Low Turnover Osteoporosis Research Articles

Bone biopsy findings in patients receiving long-term bisphosphonate therapy for glucocorticoid-induced osteoporosis.

Bisphosphonates (BPs) have been shown to reduce the incidence of vertebral fractures during the first year or two of glucocorticoid (GC) treatments and are therefore recommended as a first-line treatment for GC-induced osteoporosis (GIO). However, there are theoretical concerns about the long-term use of BPs in low-turnover osteoporosis caused by chronic GC therapy. We analyzed the trabecular microarchitecture, bone metabolism, and material strength of iliac crest bone biopsy samples from 10 female patients with rheumatoid arthritis who received an average of 6.7years of BP therapy for GIO (GIOBP group), compared with those of 10 age- and bone mineral density (BMD)-matched non-rheumatoid arthritis postmenopausal women (reference group). Patients in the GIOBP group had a significantly greater fracture severity index, as calculated from the number and the extent of vertebral fractures compared with the reference patients. Micro-computed tomography analysis showed that the degree of mineralization and trabecular microarchitecture were significantly lower in the GIOBP group than in the reference patients. Patients in the GIOBP group exhibited lower bone contact stiffness, determined by micro-indentation testing, than in the reference group. The contact stiffness of the bone was negatively correlated with the fracture severity index and the daily prednisolone dosage. Immunohistochemistry and serum bone turnover markers showed decreased osteoclastic activity, impaired mineralization, and an increased fraction of empty lacunae in the GIOBP group. Our findings indicate that patients receiving long-term BP for GIO are still at high risk for fragility fractures because of poor bone quality.

Klotho upregulates the interaction between RANK and TRAF6 to facilitate RANKL-induced osteoclastogenesis via the NF-κB signaling pathway.

Backgroundα-Klotho (Klotho) plays a wide range of roles in pathophysiological processes, such as low-turnover osteoporosis observed in klotho mutant mice (kl/kl mice). However, the precise function and underlying mechanism of klotho during osteoclastogenesis are not fully understood. Here, we investigated the effects of klotho on osteoclastogenesis induced by receptor activator of nuclear factor kappa-B ligand (RANKL).MethodsThe effects of klotho deficiency on osteoclastogenesis were explored using kl/kl mice both in vivo and in vitro. In in vitro experiments, lentivirus transfection, real-time quantitative PCR (RT-qPCR) analysis, western blot analysis, immunostaining, RNA-seq analysis, differential pathway analysis, Energy-based protein docking analysis and co-immunoprecipitation were used for deeply investigating the effects of klotho on RANKL-induced Osteoclastogenesis and the underlying mechanism.ResultsWe found that klotho deficiency impaired osteoclastogenesis. Furthermore, in vitro studies revealed that klotho facilitated osteoclastogenesis and upregulated the expression of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) during osteoclastogenesis. Mechanistically, we confirmed that klotho co-localized with nuclear factor kappa B (RANK) and facilitated the interaction between activated RANK and TNFR-associated factor 6 (TRAF6), thus klotho exerts its function in osteoclastogenesis through the activation of the NF-κB signaling pathway.ConclusionsKlotho promotes RANKL-induced osteoclastogenesis through upregulating the interaction between RANK and TARF6, Targeting on klotho may be an attractive therapeutic method for osteopenic diseases.

Bone matrix mineralization increases with age and remains elevated after Teriparatide treatment in WNT1 or PLS3 mutation-related low-turnover osteoporosis: A transiliac bone biopsy study

Bone matrix mineralization increases with age and remains elevated after Teriparatide treatment in WNT1 or PLS3 mutation-related low-turnover osteoporosis: A transiliac bone biopsy study

An evaluation of the rebound phenomenon during denosumab therapy in children with low turnover osteoporosis

An evaluation of the rebound phenomenon during denosumab therapy in children with low turnover osteoporosis

PLS3 sequencing in childhood-onset primary osteoporosis identifies two novel disease-causing variants

