Major alterations in body composition, such as with obesity and weight loss, have complex effects on the measurement of bone mineral density (BMD) by dual-energy x-ray absorptiometry (DXA). The effects of altered body fat on quantitative computed tomography (QCT) measurements are unknown.
We scanned a spine phantom by DXA and QCT before and after surrounding with sequential fat layers (up to 12 kg). In addition, we measured lumbar spine and proximal femur BMD by DXA and trabecular spine BMD by QCT in 13 adult volunteers before and after a simulated 7.5 kg increase in body fat.
With the spine phantom, DXA BMD increased linearly with sequential fat layering at the normal (p<0.01) and osteopenic (p<0.01) levels, but QCT BMD did not change significantly. In humans, fat layering significantly reduced DXA spine BMD values (mean ± SD: −2.2 ± 3.7%, p=0.05) and increased the variability of measurements. In contrast, fat layering increased QCT spine BMD in humans (mean ± SD: 1.5 ± 2.5%, p=0.05). Fat layering did not change mean DXA BMD of the femoral neck or total hip in humans significantly, but measurements became less precise. Associations between baseline and fat-simulation scans were stronger for QCT of the spine (r2 = 0.97) than for DXA of the spine (r2 = 0.87), total hip (r2 = 0.80), or femoral neck (r2 = 0.75). Bland-Altman plots revealed that fat-associated errors were greater for DXA spine and hip BMD than for QCT trabecular spine BMD.
Fat layering introduces error and decreases the reproducibility of DXA BMD spine and hip measurements in human volunteers. Although overlying fat also affects QCT BMD measurements, the error is smaller and more uniform than with DXA BMD. Caution must be used when interpreting BMD changes in humans whose body composition is changing.
bone densitometry; bone QCT; body composition
Guidelines recommend bone density screening with dual-energy x-ray absorptiometry (DXA) in women 65 years or older, but <30% of eligible women undergo DXA testing. There is a need to identify a systematic, effective, and generalizable way to improve osteoporosis screening.
Group randomized, controlled trial of women ≥65 years old with no DXA in the past 4 years, randomized to receive intervention materials (patient osteoporosis brochure and a letter explaining how to self-schedule a DXA scan) vs. usual care (control). Outcome of interest was DXA completion.
Of 2997 women meeting inclusion criteria, 977 were randomized to the intervention group. A total of 17.3% of women in the intervention group completed a DXA, compared to 5.2% in the control group (12.1% difference, p<0.0001). When including only those medically appropriate, we found a difference of 19% between the two groups (p<0.0001). DXA receipt was greater in main clinic patients compared to satellite clinic patients (20.9% main clinic vs. 10.1% satellite clinic). The cost to print and mail the intervention was $0.79 per patient, per mailing. The number of women to whom intervention needed to be mailed to yield one extra DXA performed was 9, at a cost of $7.11.
DXA scan completion was significantly improved through use of a mailed osteoporosis brochure and the availability for patients to self-schedule. This simple approach may be an effective component of a multi-faceted quality improvement program to increase rates of osteoporosis screening.
osteoporosis; screening; DXA; randomized controlled trial
Sox9 is an essential transcription factor for the differentiation of the chondrocytic lineage during embryonic development. To test whether Sox9 continues to play a critical role in cartilaginous tissues in the adult mice, we used an inducible, genetic strategy to disrupt the Sox9 gene postnatally in these tissues. The postnatal inactivation of Sox9 led to stunted growth characterized by decreased proliferation, increased cell death, and de-differentiation of growth plate chondrocytes. Upon postnatal Sox9 inactivation in the articular cartilage, the sulfated proteoglycan and aggrecan content of the uncalcified cartilage were rapidly depleted and the degradation of aggrecan was accompanied by higher ADAMTS5 immunostaining and increased detection of the aggrecan neoepitope, NITEGE. In spite of the severe loss of Collagen 2a1 mRNA, the Collagen II protein persisted in the articular cartilage, and no histopathological signs of osteoarthritis were observed. The homeostasis of the intervertebral disk (IVD) was dramatically altered upon Sox9 depletion, resulting in disk compression and subsequent degeneration. Inactivation of Sox9 in the IVD markedly reduced the expression of several genes encoding extracellular matrix proteins, as well as some of the enzymes responsible for their posttranslational modification. Furthermore, the loss of Sox9 in the IVD decreased the expression of cytokines, cell surface receptors, and ion channels suggesting that Sox9 coordinates a large genetic program that is instrumental for the proper homeostasis of the cells contained in the intervertebral disk postnatally. Our results indicate that Sox9 has an essential role in the physiological control of cartilaginous tissues in adult mice.
