increased by 14 weeks. The increase in trabecular bone was due to an increase in active osteoblasts on the bone surface, without detectable alterations in osteoclast number or activity. Consistent with the histomorphometric results, we observed an increase in gene expression of the bone formation markers alkaline phosphatase (Alp) and bone sialoprotein (Bsp) in bone and increase in serum ALP, but no change in the osteoclast regulators receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) or the resorption marker carboxy-terminal collagen crosslinks (CTX). More colony forming units-alkaline phosphatase and -osteoblast (CFU-ALP, CFU-O respectively) but not CFU-fibroblast (CFU-F) formed in ERRγ +/− versus ERRγ +/+ stromal cell cultures, suggesting that ERRγ negatively regulates osteoblast differentiation and matrix mineralization but not mesenchymal precursor number. By co-immunoprecipitation experiments, we found that ERRγ and RUNX2 interact in an ERRγ DNA binding domain (DBD)-dependent manner. Treatment of post-confluent differentiating bone marrow stromal cell cultures with Runx2 antisense oligonucleotides resulted in a reduction of CFU-ALP/CFU-O in ERRγ +/− but not ERRγ +/+ mice compared to their corresponding sense controls. Our data indicate that ERRγ is not required for skeletal development but is a sex-dependent negative regulator of postnatal bone formation, acting in a RUNX2- and apparently differentiation stage-dependent manner.

Mutation in Osteoactivin Decreases Bone Formation in Vivo and Osteoblast Differentiation in Vitro

We have previously identified osteoactivin (OA), encoded by Gpnmb, as an osteogenic factor that stimulates osteoblast differentiation invitro. To elucidate the importance of OA in osteogenesis, we characterized the skeletal phenotype of a mouse model, DBA/2J (D2J) with a loss-of-function mutation in Gpnmb. Microtomography of D2J mice showed decreased trabecular mass, compared to that in wild-type mice [DBA/2J-Gpnmb(+)/SjJ (D2J/Gpnmb(+))]. Serum analysis showed decreases in OA and the bone-formation markers alkaline phosphatase and osteocalcin in D2J mice. Although D2J mice showed decreased osteoid and mineralization surfaces, their osteoblasts were increased in number, compared to D2J/Gpnmb(+) mice. We then examined the ability of D2J osteoblasts to differentiate in culture, where their differentiation and function were decreased, as evidenced by low alkaline phosphatase activity and matrix mineralization. Quantitative RT-PCR analyses confirmed the decreased expression of differentiation markers in D2J osteoblasts. Invitro, D2J osteoblasts proliferated and survived significantly less, compared to D2J/Gpnmb(+) osteoblasts. Next, we investigated whether mutant OA protein induces endoplasmic reticulum stress in D2J osteoblasts. Neither endoplasmic reticulum stress markers nor endoplasmic reticulum ultrastructure were altered in D2J osteoblasts. Finally, we assessed underlying mechanisms that might alter proliferation of D2J osteoblasts. Interestingly, TGF-β receptors and Smad-2/3 phosphorylation were up-regulated in D2J osteoblasts, suggesting that OA contributes to TGF-β signaling. These data confirm the anabolic role of OA in postnatal bone formation.

Appliance‐induced osteopenia of dentoalveolar bone in the rat: effect of reduced bone strains on serum bone markers and the multifunctional hormone leptin

To understand, in greater detail, the molecular mechanisms regulating the complex relationship between mechanical strain and alveolar bone metabolism during orthodontic treatment, passive cross-arch palatal springs were bonded to the maxillary molars of 6-wk-old rats, which were killed after 4 and 8 d. Outcome measures included serum assays for markers of bone formation and resorption and for the multifunctional hormone leptin, and histomorphometry of the inter-radicular bone. The concentration of the bone-formation marker alkaline phosphatase (ALP) was significantly reduced at both time points in the appliance group, accompanied by a 50% reduction in inter-radicular bone volume; however, osteocalcin (bone Gla protein) levels remained unaffected. Bone collagen deoxypyridinoline (DPD) crosslinks increased 2.3-fold at 4 d only, indicating a transient increase in bone resorption; in contrast, the level of the osteoclast-specific marker, tartrate-resistant acid phosphatase 5b (TRACP 5b), was unchanged. Leptin levels closely paralleled ALP reductions at both time points, suggesting an important role in the mechanostat negative-feedback loop required to normalize bone mass. These data suggest that an orthodontic appliance, in addition to remodeling the periodontal ligament (PDL)-bone interface, may exert unexpected side-effects on the tooth-supporting alveolar bone, and highlights the importance of recognizing that bone strains can have negative, as well as positive, effects on bone mass.

Effects of a reduced calcium, phosphorus and protein intake and of benzoic acid on calcium and phosphorus metabolism of growing pigs

In order to minimise environmental pollution, many pig feeds contain low phosphorus and protein concentrations as well as benzoic acid (BA), an additive which reduces ammonia formation in the slurry. Since both a low P intake and metabolic acidosis compromise bone mineralisation, the effect of a diet with a low concentration of calcium (Ca), phosphorus (P) and crude protein (CP) and the effect of BA on Ca and P metabolism were examined in a 2 × 2 two-factorial experiment using pigs from 13 to 64 kg body weight (BW). Compared to the control piglet and grower diets (8.7 and 6.9 g Ca; 6.9 and 5.3 g P; 172 and 156 g CP per kg, respectively), the intake of the low nutrient piglet and grower diets (5.3 g Ca; 4.3 and 4.0 g P; 154 and 147 g CP per kg, respectively, both supplemented with 1500 U/kg microbial phytase) reduced (P<0.01) Ca and P retention by 27% and 24%, respectively, reduced (P<0.05) the growth rate of the piglets by 7%, and decreased (P<0.05) the bone breaking strength and bone mineral content (P<0.01) by 5% in the animals which were slaughtered at 64 kg BW. Benzoic acid (5 and 10 g per kg piglet and grower diet, respectively) did not influence (P>0.05) the apparent digestibility of Ca, increased the apparent digestibility of P (P<0.05) by 5% and increased the urinary Ca and P output (P<0.01) by 70% and 83%, respectively, but had no effect (P>0.05) on the proportion of ingested Ca and P which was retained. Furthermore, BA increased (P<0.01) the serum activity of the bone formation marker alkaline phosphatase at 25 and 40 kg BW by 17% and 13%, respectively and decreased (P<0.01) the concentration of the bone resorption marker serum crosslaps at 25 kg BW by 12%, implying that BA affected bone metabolism at 25 and 40 kg BW. Since BA neither affected the blood variables at 60 kg nor the bone breaking strength and bone mineral content, any possible negative effect of BA on bone metabolism of the piglets and of the young growing animals thus seems to have disappeared during the last period of the grower period. In conclusion, the slight metabolic acidosis caused by BA had no lasting negative effects on the bones of the growing pigs.