SummaryAltogether 95 children with primary bone fragility were screened for variants in PLS3, the gene underlying X-linked osteoporosis. Two children with multiple peripheral and spinal fractures and low BMD had novel disease-causing PLS3 variants. Children with milder phenotypes had no pathogenic variants. PLS3 screening is indicated in childhood-onset primary osteoporosis.IntroductionThe study aimed to determine the role of pathogenic PLS3 variants in children’s bone fragility and to elucidate the associated phenotypic features.MethodsTwo cohorts of children with bone fragility were screened for variants in PLS3, the gene underlying X-linked osteoporosis. Cohort I comprised 31 patients with childhood-onset primary osteoporosis of unknown etiology. Cohort II comprised 64 children who had sustained multiple fractures but were otherwise healthy. Clinical and radiological data were reviewed. Peripheral blood DNA was Sanger sequenced for coding exons and flanking intronic regions of PLS3.ResultsIn two patients of cohort I, where other common genetic causes had been excluded, we identified two novel disease-causing PLS3 variants. Patient 1 was a male with bilateral femoral fractures at 10 years, low BMD (Z-score −4.1; 18 years), and multiple vertebral compression fractures. He had a novel nonsense variant in PLS3. Patient 2 was a girl with multiple long bone and vertebral fractures and low BMD (Z-score −6.6 at 6 years). She had a de novo missense variant in PLS3; whole exome sequencing and array-CGH identified no other genetic causes. Iliac crest bone biopsies confirmed low-turnover osteoporosis in both patients. In cohort II, no pathogenic PLS3 variants were identified in any of the subjects.ConclusionsTwo novel disease-causing variants in PLS3 were identified in a boy and a girl with multiple peripheral and spinal fractures and very low BMD while no pathogenic variants were identified in children with less severe skeletal fragility. PLS3 screening is warranted in male and female patients with childhood-onset primary osteoporosis.

Sheep model for osteoporosis: The effects of peripheral hormone therapy on centrally induced systemic bone loss in an osteoporotic sheep model

Hypothalamic-pituitary disconnection (HPD) leads to low bone turnover followed by bone loss and reduced biomechanical properties in sheep. To investigate the role of peripheral hormones in this centrally induced systemic bone loss model, we planned a hormone replacement experiment. Therefore, estrogen (OHE), thyroxin (OHT) or a combination of both (OHTE) was substituted in ovariectomized HPD sheep, as both hormones are decreased in HPD sheep and are known to have a significant but yet not fully understood impact on bone metabolism. Bone turnover and structural parameters were analyzed in comparison to different control groups – untreated sheep (C), ovariectomized (O) and ovariectomized+HPD sheep (OH). We performed histomorphometric and HR-pQCT analyses nine months after the HPD procedure, as well as biomechanical testing of all ewes studied. In HPD sheep (OH) the low bone turnover led to a significant bone loss. Treatment with thyroxin alone (OHT) mainly increased bone resorption, leading to a further reduction in bone volume. In contrast, the treatment with estrogen alone (OHE) and the combined treatment with estrogen and thyroxin (OHTE) prevented HPD-induced bone loss completely. In conclusion, peripheral hormone substitution was able to prevent HPD-induced low-turnover osteoporosis in sheep. But only the treatment with estrogen alone or in combination with thyroxin was able to completely preserve bone mass and structure. These findings demonstrate the importance of peripheral hormones for a balanced bone remodeling and a physiological bone turnover.

Teriparatide Treatment in Patients With WNT1 or PLS3 Mutation-Related Early-Onset Osteoporosis: A Pilot Study.

We previously identified 2 Finnish families with dominantly inherited, low-turnover osteoporosis caused by mutations in WNT1 or PLS3. This prospective, longitudinal, uncontrolled study was undertaken to evaluate whether these patients respond to teriparatide. We recruited 6 adults (median age, 54 years); 3 with a WNT1 missense mutation, c.652T>G, and 3 with a PLS3 splice mutation, c.73-24T>A, to receive teriparatide 20 μg daily for 24 months. Five patients had previously used bisphosphonates. Outcome measures included lumbar spine and hip bone mineral density (BMD) by dual-energy X-ray absorptiometry, distal radius peripheral quantitative computed tomography, spinal radiography, serum bone turnover markers, paired iliac crest biopsies. All patients showed increases in formation markers procollagen type 1 amino-terminal propeptide (90% to 398%) and osteocalcin (50% to 280%) and in resorption markers cross-linked C-terminal telopeptide of type I collagen (58% to 457%) and tartrate-resistant acid phosphatase 5b (20% to 68%) in first 6 months. Lumbar spine BMD increased 5.2% to 7.9% in 5 patients and femoral neck BMD 2.6% to 7.8% in 4 patients in 24 months. Distal radius cortical volumetric BMD decreased 5.4% to 26.1%. In histomorphometric analyses, osteoid indices increased more consistently in patients with WNT1 vs PLS3 mutation. Eroded surface decreased 44% to 100% in all patients. Adipocyte number increased in 5 patients studied. Patients with WNT1 or PLS3 mutation-related osteoporosis responded to teriparatide treatment. Future studies are needed to evaluate whether observed changes translate to fracture resistance.