Sox9; aggrecan; osteoarthritis; intervertebral; Collagen II
Idiopathic osteoporosis (IOP) in premenopausal women is characterized by fragility fractures at low or normal bone mineral density (BMD) in otherwise healthy women with normal gonadal function. Histomorphometric analysis of transiliac bone biopsy samples has revealed microarchitectural deterioration of cancellous bone and thinner cortices. To examine bone material quality, we measured the bone mineralization density distribution (BMDD) in biopsy samples by quantitative backscattered electron imaging (qBEI), and mineral/matrix ratio, mineral crystallinity/maturity, relative proteoglycan content and collagen cross-link ratio at actively bone forming trabecular surfaces by Raman and Fourier Transform Infrared (FTIRM) microspectroscopic techniques. The study groups included: premenopausal women with idiopathic fractures (IOP, n=45), or idiopathic low BMD (Z-score ≤-2.0 at spine and/or hip) but no fractures (ILBMD, n=19), and healthy controls (CONTROL, n=38). BMDD of cancellous bone showed slightly lower mineral content in IOP (both Cn.CaMean and Cn.CaPeak are 1.4% lower) and in ILBMD (both are 1.6% lower, p<0.05) versus CONTROL, but no difference between IOP and ILBMD. Similar differences were found when affected groups were combined versus CONTROL. The differences remained significant after adjustment for mineralizing surface (MS/BS), suggesting that the reduced mineralization of bone matrix cannot be completely accounted for by differences in bone turnover. Raman and FTIRM analysis at forming bone surfaces showed no differences between combined IOP/ILBMD groups versus CONTROL, with the exceptions of increased proteoglycan content per mineral content and increased collagen cross-link ratio. When the two affected subgroups were considered individually, mineral/matrix ratio and collagen cross-link ratio were higher in IOP than ILBMD. In conclusion, our findings suggest that bone material properties differ between premenopausal women with IOP/ILBMD and normal controls. In particular, the altered collagen properties at sites of active bone formation support the hypothesis that affected women have osteoblast dysfunction that may play a role in bone fragility.
Osteoclasts are bone specific polykarons derived from myeloid precursors under the stimulation of MCSF and RANKL. E proteins are basic helix-loop-helix (bHLH) transcription factors that modulate lymphoid versus myeloid cell fate decisions. To study the role of E proteins in osteoclasts, myeloid specific E protein gain-of-function transgenic mice were generated. These mice have high bone mass due to decreased osteoclast numbers and increased osteoclast apoptosis leading to overall reductions in resorptive capacity. The molecular mechanism of decreased osteoclast numbers and resorption is due, in part, to elevated expression of CD38, a regulator of intracellular calcium pools with known anti-osteoclastogenic properties, which increases sensitivity to apoptosis. In vivo, exogenous RANKL stimulation can overcome this inhibition to drive osteoclastogenesis and bone loss. In vitro derived ET2 osteoclasts are more spread and more numerous with increases in RANK, TREM2, and NFATc1 compared to wild type. However, their resorptive capacity does not increase accordingly. Thus, E proteins participate in osteoclast maturation and survival in homeostatic bone remodeling.
