Serum Markers Of Bone Formation Research Articles

Oral daily PTH(1-34) tablets (EB613) in postmenopausal women with low BMD or osteoporosis: a randomized, placebo-controlled, six-month, phase 2 study.

Anabolic treatment is indicated for high and very-high risk patients with osteoporosis, but acceptance is limited because current anabolic medications require subcutaneous injections. The purpose of this study was to assess the effects of a novel orally administered parathyroid hormone (PTH) tablet on serum markers of bone formation [N-terminal propeptide of Type I procollagen (PINP) and osteocalcin (OC)] and bone resorption [crosslinked C-telopeptide (CTX)], bone mineral density (BMD) and safety in postmenopausal women with low BMD or osteoporosis. In this 6-month, double-blind, placebo-controlled study, 161 patients were randomized to oral PTH tablets containing 0.5, 1.0, 1.5, or 2.5mg or placebo daily. Biochemical markers were assessed at 1, 2, 3 and 6months and BMD of lumbar spine, total hip and femoral neck was measured at 6months. Biochemical marker changes were dose dependent with minimal or no effect at the two lowest doses. At the highest dose (2.5mg once daily), serum PINP and OC levels increased 30% within 1month after oral PTH initiation (p < 0.0001), remained elevated through 3months and were back to baseline at 6months. In contrast, serum CTX levels declined 16% and 21% below baseline at 3 and 6months respectively (both p ≤ 0.02). At 6months, 2.5mg tablets increased mean BMD vs placebo of the lumbar spine by 2.7%, total hip by 1.8%, and femoral neck by 2.8% (all p ≤ 0.01). There were no drug-related serious adverse events. The most common adverse events were headache, nausea, and dizziness. In contrast to subcutaneous PTH, the oral PTH tablet appears to increase BMD rapidly by the dual mechanism of stimulating formation and inhibiting bone resorption. This might be the first effective oral anabolic alternative to subcutaneous administration for the treatment of low BMD or osteoporosis.

Lysine-rich rice partially enhanced the growth and development of skeletal system with better skeletal microarchitecture in young rats

Rice is the primary staple food for half of the world's population but is low in lysine content. Previously, we developed transgenic rice with enhanced free lysine content in rice seeds (lysine-rich rice), which was shown safe for consumption and improved the growth in rats. However, the effects of lysine-rich rice on skeletal growth and development remained unknown. In this study, we hypothesized that lysine-rich rice improved skeletal growth and development in weaning rats. Male weaning Sprague–Dawley rats received lysine-rich rice (HFL) diet, wild-type rice (WT) diet, or wild-type rice with various contents of lysine supplementation diet for 70 days. Bone microarchitectures were examined by microcomputed tomography, bone strength was investigated by mechanical test, and dynamics of bone growth were examined by histomorphometric analysis. In addition, we explored the molecular mechanism of lysine and skeletal growth through biochemical testing of growth hormone, bone turnover marker, and amino acid content of rat serum analysis, as well as in a cell culture system. Results indicated that the HFL diet improved rats' bone growth, strength, and microarchitecture compared with the WT diet group. In addition, the HFL diet increased the serum essential amino acids, growth hormone (insulin-like growth factor-1), and bone formation marker concentrations. The cell culture model showed that lysine deficiency reduced insulin-like growth factor-1 and Osterix expression, Akt/mammalian target of rapamycin signaling, and matrix mineralization, and inhibited osteoblast differentiation associated with bone growth. Our findings showed that lysine-rich rice improved skeletal growth and development in weaning rats. A further increase of rice lysine content is highly desirable to fully optimize bone growth and development.

The effects of PPARγ inhibitor on bones and bone marrow fat in aged glucocorticoid-treated female rats

Progressive bone marrow (BM) fat accumulation is a common bone loss characteristic in older populations and glucocorticoid (GC)-induced skeletal destruction that is inversely associated with bone synthesis and directly associated with increased peroxisomal proliferator-activated receptor gamma (PPARγ) expression. PPARγ inhibition is an efficient therapeutic strategy for aged- and GC-related skeletal disorders. This study aimed to evaluate the effect of PPARγ inhibition on aged GC-treated female rats. It was hypothesised that bisphenol A diglycidyl ether (BADGE) could inhibit marrow adiposity and improve osteogenesis by inhibiting PPARγ, thereby preventing GC-induced osteoporosis (GIO). Female Sprague–Dawley rats (n = 32, age = 18 months) were randomly allocated to one of the following groups: (1) control, (2) BADGE (30 mg/kg/day, intraperitoneal), (3) methylprednisolone (MP; 30 mg/kg/day, subcutaneous), and (4) MP + BADGE. After eight weeks of treatment, bone density (BD) and trabecular bone microarchitectures were quantified by micro-computed tomography (CT), and BM adipocytes were quantified by histopathology. Additionally, mRNA and protein expression of adipogenic and osteogenic markers were quantified by reverse transcription-quantitative polymerase chain reaction. Furthermore, serum bone turnover biomarker levels were quantified by enzyme-linked immunosorbent assay. MP treatment led to marrow adipogenesis and bone deterioration. However, rats treated with MP + BADGE showed lower marrow adipogenesis, as indicated by smaller marrow adipocyte diameter, decreased density and area percentages, reduced expression of marrow adipogenic genes and proteins, improved BD and trabecular microarchitectures, increased expression of osteogenic genes and proteins, and higher levels of serum bone formation markers. These results were consistent with the differences observed between control and BADGE mono-treated rats. In conclusion, BADGE treatment attenuates BM adiposity and improves bone formation in aged GC-treated female rats by inhibiting PPARγ. Therefore, PPARγ might be a potential target for treating GIO in older populations.