Skeletal Characteristics of WNT1 Osteoporosis in Children and Young Adults.

WNT proteins comprise a 19-member glycoprotein family that act in several developmental and regenerative processes. In bone, WNT proteins regulate osteoblast differentiation and maintain bone health by activating the canonical WNT/β-catenin pathway. We reported a heterozygous missense mutation c.652T>G (p.C218G) in WNT1 exon 4 as the cause for severe early-onset, autosomal dominant osteoporosis. The initial study concerned a large Finnish family with 10 affected adults. Here we report clinical findings of the WNT1 osteoporosis in 8 children and young adults (median age 14 years; range 10 to 30 years) in two families, all with the p.C218G mutation in WNT1. Clinical assessments showed no apparent dysmorphia or features similar to typical osteogenesis imperfecta (OI). Biochemistry revealed no changes in parameters of calcium metabolism and bone turnover markers. Fracture frequencies varied, but all subjects had sustained at least one fracture and 4 had a pathological fracture history. Plain radiographs showed osteopenic appearance, loss in vertebral height, and thin diaphyses of the long bones. Bone densitometry showed the BMD to be below normal median in all subjects and the bone mass deficit seemed to be more severe in older participants. Bone histomorphometry revealed a low turnover osteoporosis in 2 subjects at ages 14 and 16 years. These findings are congruent with earlier findings in adult patients and indicate that WNT1 osteoporosis causes significant skeletal changes already in early childhood and impairs bone mass gain during pubertal years. Genetic testing of children or close relatives of affected individuals is recommended for appropriate preventive measures. © 2016 American Society for Bone and Mineral Research.

Parameters of bone health and fracture risk in older female fall victims: what do they tell us?

A common and severe osteoporotic type fracture in older women is a hip fracture. It is not clear whether bone turnover parameters measured in blood can be a useful tool to predict fracture risk in older persons. The aim of the current study was to assess the association between serum vitamin D (25OHD) levels, parathyroid hormone (PTH), total osteocalcin, carboxy-terminal collagen crosslinks (CTX) and hip fractures in older fallers. A single centre, prospective cohort study of bone parameters was carried out in 400 female patients aged > 70 years including 200 with a hip fracture and 200 without fractures, admitted after a fall between January 2005 and December 2007. Serum total osteocalcin levels were significantly lower in the fracture group compared to the non-fracture group (20.4ng/ml vs 26.1ng/ml, respectively, p = 0.01). This finding remained significant after exclusion of the patients on bisphosphonates (p = 0.003). There were no significant differences in 25OHD, PTH or CTX levels between the two groups. In the current study there was an association between the presence of a hip fracture and lower total serum osteocalcin concentrations. This could be indicative of low bone turnover osteoporosis in these women. An association for other bone turnover markers was lacking.

Effect of Locally Applied Fluvastatin in Low-turnover Osteoporosis Model Mouse with Femur Bone Defect

Effect of Locally Applied Fluvastatin in Low-turnover Osteoporosis Model Mouse with Femur Bone Defect

Ursodeoxycholic acid decreases bilirubin-induced osteoblast apoptosis.