Osteoclast; E proteins; CD38; transcription factor
Bone water (BW) plays a pivotal role in nutrient transport and conferring bone with its viscoelastic mechanical properties. BW is partitioned between the pore spaces of the Haversian and lacuno-canalicular system, and water predominantly bound to the matrix proteins (essentially collagen). The general model of BW is that the former predominantly experiences fast isotropic molecular reorientation, whereas water in the bone matrix undergoes slower anisotropic rotational diffusion. Here, we provide direct evidence for the correctness of this model and show that unambiguous quantification in situ of these two functionally and dynamically different BW fractions is possible.
The approach chosen relies on nuclear magnetic resonance (NMR) of deuterium (2H) that unambiguously separates and quantifies the two fractions on the basis of their distinguishing microdynamic properties. Twenty-four specimens of the human tibial cortex from six donors (3 male, 3 female, ages 27-83 years) were cored and 2H spectra recorded at 62 MHz (9.4 Tesla) on a Bruker Instruments DMX 400 spectrometer after exchange of native BW with 2H2O. Spectra consisted of a doublet signal resulting from quadrupole interaction of water bound to collagen. Doublet splittings were found to depend on the orientation of the osteonal axis with respect to the magnetic field direction (8.2 and 4.3 kHz for parallel and perpendicular orientation, respectively). In contrast, the isotropically reorienting pore-resident water yielded a single resonance line superimposed on the doublet. Nulling of the singlet resonance allowed separation of the two fractions. The results indicate that in human cortical bone 60-80% of detectable BW is collagen-bound. Porosity determined as the difference between total BW and collagen bound water fraction was found to strongly parallel μCT based measurements (R2 = 0.91). Our method provides means for direct validation of emerging relaxation-based measurements of cortical bone porosity by proton MRI.
cortical bone; bone water; porosity; deuterium NMR; MRI
Osteoclastic bone resorption depends upon the cell’s ability to organize its cytoskeleton via the αvβ3 integrin and osteoclastogenic cytokines. Since paxillin associates with αvβ3, we asked if it participates in skeletal degradation. Unlike deletion of other αvβ3-associated cytoskeleton-regulating molecules, which impairs the cell’s ability to spread, paxillin-deficient (Pax−/−) osteoclasts, generated from embryonic stem cells, “superspread” in response to RANK ligand (RANKL) and form large, albeit dynamically atypical, actin bands. Despite their increased size, Pax−/− osteoclasts resorb bone poorly, excavating pits approximately 1/3 normal depth. Ligand-occupied αvβ3 or RANKL promotes paxillin serine and tyrosine phosphorylation, the latter via c-Src. The abnormal Pax−/− phenotype is rescued by WT paxillin but not that lacking its LD4 domain. In keeping with the appearance of mutant osteoclasts, WT paxillin, overexpressed in WT cells, contracts the cytoskeleton. Most importantly, the abnormal phenotype of Pax−/− osteoclasts likely represents failed RANKL-mediated delivery of myosin IIA to the actin cytoskeleton via the paxillin LD4 domain but is independent of tyrosine phosphorylation. Thus, in response to RANKL, paxillin associates with myosin IIA to contract the osteoclast cytoskeleton thereby promoting its bone-degrading capacity.
osteoclasts; paxillin; myosin IIA; cytoskeleton; resorption
Bone homeostasis requires stringent regulation of osteoclasts, which secrete proteolytic enzymes to degrade the bone matrix. Despite recent progress in understanding how bone resorption occurs, the mechanisms regulating osteoclast secretion, and in particular the trafficking route of cathepsin K vesicles, remain elusive. Using a genetic approach, we describe the requirement for PKCδ in regulating bone resorption by affecting cathepsin K exocytosis. Importantly, PKCδ deficiency does not perturb formation of the ruffled border or trafficking of lysosomal vesicles containing the v-ATPase. Mechanistically, we find that cathepsin K exocytosis is controlled by PKCδ through modulation of the actin bundling protein MARCKS. The relevance of our finding is emphasized in vivo as PKCδ−/− mice exhibit increased bone mass and are protected from pathological bone loss in a model of experimental post-menopausal osteoporosis. Collectively, our data provide novel mechanistic insights into the pathways that selectively promote secretion of cathepsin K lysosomes independently of ruffled border formation, providing evidence for the presence of multiple mechanisms that regulate lysosomal exocytosis in osteoclasts.