Preventive effect of Total ethanolic extract of Butea monosperma on bone loss in osteopenic rats

Aim: The intent of the study was to assess the bone conserving effect of Butea monosperma total extract (BTE) prepared from the stem-bark, in ovariectomy (Ovx)-induced bone loss in Sprague Dawley (SD) rats.&#x0D; Methods: Sprague-Dawley rats were ovariectomized (Ovx) and BTE treatment with three different doses (100mg, 250mg and 500mg/kg body weight) commenced a day after Ovx and continued for 12 weeks. Skeletal parameters including trabecular and cortical bone microarchitecture, bone mineral density, bone mineral content, biomechanical strength, bone turnover markers (serum procollagen 1 N-terminal propeptide/ P1NP and cross-linked C-telopeptide of type I collagen/CTX-1), new bone formation rate (periosteal bone formation rate and mineral apposition rate), and expression of osteogenic genes were assessed. Estrogenicity of BTE was assessed by studying uterus at the end of the treatments.&#x0D; Results: Ovx rats treated with BTE prevented the alterations in trabecular and cortical microarchitecture, maintained bone mineral density, bone mineral content, and increased bone biomechanical strength in comparison with Ovx rats. Treatment of BTE also enhanced new bone formation rate, increased the serum bone formation marker (P1NP), decreased the serum bone resorption marker (CTX-1), and upregulated the osteogenic genes in bones. BTE was devoid of estrogen-like effect in uterus.&#x0D; Conclusion: BTE protects against Ovx-induced bone loss by inhibiting bone resorption and increasing new bone formation. The most effective bone conserving dose of BTE was 250mg/kg. This study demonstrates the potential of BTE in preventing/delaying osteoporosis development in postmenopausal women.&#x0D; Keywords: Butea total extract, ovariectomy, bone formation, bone mineral density.

Attenuation of Alzheimer’s brain pathology in 5XFAD mice by PTH1-34, a peptide of parathyroid hormone

BackgroundAlzheimer’s disease (AD) and osteoporosis are two distinct diseases but often occur in the same patient. Their relationship remains poorly understood. Studies using Tg2576 AD animal model demonstrate bone deficits, which precede the brain phenotypes by several months, arguing for the independence of bone deficits on brain degeneration and raising a question if the bone deficits contribute to the AD development. To address this question, we investigated the effects of PTH1-34, a peptide of parathyroid hormone analog and a well-recognized effective anabolic therapy drug for patients with osteoporosis, on 5XFAD animal model.Methods5XFAD mice, an early onset β-amyloid (Aβ)-based AD mouse model, were treated with PTH1-34 intermittently [once daily injection of hPTH1–34 (50 μg/Kg), 5 days/week, starting at 2-month old (MO) for 2–3 month]. Wild type mice (C57BL/6) were used as control. The bone phenotypes were examined by microCT and evaluated by measuring serum bone formation and resorption markers. The AD relevant brain pathology (e.g., Aβ and glial activation) and behaviors were assessed by a combination of immunohistochemical staining analysis, western blots, and behavior tests. Additionally, systemic and brain inflammation were evaluated by serum cytokine array, real-time PCR (qPCR), and RNAscope.ResultsA reduced trabecular, but not cortical, bone mass, accompanied with a decrease in bone formation and an increase in bone resorption, was detected in 5XFAD mice at age of 5/6-month old (MO). Upon PTH1-34 treatments, not only these bone deficits but also Aβ-associated brain pathologies, including Aβ and Aβ deposition levels, dystrophic neurites, glial cell activation, and brain inflammatory cytokines, were all diminished; and the cognitive function was improved. Further studies suggest that PTH1-34 acts on not only osteoblasts in the bone but also astrocytes in the brain, suppressing astrocyte senescence and expression of inflammatory cytokines in 5XFAD mice.ConclusionsThese results suggest that PTH1-34 may act as a senolytic-like drug, reducing systemic and brain inflammation and improving cognitive function, and implicate PTH1-34’s therapeutic potential for patients with not only osteoporosis but also AD.