Low bone turnover osteoporosis is common in cholestatic diseases. Ursodeoxycholic acid (UDCA) counteracts the damaging effects of bilirubin or lithocholic acid (LCA) on osteoblast viability, proliferation and mineralisation. UDCA is anti-apoptotic in various cell lines, but this effect in bone cells is unknown. Therefore, the consequences of bilirubin and LCA on apoptosis, and whether UDCA has anti-apoptotic effects have been assessed on osteoblasts. Human osteoblasts (hOB) and osteosarcoma cell line (Saos-2) were treated with camptothecin as a pro-apoptotic agent, and UDCA, LCA and bilirubin. Apoptosis was determined by DNA fragmentation, flow cytometry, caspase-3 activity and expression of pro-apoptotic (Bcl-2-associated X protein BAX) and anti-apoptotic (BCL2 and BCL2-like 1 protein, BCL2L) genes. Both LCA (10μM) and bilirubin (50μM) induced apoptosis as indicated by DNA fragmentation (4·7- and 3·7-fold, respectively, P<0·001), caspase-3 activity and flow cytometry in Saos-2 and hOB. UDCA (10μM) reduced the apoptotic effects of camptothecin (0·5μM) by 61%, (P<0·001) and counteracted the apoptotic effects of LCA and bilirubin determined by DNA fragmentation (56% and 60%, respectively, P<0·001), cytometry and caspase-3 activity in Saos-2, with lower effects in hOB. UDCA (10μM) downregulated BAX (75%), upregulated BCL2L (10-fold, P<0·01) genes, and neutralised BAX upregulation (P<0·01) and BCL2L downregulation (P<0·01) induced by LCA and bilirubin. Bilirubin and LCA induce apoptosis in osteoblastic cells. UDCA counteracts the apoptotic consequences of these two substances, and therefore, it may have further beneficial effects on the decreased bone formation in the cholestasis.

Diabetic osteopenia by decreased β-catenin signaling is partly induced by epigenetic derepression of sFRP-4 gene.

In diabetics, methylglyoxal (MG), a glucose-derived metabolite, plays a noxious role by inducing oxidative stress, which causes and exacerbates a series of complications including low-turnover osteoporosis. In the present study, while MG treatment of mouse bone marrow stroma-derived ST2 cells rapidly suppressed the expression of osteotrophic Wnt-targeted genes, including that of osteoprotegerin (OPG, a decoy receptor of the receptor activator of NF-kappaB ligand (RANKL)), it significantly enhanced that of secreted Frizzled-related protein 4 (sFRP-4, a soluble inhibitor of Wnts). On the assumption that upregulated sFRP-4 is a trigger that downregulates Wnt-related genes, we sought out the molecular mechanism whereby oxidative stress enhanced the sFRP-4 gene. Sodium bisulfite sequencing revealed that the sFRP-4 gene was highly methylated around the sFRP-4 gene basic promoter region, but was not altered by MG treatment. Electrophoretic gel motility shift assay showed that two continuous CpG loci located five bases upstream of the TATA-box were, when methylated, a target of methyl CpG binding protein 2 (MeCP2) that was sequestered upon induction of 8-hydroxy-2-deoxyguanosine, a biomarker of oxidative damage to DNA. These in vitro data suggest that MG-derived oxidative stress (not CpG demethylation) epigenetically and rapidly derepress sFRP-4 gene expression. We speculate that under persistent oxidative stress, as in diabetes and during aging, osteopenia and ultimately low-turnover osteoporosis become evident partly due to osteoblastic inactivation by suppressed Wnt signaling of mainly canonical pathways through the derepression of sFRP-4 gene expression.

The Oxytocin–Bone Axis

We recently demonstrated a direct action of oxytocin (OT) on skeletal homeostasis, mainly mediated through stimulation of osteoblasts (OBs) formation and through the reciprocal modulation of osteoclast (OCs) formation and function. Thus, mice lacking the hormone or its receptor develop a low turnover osteoporosis that worsens with age in both sexes. The skeletons of OT (Ot) and OT receptor (Oxtr) null mice display a pronounced decrease in vertebral and femoral trabecular volume. At the cellular level, OBs from Ot KO and Oxtr KO mice exhibit lower mineralization activity and, at the mRNA level, all master genes for osteoblast differentiation are down-regulated. Moreover, OT has dual effects on OCs: it increases osteoclast formation both directly, by activating nuclear factor kB (NFkB) and mitogen-activated protein kinase (MAPK) signalling and, indirectly, through the up-regulation of receptor activator nuclear factor-kappaB ligand synthesis by OBs. On the other hand, it inhibits bone resorption by triggering cytosolic Ca(2+) release and nitric oxide synthesis in mature OCs. OT is locally produced by osteoblasts acting as paracrine-autocrine regulators of bone formation modulated by oestrogens. The oestrogen signal involved in this feedforward circuit is nongenomic because it requires an intact MAPK kinase signal transduction pathway, instead of the classical nuclear translocation of oestrogen receptor. The ability of oestrogen to increase bone mass in vivo is to some extent OXTR-dependent. Thus, Oxtr KO mice injected 17β-oestradiol did not show any effects on bone formation parameters, whereas the same treatment increases trabecular and cortical bone in wild-type mice. An intact OT autocrine-paracrine circuit appears to be essential for optimal skeletal remodelling.