The local variability of microarchitecture of human trabecular calcaneus bone is investigated using high resolution microCT scanning. The fabric tensor is employed as the measure of the microarchitecture of the pore structure of a porous medium. It is hypothesized that a fabric tensor-dependent poroelastic ultrasound approach will more effectively predict the data variance than will porosity alone. The specific aims of the present study are i) to quantify the morphology and local anisotropy of the calcaneus microarchitecture with respect to anatomical directions, ii) to determine the interdependence, or lack thereof, of microarchitecture parameters, fabric, and volumetric bone mineral density (vBMD), and iii) to determine the relative ability of vBMD and fabric measurements in evaluating the variance in ultrasound wave velocity measurements along orthogonal directions in the human calcaneus. Our results show that the microarchitecture in the analyzed regions of human calcanei is anisotropic, with a preferred alignment along the posterior-anterior direction. Strong correlation was found between most scalar architectural parameters and vBMD. However, no statistical correlation was found between vBMD and the fabric components, the measures of the pore microstructure orientation. Therefore, among the parameters usually considered for cancellous bone (i.e., classic histomorphometric parameters such as porosity, trabecular thickness, number and separation), only fabric components explain the data variance that cannot be explained by vBMD, a global mass measurement, which lacks the sensitivity and selectivity to distinguish osteoporotic from healthy subjects because it is insensitive to directional changes in bone architecture. This study demonstrates that a multi-directional, fabric-dependent poroelastic ultrasound approach has the capability of characterizing anisotropic bone properties (bone quality) beyond bone mass, and could help to better understand anisotropic changes in bone architecture using ultrasound.
CCN family 2/connective tissue growth factor (CCN2/CTGF) promotes endochondral ossification. However, the role of CCN2 in the replacement of hypertrophic cartilage with bone is still unclear. The phenotype of Ccn2 null mice, having an expanded hypertrophic zone, indicates that the resorption of the cartilage extracellular matrix is impaired therein. Therefore, we analyzed the role of CCN2 in osteoclastogenesis because cartilage extracellular matrix is resorbed mainly by osteoclasts during endochondral ossification. Expression of the Ccn2 gene was upregulated in mouse macrophage cell line RAW264.7 on day 6 after treatment of glutathione S transferase (GST) fusion mouse receptor activator of NF-κB ligand (GST-RANKL), and a combination of recombinant CCN2 (rCCN2) and GST-RANKL significantly enhanced tartrate-resistant acid phosphatase (TRACP)–positive multinucleated cell formation compared with GST-RANKL alone. Therefore, we suspected the involvement of CCN2 in cell-cell fusion during osteoclastogenesis. To clarify the mechanism, we performed real-time PCR analysis of gene expression, coimmunoprecipitation analysis, and solid-phase binding assay of CCN2 and dendritic cell–specific transmembrane protein (DC-STAMP), which is involved in cell-cell fusion. The results showed that CCN2 induced and interacted with DC-STAMP. Furthermore, GST-RANKL–induced osteoclastogenesis was impaired in fetal liver cells from Ccn2 null mice, and the impaired osteoclast formation was rescued by the addition of exogenous rCCN2 or the forced expression of DC-STAMP by a retroviral vector. These results suggest that CCN2 expressed during osteoclastogenesis promotes osteoclast formation via induction of and interaction with DC-STAMP.