Kefir peptides ameliorate osteoporosis in AKR1A1 knockout mice with vitamin C deficiency by promoting osteoblastogenesis and inhibiting osteoclastogenesis

The AKR1A1 protein is a member of the aldo-keto reductase superfamily that catalyzes the transformation of D-glucuronate to L-gulonate in the synthesis of L-ascorbic acid (vitamin C, Vit C). We previously demonstrated that AKR1A1 knockout mice (AKR1A1eGFP/eGFP) with Vit C deficiency exhibited aberrant bone formation and osteoporosis. In this study, we aimed to evaluate the osteoprotective effects of kefir peptides (KPs) in AKR1A1eGFP/eGFP mice and uncover the underlying mechanism of KPs in the modulation of bone remodeling. Six male CD-1 mice and 24 male AKR1A1eGFP/eGFP mice were used in this study, in which the AKR1A1eGFP/eGFP mice were randomly divided into four groups (n = 6). KPs treatment for 12 weeks exerted several effects in AKR1A1eGFP/eGFP mice including the reduction of serum proinflammatory cytokines (IL-1β, IL-6, TNF-α), bone resorption markers (CTX-1, RANKL), and the increase of serum bone formation markers (P1NP, OPG, OC). μ-CT analysis indicated that KPs prevented the bone loss in the femurs of AKR1A1eGFP/eGFP mice by significantly increasing the trabecular parameters of bone mineral density, bone volume and bone number. Nanoindentation analysis demonstrated that KPs enhanced the elasticity and hardness of femoral cortical bones in AKR1A1eGFP/eGFP mice. KPs promoted bone marrow mesenchymal stem cells (BMMSCs)-derived osteoblast differentiation and mineralization by upregulating positive regulators of osteoblastogenesis (Runx2, β-catenin, BMP-2, NFATc1). Conversely, KPs inhibited bone marrow macrophages (BMMs)-derived osteoclast differentiation and bone resorption, which was demonstrated by the facts that KPs suppressed RANKL-induced p38, NF-κB, Akt, PLCγ2 and CREB-1 phosphorylation, decreased the nuclear translocation of NFATc1 and c-Fos. Our findings demonstrate the efficacy of KPs in the prevention of osteoporosis in AKR1A1eGFP/eGFP mice and also unveil the dual effects of KPs in osteogenic promotion and osteoclastic inhibition. This study supports the use of KPs as nutritional supplements for the prevention of osteoporosis.

The Consumption of Two Eggs per Day for 12 Weeks Does Not Significantly Improve Biomarkers of Bone Metabolism or Inflammation in College-Aged Females Running Cross Country

ObjectivesThe primary aim of this study was to investigate the effects of increased dietary cholesterol intake through the consumption of two eggs per day on biomarkers of bone metabolism and inflammation in female division one cross country and track athletes.MethodsFifteen female Division I student-athletes from university's women's Cross Country/Track and Field Team (age 20.2 + 1.0 yrs.) participated in this study. Prior to the study, subjects were asked to refrain from all egg-based food products for two weeks. After this initial period, subjects were instructed to consume two eggs per day in the form of their choice for 12 weeks. Fasting venous blood samples were collected in the morning hours (0700–1000) following a 12-hour fast and absence of intense exercise training within the last 24 hrs at baseline and after 12 weeks. Serum was collected and immediately stored at -80°C until analysis. Serum bone formation markers; N-terminal propeptide of type I procollagen (PINP) and osteocalcin (OC) and bone resorption marker, C-terminal of telopeptides of type I procollagen (CTXI), and serum inflammatory biomarkers; tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1 β), and IL-6 were measured using ELISA kits.ResultsThere were no significant changes in any of the bone metabolism biomarkers, but they were trending in a positive direction. Mean serum CTXI levels were slightly lower at 0.36 vs 0.30 ng/mL after the intervention, but not significantly (p = 0.083). Serum OC levels didn't change 0.35 vs 0.38 ng/mL nor did serum PINP levels, 40.89 vs 42.72 ng/mL after the intervention. For the biomarkers of inflammation we did observe a significant increase in serum IL-6, 3.82 vs 4.27 pg/mL (p = 0.037) after the intervention. However, there were no significant changes in TNF-α, 10.27 vs 9.70 pg/mL or IL-1β, 5.97 vs 6.05 pg/mL after the intervention.ConclusionsThe current study shows no substantial changes in serum biomarkers of bone metabolism or inflammation after intervention with consuming two eggs per day for 12 weeks. However, there was a significant increase in IL-6 after the intervention.Funding SourcesSupported in part by an Internal Seed Research Grant from the University of Massachusetts Lowell.