Metaphyseal fracture healing in a sheep model of low turnover osteoporosis induced by hypothalamic–pituitary disconnection (HPD)

We recently established a large animal model of osteoporosis in sheep using hypothalamic-pituitary disconnection (HPD). As central regulation is important for bone metabolism, HPD-sheep develop severe osteoporosis because of low bone turnover. In this study we investigated metaphyseal fracture healing in HPD-sheep. To elucidate potential pathomechanisms, we included a treatment group receiving thyroxine T4 and 17β-estradiol. Because clinically osteoporotic fractures often occur in the bone metaphysis, HPD-sheep and healthy controls received an osteotomy in the distal femoral condyle. Half of the HPD-sheep were systemically treated with thyroxine T4 and 17β-estradiol during the healing period. Fracture healing was evaluated after 8 weeks using pQCT, µCT, and histomorphometrical analysis. Bone mineral density (BMD) and bone volume/total volume (BV/TV) were considerably reduced by 30% and 36%, respectively, in the osteotomy gap of the HPD-sheep compared to healthy sheep. Histomorphometry also revealed a decreased amount of newly formed bone (-29%) and some remaining cartilage in the HPD-group, suggesting that HPD disturbed fracture healing. Thyroxine T4 and 17β-estradiol substitution considerably improved bone healing in the HPD-sheep. Our results indicate that fracture healing requires central regulation and that thyroxine T4 and 17β-estradiol contribute to the complex pathomechanisms of delayed metaphyseal bone healing in HPD-sheep.

THU0079 Anti-IL-6 Receptor Antibody Suppresses Systemic Bone Loss by not Only Normalizing Bone Resorption but also Enhancing Bone Formation in a Mouse Model of Collagen-Induced Arthritis

BackgroundPatients with rheumatoid arthritis (RA) have a high risk of osteoporosis and osteoporotic fracture. In primary osteoporosis (either high bone turnover osteoporosis [type I] or low bone turnover osteoporosis [type...

Metaphyseal fracture healing in a sheep model of low turnover osteoporosis induced by hypothalamic[ndash]pituitary disconnection

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

Sheep model for osteoporosis: Sustainability and biomechanical relevance of low turnover osteoporosis induced by hypothalamic–pituitary disconnection

Hypothalamo-pituitary disconnection (HPD) leads to low bone turnover and osteoporosis in sheep. To determine the sustainability of bone loss and its biomechanical relevance, we studied HPD-sheep 24 months after surgery (HPD + OVX-24) in comparison to untreated control (Control), ovariectomized sheep (OVX), and sheep 12 months after HPD (HPD + OVX-12). We performed histomorphometric, HR-pQCT, and qBEI analyses, as well as biomechanical testing of all ewes studied. Twenty-four months after HPD, histomorphometric analyses of the iliac crest showed a significant reduction of BV/TV by 60% in comparison to Control. Cortical thickness of the femora measured by HR-pQCT did not change between 12 and 24 months after HPD but remained decreased by 30%. These structural changes were caused by a persisting depression of osteoblast and osteoclast cellular activity. Biomechanical testing of the femora showed a significant reduction of bending strength, whereas calcium content and distribution was found to be unchanged. In conclusion, HPD surgery leads to a persisting low turnover status with negative turnover balance in sheep followed by dramatic cortical and trabecular bone loss with consequent biomechanical impairment.