Osteoclasts; CCN Family 2/Connective Tissue Growth Factor (CCN2/CTGF); Receptor Activator of Nf-κB Ligand (RANKL); Dendritic Cell; Specific Transmembrane Protein (DC-STAMP)
The surface properties of materials contribute to host cellular response and play a significant role in determining the overall success or failure of an implanted biomaterial. Rough titanium (Ti) surface microtopography and high surface free energy have been shown to enhance osteoblast maturation in vitro and increase bone formation in vivo. While the surface properties of Ti are known to affect osteoblast response, host bone quality also plays a significant role in determining successful osseointegration. One factor affecting host bone quality is patient age. We examined both in vitro and in vivo whether response to Ti surface features was affected by animal age. Calvarial osteoblasts isolated from 1-, 3-, and 11-month-old rats all displayed a reduction in cell number and increases in alkaline phosphatase specific activity and osteocalcin in response to increasing Ti surface microtopography and surface energy. Further, osteoblasts from the three ages examined displayed increased production of osteocalcin and local factors osteoprotegerin, VEGF-A, and active TGF-β1 in response to increasing Ti surface roughness and surface energy. Latent TGF-β1 only increased in cultures of osteoblasts from 1- and 3-month-old rats. Treatment with the systemic osteotropic hormone 1α,25(OH)2D3 further enhanced the response of osteoblasts to Ti surface features for all three age groups. However, osteoblasts derived from 11-month-old animals had a reduced response to 1α,25(OH)2D3 as compared to osteoblasts derived from 1-or 3-month-old animals. These results were confirmed in vivo. Ti implants placed in the femoral intramedullary canal of old (9-month) mice yielded lower bone-to-implant contract and neovascularization in response to Ti surface roughness and energy compared to younger (2-month) mice. These results show that rodent osteoblast maturation in vitro as well as new bone formation in vivo is reduced with age. Whether comparable age differences exist in humans needs to be determined.
Bisphosphonates have been shown to reduce mortality in patients with osteoporotic fractures, but the mechanism is unclear. Bisphosphonates have immunomodulatory effects that may influence the development of vascular disease. We sought to determine if bisphosphonate use is associated with a reduced risk of myocardial infarction (MI) in a rheumatoid arthritis (RA) population with high prevalence of bisphosphonate use and vascular disease. Adult patients with RA enrolled in the National Data Bank for Rheumatic Diseases, a longitudinal study of RA patients enrolled continuously from U.S. rheumatology practices between 2003 and 2011, were included in the analysis (n = 19,281). Patients completed questionnaires every 6 months. including questions on medication use, demographic information, clinical information, and health status. MIs were confirmed by a central adjudicator. Among the 5689 patients who were treated with bisphosphonates at some time during the study period, the risk of MI while on bisphosphonate compared to when not on bisphosphonate was 0.56 (95% confidence interval [CI], 0.37–0.86; p < 0.01) after adjustment for multiple confounders. In models including all 19,281 treated and untreated patients, the adjusted risk of first MI was 0.72 (95% CI, 0.54–0.96; p = 0.02) and of all MIs it was 0.72 (95% CI, 0.53–0.97; p = 0.03) in bisphosphonate users compared to nonusers. This finding suggests a potential mechanism for the mortality reduction observed with bisphosphonate medications.