Spatiotemporal responses of trabecular and cortical bone to complete spinal cord injury in skeletally mature rats

ObjectiveCharacterise the spatiotemporal responses of trabecular and cortical bone to complete spinal cord injury (SCI) in the skeletally mature rat in the acute (4-week) period following injury. MethodsThe spinal cord of 5-month old male rats was transected at the T9 level. Outcome measures were assessed using micro-computed tomography, three-point bending and serum markers at 1-, 2-, and 4-weeks post-transection. Comparison was made with time-0 and sham animals. ResultsLower levels of circulating serum bone formation markers and higher bone resorption markers suggested uncoupled bone turnover as early at 1-week post-transection. Micro-computed tomography showed metaphyseal and epiphyseal trabecular bone loss was observed only at 4-weeks post-transection. The bone loss was site-specific with a more severe reduction in trabecular BV/TV observed in the metaphyseal (50%) relative to epiphyseal (19%) region. Metaphyseal trabecular bone exhibited a 54% reduction in connectivity density while the epiphyseal trabecular bone was unaffected. Cortical bone deficits were not seen over the time periods examined. ConclusionsThe study demonstrates that the skeletally mature spinal cord transected rat model replicates the biphasic pattern of osteoporotic changes observed in the human SCI population, providing a relevant model for testing the efficacy of interventions against SCI-induced osteoporosis.

Bone Turnover Markers After Six Nights of Insufficient Sleep and Subsequent Recovery Sleep in Healthy Men.

The goal of this study was to determine the bone turnover marker (BTM) response to insufficient and subsequent recovery sleep, independent of changes in posture, body weight, and physical activity. Healthy men (N = 12) who habitually slept 7-9h/night were admitted to an inpatient sleep laboratory for a baseline 8h/night sleep opportunity followed by six nights of insufficient sleep (5h/night). Diet, physical activity, and posture were controlled. Serum markers of bone formation (osteocalcin, PINP) and resorption (β-CTX) were obtained over 24 h at baseline and on the last night of sleep restriction, and on fasted samples obtained daily while inpatient and five times after discharge over 3weeks. Maximum likelihood estimates in a repeated measures model were used to assess the effect of insufficient and subsequent recovery sleep on BTM levels. There was no statistically or clinically significant change in PINP (p = 0.53), osteocalcin (p = 0.66), or β-CTX (p = 0.10) in response to six nights of insufficient sleep. There were no significant changes in BTMs from the inpatient stay through 3weeks of recovery sleep (all p [Formula: see text] 0.63). On average, body weight was stable during the inpatient stay (Δweight = - 0.55 ± 0.91kg, p = 0.06). No significant changes in serum BTMs were observed after six nights of insufficient or subsequent recovery sleep in young healthy men. Changes in weight and physical activity may be required to observe significant BTM change in response to sleep and circadian disruptions. Clinical Trials Registration Registered at ClinicalTrials.gov (NCT03733483) on November 7, 2018.

Ovariectomy-Induced Dysbiosis May Have a Minor Effect on Bone in Mice.

We determined the bone mineral density (BMD) and the expression of serum bone formation marker (procollagen type I N-terminal propeptide: PINP) and bone resorption marker (C-terminal telopeptide of collagen: CTX) by ELISA to evaluate ovariectomy-induced osteoporosis in ovariectomized (OVX) mice. The intestinal microbiota of the mice was assessed using 16S rRNA gene sequencing. OVX mice exhibited a lower BMD of 87% with higher serum levels of CTX and PINP compared to sham-operated (sham) mice. The cecum microbiome of OVX mice showed lower bacterial diversity than that of sham mice. TNFα mRNA levels in the colon were 1.6 times higher, and zonula occludens-1 mRNA and protein expression were lower in OVX mice than in sham mice, suggesting that ovariectomy induced inflammation and increased intestinal permeability. Next, we used antibiotic treatment followed by fecal microbiota transplantation (FMT) to remodel the gut microbiota in the OVX mice. A decrease in PINP was observed in antibiotic-treated mice, while there was no change in BMD or CTX between mice with and without antibiotic treatment. Oral transplantation of the luminal cecal content of OVX or sham mice to antibiotic-treated mice did not affect the BMD or PINP and CTX expression. Additionally, transplantation of the luminal contents of OVX or sham mice to antibiotic-treated OVX mice had similar effects on BMD, PINP, and CTX. In conclusion, although ovariectomy induces dysbiosis in the colon, the changes in the gut microbiota may only have a minor role in ovariectomy-induced osteoporosis.