Effect of Low-Intensity Pulsed Ultrasound on Bone-Healing Process in Murine Low-Turnover Osteoporosis Model

The effects of postmenopausal osteoporosis for bone defect healing processes have already been reported, but not for senile osteoporosis caused by low-turnover metabolism. Low-Intensity-Pulsed Ultrasound (LIPUS) is known to promote bone defect healing in high-turnover osteoporosis an animal model. The aim of this study was to investigate the effect of LIPUS on the bone-healing process in a low-turnover osteoporosis model using the Senescence-Accelerated Mouse Prone 6 (SAMP6) strain of mice. Twenty-week-old SAMP6 and Senescence-Accelerated Mouse Resistant (SAMR1) mice were used as senile osteoporosis and normal aging models, respectively. Bone defects (diameter, 0.9 mm) were created in the both SAMP6 and SAMR1 femurs. At 7days after surgery, the LIPUS irradiation groups of SAMR1 (R1LG) and SAMP6 (P6LG) were exposed to LIPUS (1.0 MHz, 320 mW, 15 min/day) for 6 days. The non-irradiation groups of SAMR1 (R1CG) and SAMP6 (P6CG) were used as controls. All groups were sacrificed at 14 days after creation of bone defects. Radiological analysis, histological evaluation and immunohistochemical staining for osteocalcin (OC) were performed. From the radiological evaluation, the new bone of defected area in SAMR1 group showed cortical bone-like structure, but that in the SAMP6 group showed trabecular bone-like structures. The increase in bone area in P6LG was greater than that in P6CG according to chronological change analysis using X-ray micro-CT (p < 0.01). Histological analysis revealed outward new bone formation originating in the periosteum in P6LG. Positive reaction for OC was localized on the surface of new bone in P6CG, whereas that in P6LG was observed over the whole region of new bone, from the outer to the bone marrow side. These results showed that LIPUS accelerates healing on low-turnover osteoporosis by promoting bone formation from periosteum and supplementing reduced bone formation from bone marrow.

Low turnover osteoporosis in sheep induced by hypothalamic-pituitary disconnection

The hypothalamus is of critical importance in regulating bone remodeling. This is underscored by the fact that intracerebroventricular-application of leptin in ewe leads to osteopenia. As a large animal model of osteoporosis, this approach has some limitations, such as high technical expenditure and running costs. Therefore we asked if a surgical ablation of the leptin signaling axis would have the same effects and would thereby be a more useful model. We analyzed the bone phenotype of ewe after surgical hypothalamo-pituitary disconnection (HPD + OVX) as compared to control ewe (OVX) after 3 and 12 months. Analyses included histomorphometric characterization, micro-CT and measurement of bone turnover parameters. Already 3 months after HPD we found osteopenic ewe with a significantly decreased bone formation (69%) and osteoclast activity (49%). After a period of 12 months the HPD group additionally developed an (preclinical) osteoporosis with significant reduction (33%) of femoral cortical thickness, as compared to controls (OVX). Taken together, HPD leads after 12 month to osteoporosis with a reduction in both trabecular and cortical bone caused by a low bone turnover situation, with reduced osteoblast and osteoclast activity, as compared to controls (OVX). The HPD-sheep is a suitable large animal model of osteoporosis. Furthermore our results indicate that an intact hypothalamo-pituitary axis is required for activation of bone turnover.

The Effects of Smoke Carcinogens on Bone

The greatest cause of preventable morbidity and mortality is smoking, and one of the often-underappreciated effects of smoking is profound bone loss. The existing clinical paradigm for smoking is that there is a low turnover osteoporosis. This review highlights findings from recent clinical trials and animal research demonstrating either support or conflict with the existing paradigm. Clinically, it is noted that markers of bone formation are often normal in smokers; these clinical findings conflict with well-conducted animal research demonstrating that carcinogens acting on the aryl hydrogen receptor can significantly reduce osteoblast formation and function. Regarding bone resorption, highlights from recent clinical studies suggest that bone remodeling is increased in smokers. Directly contradicting this enhanced osteoclastogenesis are several animal studies all demonstrating significant inhibition of osteoclast formation and function upon exposure to smoke carcinogens. Future research is needed to clarify whether smoking is truly a low bone remodeling osteoporosis, or an osteoclast-driven bone destruction, with inappropriately normal bone formation.