Bisphosphonate; Myocardial Infarction; Rheumatoid Arthritis
This prospective case-cohort study aimed to map the distribution of bone density in the proximal femur and examine its association with hip fracture. We analyzed baseline quantitative computed tomography (QCT) scans in 250 men aged 65 yrs or older, which comprised a randomly-selected subcohort of 210 men and 40 cases of first hip fracture during a mean follow-up period of 5.5 yr. We quantified cortical, trabecular and integral vBMD and cortical thickness (CtTh) in four quadrants of cross sections along the length of the femoral neck (FN), intertrochanter (IT) and trochanter (TR). In most quadrants, vBMDs and CtTh were significantly (P<0.05) lower in cases compared to the subcohort and these deficits were present across the entire proximal femur. To examine the association of QCT measurements with hip fracture, we merged the two quadrants in the medial and lateral aspects of the FN, IT and TR. At most sites, QCT measurements were associated significantly (p<0.001) with hip fracture, the hazard ratio (HR) adjusted for age, BMI and clinical site for one standard deviation decrease ranged between 2.28 (95% CI 1.44–3.63) to 6.91 (95% CI 3.11–15.53). After additional adjustment for TH aBMD, trabecular vBMDs at the FN, TR and TH were still associated with hip fracture significantly (p<0.001), the HRs ranged from 3.21 (95% CI 1.65–6.24) for the supero-lateral FN to 6.20 (95% CI 2.71–14.18) for medial TR. QCT measurements alone or in combination did not predict fracture significantly (p>0.05) better than TH aBMD. With an AUC of 0.901 (95% CI 0.852–0.950), the regression model combining TH aBMD, age and trabecular vBMD predicted hip fracture significantly (p<0.05) better than TH aBMD alone or TH aBMD plus age. These findings confirm that both cortical and trabecular bone contribute to hip fracture risk and highlight trabecular vBMD at the FN and TR as an independent risk factor.
Low 25-hydroxyvitamin D (VitD), low sex hormones (SH), and high sex hormone binding globulin (SHBG) levels are common in older men. We tested the hypothesis that combinations of low VitD, low SH, and high SHBG would have a synergistic effect on bone mineral density (BMD), bone loss, and fracture risk in older men. Participants were a random subsample of 1468 men (mean age 74) from the Osteoporotic Fractures in Men Study (MrOS) plus 278 MrOS men with incident non-spine fractures studied in a case-cohort design. “Abnormal” was defined as lowest quartile for VitD (<20 ng/ml), bioavailable testosterone (BioT, <163 ng/dl), and bioavailable estradiol (BioE, <11 pg/ml); and highest quartile for SHBG (>59 nM).
Overall, 10% had isolated VitD deficiency; 40% had only low SH or high SHBG; 15% had both SH/SHBG and VitD abnormality, and 35% had no abnormality. Compared to men with all normal levels, those with both SH/SHBG and VitD abnormality tended to be older, more obese, and to report less physical activity. Isolated VitD deficiency, and low BioT with or without low VitD, was not significantly related to skeletal measures. The combination of VitD deficiency with low BioE and/or high SHBG was associated with significantly lower baseline BMD and higher annualized rates of hip bone loss than SH abnormalities alone or no abnormality. Compared to men with all normal levels, the multivariate-adjusted hazard ratio (95% CI) for incident non-spine fracture during 4.6 yr median follow-up was 1.2 (0.8–1.8) for low VitD alone; 1.3 (0.9–1.9) for low BioE and/or high SHBG alone; and 1.6 (1.1–2.5) for low BioE/high SHBG plus low VitD.
In summary, adverse skeletal effects of low sex steroid levels were most pronounced in older men with low VitD levels. The presence of low VitD in the presence of low BioE/high SHBG may contribute substantially to poor skeletal health.
bone loss; fracture; older men; sex hormones; vitamin
Wnt signaling is essential for osteogenesis and also functions as an adipogenic switch, but it is not known if interrupting wnt signaling via knockout of β-catenin from osteoblasts would cause bone marrow adiposity. Here, we determined whether postnatal deletion of β-catenin in preosteoblasts, through conditional cre expression driven by the osterix promoter, causes bone marrow adiposity. Postnatal disruption of β-catenin in the preosteoblasts led to extensive bone marrow adiposity and low bone mass in adult mice. In cultured bone marrow-derived cells isolated from the knockout mice, adipogenic differentiation was dramatically increased, whereas osteogenic differentiation was significantly decreased. As myoblasts, in the absence of wnt/β-catenin signaling, can be reprogrammed into the adipocyte lineage, we sought to determine whether the increased adipogenesis we observed partly resulted from a cell-fate shift of preosteoblasts that had to express osterix, (lineage-committed early osteoblasts), from the osteoblastic to the adipocyte lineage. Using lineage tracing both in vivo and in vitro we demonstrated that the loss of β-catenin from preosteoblasts caused a cell-fate shift of these cells from osteoblasts to adipocytes, a shift that may at least partly contribute to the bone marrow adiposity and low bone mass in the knockout mice. These novel findings indicate that wnt/β-catenin signaling exerts control over the fate of lineage-committed early osteoblasts, with respect to their differentiation into osteoblastic vs. adipocytic populations in bone, and thus offers potential insight into the origin of bone marrow adiposity.