25-hydroxycholecalciferol reverses heat induced alterations in bone quality in finisher broilers associated with effects on intestinal integrity and inflammation

BackgroundAlterations in ambient temperature have been associated with multiple detrimental effects on broilers such as intestinal barrier disruption and dysbiosis resulting in systemic inflammation. Inflammation and 25-hydroxycholecalciferol (25-OH-D3) have shown to play a negative and positive role, respectively, in the regulation of bone mass. Hence the potential of 25-OH-D3 in alleviating heat induced bone alterations and its mechanisms was studied.ResultsHeat stress (HS) directly induced a decrease in tibia material properties and bone mass, as demonstrated by lower mineral content, and HS caused a notable increase in intestinal permeability. Treatment with dietary 25-OH-D3 reversed the HS-induced bone loss and barrier leak. Broilers suffering from HS exhibited dysbiosis and increased expression of inflammatory cytokines in the ileum and bone marrow, as well as increased osteoclast number and activity. The changes were prevented by dietary 25-OH-D3 administration. Specifically, dietary 25-OH-D3 addition decreased abundance of B- and T-cells in blood, and the expression of inflammatory cytokines, especially TNF-α, in both the ileum and bone marrow, but did not alter the diversity and population or composition of major bacterial phyla. With regard to bone remodeling, dietary 25-OH-D3 supplementation was linked to a decrease in serum C-terminal cross-linked telopeptide of type I collagen reflecting bone resorption and a concomitant decrement in osteoclast-specific marker genes expression (e.g. cathepsin K), whereas it did not apparently change serum bone formation markers during HS.ConclusionsThese data underscore the damage of HS to intestinal integrity and bone health, as well as that dietary 25-OH-D3 supplementation was identified as a potential therapy for preventing these adverse effects.

Myeloid Lineage Ablation of Phlpp1 Regulates M-CSF Signaling and Tempers Bone Resorption in Female Mice.

Prior work demonstrated that Phlpp1 deficiency alters trabecular bone mass and enhances M-CSF responsiveness, but the cell types and requirement of Phlpp1 for this effect were unclear. To understand the function of Phlpp1 within myeloid lineage cells, we crossed Phlpp1 floxed mice with mice harboring LysM-Cre. Micro-computed tomography of the distal femur of 12-week-old mice revealed a 30% increase in bone volume per total volume of Phlpp1 female conditional knockouts, but we did not observe significant changes within male Phlpp1 cKOLysM mice. Bone histomorphmetry of the proximal tibia further revealed that Phlpp1 cKOLysM females exhibited elevated osteoclast numbers, but conversely had reduced levels of serum markers of bone resorption as compared to littermate controls. Osteoblast number and serum markers of bone formation were unchanged. In vitro assays confirmed that Phlpp1 ablation enhanced osteoclast number and area, but limited bone resorption. Additionally, reconstitution with exogenous Phlpp1 suppressed osteoclast numbers. Dose response assays demonstrated that Phlpp1−/− cells are more responsive to M-CSF, but reconstitution with Phlpp1 abrogated this effect. Furthermore, small molecule-mediated Phlpp inhibition enhanced osteoclast numbers and size. Enhanced phosphorylation of Phlpp substrates—including Akt, ERK1/2, and PKCζ—accompanied these observations. In contrast, actin cytoskeleton disruption occurred within Phlpp inhibitor treated osteoclasts. Moreover, Phlpp inhibition reduced resorption of cells cultured on bovine bone slices in vitro. Our results demonstrate that Phlpp1 deficiency within myeloid lineage cells enhances bone mass by limiting bone resorption while leaving osteoclast numbers intact; moreover, we show that Phlpp1 represses osteoclastogenesis and controls responses to M-CSF.

Effect of TNF inhibitors with bisphosphonates vs bisphosphonates alone on bone mineral density and bone and cartilage biomarkers at 1 year in patients with rheumatoid arthritis: A prospective study.

The present study aimed prospectively to investigate the effect of a combination of tumour necrosis factor inhibitors and bisphosphonates (TNFi with BP) on bone mineral density (BMD) and bone and cartilage biomarkers compared to that of BP alone at 1 year in patients with rheumatoid arthritis (RA). Two groups of patients with RA and osteoporosis were enrolled. One group (37 patients) had already received BP, while the other group (37 patients) had already received TNFi with BP. The serum bone resorption and formation markers, cartilage markers, BMD in the lumbar spine, femoral neck, and distal radius were prospectively investigated at the beginning of the study and at 6 and 12 months. The percentages of change recorded for the various assessment categories were as follows in the TNFi with BP group: (1) tartrate-resistant acid phosphatase-5b had significantly decreased and osteocalcin had increased; (2) matrix metalloproteinase-3 and cartilage oligomeric matrix protein had significantly decreased; and (3) each BMD did not differ significantly between the groups. Our data suggested that TNFi with BP therapy not only suppressed cartilage degradation and bone resorption but also increased bone formation; however, this treatment did not affect the BMD at 1 year.

Depletion of Mature B Cells By Anti CD20-Directed Antibody Induces Trabecular Bone Loss in Young Mice: An Alert for Clinicians?

Targeted B cell therapy by anti-CD20 antibodies has revolutionized the treatment of B cell malignancies, immune cytopenias as well as connective tissue diseases. A growing body of evidence links B cell homeostasis and bone metabolism, including reports demonstrating severe bone loss in transgenic mice lacking IgM+ B cells. Here, we addressed the hypothesis that pharmacological depletion of mature B cells induces bone loss in an age-dependent manner.Ten-week and one-year old C57BL/6J female mice were treated with 150µg and 250µg, respectively, of mouse anti-mouse CD20 (hereafter αCD20), administered in two equal doses, two weeks apart. Four weeks after the first injection, distal femurs and L3 vertebrae (old mice only) were subjected to bone morphometric analysis using µCT. The efficiency of B cell depletion as well as immunophenotyping of the bone marrow (BM) and spleen were assessed by flow cytometry. We used ELISA to measure serum markers of bone formation (N-terminal propeptide of procollagen 1, P1NP)and bone resorption (tartrate-resistant acid phosphatase 5b, TRAcP 5b). The expression of receptor activator of nuclear factor κ-B ligand (RANKL), osteoprotegerin (OPG) and interleukin 7 (IL-7) in both BM containing bone fragment (BM-BF) and spleen was analyzed by means of RT-qPCR. To investigate the osteoclastogenic potential of various BM B cell subsets, total BM cells from wild-type untreated mice were flow-sorted and subjected to an osteoclastogenesis assay in RANKL- and macrophage colony stimulating factor (M-CSF)-containing medium.In young mice with >90% splenic B cell depletion (5 out of 7), αCD20 treatment (compared to diluent controls) induced a significant trabecular bone loss, reflected by 25% decrease in bone mineral density (30.1±2.5 vs 40±3.7 mgHA/cm3), 44% decrease in connectivity density (10.3±1.5 vs 18.9±2.8 mm-3), 13% reduction in trabecular number (2.8±0.13 vs 3.24±0.13 mm-1), and corresponding 14% increase in trabecular separation (p<0.05 for all parameters, n=5-7). However, no effect of αCD20 on bone mass was noted in the old mice (neither in femurs nor in L3 vertebrae, n=6 in each group).αCD20 treatment induced a compensatory increase in BM pro-B cells in young animals (2.8±0.5% vs 1.8±0.1%, p<0.05) which was even more pronounced in old ones. However, only the former exhibited a 2.5-fold increase in M-CSF receptor (CD115) positivity of this population (p<0.05). In that respect, our osteoclastogenic assays showed that among various BM B cell populations, only CD115+ pro-B cells could give rise to functional osteoclasts (18±6.5% OC area vs none). Moreover, serum P1NP levels were significantly lower in the young but not in the old αCD20-treated animals as compared to their corresponding diluent controls (48.2±8.7 ng/mL vs 74.5±4.6 and 27.9±8.2 ng/mL vs 30.3±10.8, respectively), indicating decreased bone formation in the former. αCD20 treatment did not affect the levels of TRAcP 5b, a surrogate marker of the total osteoclast number. Following B cell depletion, the expression of IL-7, known to induce bone loss, was upregulated in both BM-BF and spleen by the magnitude of 37% and 50%, respectively (p<0.05). Yet, the expression of RANKL and OPG in BM-BF and in spleen, were similar in control and in the αCD20 treatment groups.The current study demonstrates for the first time the adverse skeletal outcomes of pharmacological depletion of mature B cells, conceivably fueled by B cell-derived osteoclastogenesis in the presence of decreased bone formation. Since many patients treated with clinically available anti-CD20 antibodies already have other risk factors for bone loss, e.g. female gender and glucocorticoid therapy, these data also have immediate clinical implications. DisclosuresNo relevant conflicts of interest to declare.

A high-fat diet in the presence of vitamin D deficiency status is associated with a negative influence on calcaneal quantitative ultrasound parameters in young adults: a cross-sectional study

Vitamin D deficiency and a high-fat diet are considered health problems worldwide. The aims of this study were to examine the prevalence of vitamin D deficiency/insufficiency in young adults, factors related to the vitamin D status, and the influence of vitamin D deficiency and/or a high-fat diet on bone parameters. Here, we investigated the hypothesis that a high-fat diet in the presence of a vitamin D-deficient status would have a more negative influence on bone parameters than a normal-fat diet with such a status. In the present study, we targeted young Japanese adults aged 21-23 (n = 175). We conducted a diet survey based on 3-day food records, biochemical examination of serum, and quantitative ultrasound measurements at the calcaneus. As a result, the rates of vitamin D deficiency {serum 25-hydroxyvitamin D3 [25(OH)D] concentration less than 20 ng/mL} and insufficiency [serum 25(OH)D concentration less than 30 ng/mL but not less than 20 ng/mL] were 60.6 and 30.9%, respectively. A positive correlation was observed between the serum 25(OH)D level and serum bone-specific alkaline phosphatase level, which is a serum marker of bone formation (r = 0.253, P< .01) or the speed of sound (SOS) as an index of bone density (r = 0.259, P< .01). A negative correlation was observed between the ratio of fat intake to total energy intake (%E) and serum 25(OH)D levels (r = −0.206, P< .01). Furthermore, we revealed that a high-fat diet in the presence of a vitamin D deficient status reduced the SOS parameter compared with a normal-fat diet with a vitamin D-deficient status (P< .05).

Propranolol Promotes Bone Formation and Limits Resorption Through Novel Mechanisms During Anabolic Parathyroid Hormone Treatment in Female C57BL/6J Mice.

Although the nonselective β-blocker, propranolol, improves bone density with parathyroid hormone (PTH) treatment in mice, the mechanism of this effect is unclear. To address this, we used a combination of in vitro and in vivo approaches to address how propranolol influences bone remodeling in the context of PTH treatment. In female C57BL/6J mice, intermittent PTH and propranolol administration had complementary effects in the trabecular bone of the distal femur and fifth lumbar vertebra (L5 ), with combination treatment achieving microarchitectural parameters beyond that of PTH alone. Combined treatment improved the serum bone formation marker, procollagen type 1 N propeptide (P1NP), but did not impact other histomorphometric parameters relating to osteoblast function at the L5 . In vitro, propranolol amplified the acute, PTH-induced, intracellular calcium signal in osteoblast-like cells. The most striking finding, however, was suppression of PTH-induced bone resorption. Despite this, PTH-induced receptor activator of nuclear factor κ-B ligand (RANKL) mRNA and protein levels were unaltered by propranolol, which led us to hypothesize that propranolol could act directly on osteoclasts. Using in situ methods, we found Adrb2 expression in osteoclasts in vivo, suggesting β-blockers may directly impact osteoclasts. Consistent with this, we found propranolol directly suppresses osteoclast differentiation in vitro. Taken together, this work suggests a strong anti-osteoclastic effect of nonselective β-blockers in vivo, indicating that combining propranolol with PTH could be beneficial to patients with extremely low bone density. © 2022 American Society for Bone and Mineral Research (ASBMR).

Effects of systemically administered abaloparatide, an osteoanabolic PTHrP analog, as an adjuvant therapy for spinal fusion in rats.

BackgroundAbaloparatide is a parathyroid hormone receptor agonist that increases bone formation and reduces vertebral and nonvertebral fracture risk in women with postmenopausal osteoporosis. Animal studies indicate abaloparatide stimulates vertebral bone formation and enhances bony bridging and biomechanical stability of fracture calluses.AimsThe current study is evaluating the potential utility for abaloparatide as an adjunct therapy for spinal fusions.Material and MethodsThe effects of 14 or 28 days of daily subcutaneous injections of abaloparatide (20 μg/kg/d) or vehicle were evaluated in 32 male Sprague‐Dawley rats starting 1 day after noninstrumented posterolateral fusion (PLF) with bone autograft. Fusion mass microarchitecture was analyzed by micro‐computed tomography (micro‐CT) and serum markers of bone formation and bone resorption were evaluated. Motion segments were scored in a blinded manner as fused or unfused by postmortem radiography and manual palpation.ResultsAbaloparatide‐treated rats showed higher bone formation (serum osteocalcin) at day 14 and 28 compared with vehicle controls, without increases in the bone resorption marker serum TRACP‐5b. Micro‐CT showed greater trabecular number in fusion masses from the abaloparatide group vs vehicle controls at day 14. Manual palpation and radiography indicated no fusions in either group at day 14, whereas 25% of vehicle‐treated rats and 50% of abaloparatide‐treated rats had bilateral fusion at day 28.Discussion and ConclusionIn summary, this rat PLF model showed that abaloparatide treatment was associated with higher levels of the bone formation marker osteocalcin, improved fusion mass architecture, and a non‐ significant 2‐fold higher fusion rate compared with vehicle.

Dimethandrolone Undecanoate, a Novel, Nonaromatizable Androgen, Increases P1NP in Healthy Men Over 28 Days.

Dimethandrolone undecanoate (DMAU) is being developed as a male contraceptive. Daily oral administration of DMAU, a potent androgen that is not aromatized, markedly suppresses serum testosterone (T) and estradiol (E2) in healthy men. E2 deficiency can increase bone resorption in men. This work aimed to assess changes in bone turnover markers with DMAU administration in a 28-day study. A randomized, double-blind, placebo-controlled study was conducted. This study took place at 2 academic medical centers. Healthy men, age 18 to50 years (n = 81), participated. Men received 0, 100, 200, or 400 mg of oral DMAU for 28 days. Serum C-terminal telopeptide of type I collagen (CTX; bone resorption marker) and procollagen type I amino-terminal propeptide (P1NP; bone formation marker) were measured on days 1 and 28. Changes in bone turnover markers and serum hormones over the treatment period were measured. On day 28, median serum T and E2 were markedly suppressed in all treatment groups vs placebo (P < .001 for both). Percentage change (%) in serum P1NP significantly differed across treatment groups (P = .007): Serum P1NP significantly increased in the 200 mg (5%, interquartile range [IQR] -7% to 27%) and 400 mg (22%, IQR -1% to 40%) groups relative to placebo (-8%, IQR -20% to 0%). Change (%) in serum CTX did not differ between groups (P = .09). DMAU administration for 28 days to healthy men leads to marked suppression of serum T and E2, yet increases P1NP, a serum marker of bone formation. Longer-term studies of the potent androgen DMAU are warranted to determine its impact on bone health in men.

The contribution of serum cortisone and glucocorticoid metabolites to detrimental bone health in patients receiving hydrocortisone therapy

BackgroundGlucocorticoid therapy is the most common cause of iatrogenic osteoporosis. Less is known regarding the effect of glucocorticoids when used as replacement therapy on bone remodelling in patients with adrenal insufficiency. Enhanced intracellular conversion of inactive cortisone to active cortisol, by 11 beta-hydroxysteroid dehydrogenase type 1(11β-HSD1) and other enzymes leading to alterations in glucocorticoid metabolism, may contribute to a deleterious effect on bone health in this patient group.MethodsStudy design: An open crossover prospective study randomizing ten hypopituitary men, with severe ACTH deficiency, to three commonly used hydrocortisone dose regimens.Measurements: Following 6 weeks of each regimen, patients underwent 24-h serum cortisol/cortisone sampling, measurement of bone turnover markers, and a 24-h urine collection for measurement of urinary steroid metabolites by gas chromatography-mass spectrometry (GC-MS). Serum cortisone and cortisol were analysed by liquid chromatography-mass spectrometry (LC-MS).ResultsDose-related and circadian variations in serum cortisone were seen to parallel those for cortisol, indicating conversion of ingested hydrocortisone to cortisone. The median area under the curve (AUC) of serum cortisone was significantly higher in patients on dose A (20 mg/10 mg) [670.5 (IQR 621–809.2)] compared to those on dose C (10 mg/5 mg) [562.8 (IQR 520.1–619.6), p = 0.01]. A negative correlation was observed between serum cortisone and bone formation markers, OC [1–49] (r = − 0.42, p = 0.03), and PINP (r = − 0.49, p = 0.01). There was a negative correlation between the AUC of night-time serum cortisone levels with the bone formation marker, OC [1–49] (r = − 0.41, p = 0.03) but there were no significant correlations between day-time serum cortisone or cortisol with bone turnover markers. There was a negative correlation between total urinary cortisol metabolites and the bone formation markers, PINP (r = − 0.39, p = 0.04), and OC [1–49] (r = − 0.35, p = 0.06).ConclusionSerum cortisol and cortisone and total urinary corticosteroid metabolites are negatively associated with bone turnover markers in patients receiving replacement doses of hydrocortisone, with nocturnal glucocorticoid exposure having a potentially greater influence on bone turnover.Trial registrationIrish Medicines Board Clinical Trial Number – CT900/459/1 and EudraCT Number – 2007-005018-37. Registration date: 07-09-2007.

Atorvastatin promotes bone formation in aged apoE\u2013/\u2013 mice through the Sirt1\u2013Runx2 axis

BackgroundStatins are the most widely used drugs in elderly patients; the most common clinical application of statins is in aged hyperlipemia patients. There are few studies on the effects and mechanisms of statins on bone in elderly mice with hyperlipemia. The study is to examine the effects of atorvastatin on bone phenotypes and metabolism in aged apolipoprotein E-deficient (apoE–/–) mice, and the possible mechanisms involved in these changes.MethodsTwenty-four 60-week-old apoE–/– mice were randomly allocated to two groups. Twelve mice were orally gavaged with atorvastatin (10 mg/kg body weight/day) for 12 weeks; the others served as the control group. Bone mass and skeletal microarchitecture were determined using micro-CT. Bone metabolism was assessed by serum analyses, qRT-PCR, and Western blot. Bone marrow-derived mesenchymal stem cells (BMSCs) from apoE–/– mice were differentiated into osteoblasts and treated with atorvastatin and silent information regulator 1 (Sirt1) inhibitor EX-527.ResultsThe results showed that long-term administration of atorvastatin increases bone mass and improves bone microarchitecture in trabecular bone but not in cortical bone. Furthermore, the serum bone formation marker osteocalcin (OCN) was ameliorated by atorvastatin, whereas the bone resorption marker tartrate-resistant acid phosphatase 5b (Trap5b) did not appear obviously changes after the treatment of atorvastatin. The mRNA expression of Sirt1, runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and OCN in bone tissue were increased after atorvastatin administration. Western blot showed same trend in Sirt1 and Runx2. The in vitro study showed that when BMSCs from apoE–/– mice were pretreated with EX527, the higher expression of Runx2, ALP, and OCN activated by atorvastatin decreased significantly or showed no difference compared with the control. The protein expression of Runx2 showed same trend.ConclusionsAccordingly, the current study validates the hypothesis that atorvastatin can increase bone mass and promote osteogenesis in aged apoE−/− mice by regulating the Sirt1–Runx2 axis.