Connexin 43 (Cx43) is the most abundant gap junction protein in bone and has been demonstrated as an integral component of skeletal homeostasis. In the present study, we sought to further refine the role of Cx43 in the response to mechanical unloading by subjecting skeletally mature mice with a bone-specific deletion of Cx43 (cKO) to three weeks of mechanical unloading via hindlimb suspension (HLS). The HLS model was selected to recapitulate the effects of skeletal unloading due to prolonged bed rest, reduced activity associated with aging, and spaceflight microgravity. At baseline, the cortical bone of cKO mice displayed an osteopenic phenotype, with expanded cortices, decreased cortical thickness, decreased bone mineral density, and increased porosity. There was no baseline trabecular phenotype. Following three weeks of HLS, wild-type (WT) mice experienced substantial declines in trabecular bone volume fraction, connectivity density, trabecular thickness, and trabecular tissue mineral density. These deleterious effects were attenuated in cKO mice. Conversely, there was a similar and significant amount of cortical bone loss in both WT and cKO. Interestingly, mechanical testing revealed a greater loss of strength and rigidity for cKO during HLS. Analysis of double-label quantitative histomorphometry data demonstrated a substantial decrease in bone formation rate, mineralizing surface, and mineral apposition rate at both the periosteal and endocortical surfaces of the femur following unloading of WT mice. This suppression of bone formation was not observed in cKO mice, where parameters were maintained at baseline levels. Taken together, the results of the present study indicate that Cx43 deficiency desensitizes bone to the effects of mechanical unloading, and that this may be due to an inability of mechanosensing osteocytes to effectively communicate the unloading state to osteoblasts to suppress bone formation. Cx43 may represent a novel therapeutic target for investigation as a countermeasure for age-related and unloading-induced bone loss.
connexin 43; unloading; hindlimb suspension; gap junction; bone loss
Most patients with autosomal dominant pseudohypoparathyroidism type Ib (AD-PHP-Ib) carry maternally inherited microdeletions upstream of GNAS that are associated with loss of methylation restricted to GNAS exon A/B. Only few AD-PHP-Ib patients carry microdeletions within GNAS that are associated with loss of all maternal methylation imprints. These epigenetic changes are often indistinguishable from those observed in patients affected by an apparently sporadic PHP-Ib form that has not yet been defined genetically. We have now investigated six female patients affected by PHP-Ib (four unrelated and two sisters) with complete or almost complete loss of GNAS methylation, whose healthy children (11 in total) showed no epigenetic changes at this locus. Analysis of several microsatellite markers throughout the 20q13 region made it unlikely that PHP-Ib is caused in these patients by large deletions involving GNAS or by paternal uniparental isodisomy or heterodisomy of chromosome 20 (patUPD20). Microsatellite and single-nucleotide variation (SNV) data revealed that the two affected sisters share their maternally inherited GNAS alleles with unaffected relatives that lack evidence for abnormal GNAS methylation, thus excluding linkage to this locus. Consistent with these findings, healthy children of two unrelated sporadic PHP-Ib patients had inherited different maternal GNAS alleles, also arguing against linkage to this locus. Based on our data, it appears plausible that some forms of PHP-Ib are caused by homozygous or compound heterozygous mutation(s) in an unknown gene involved in establishing or maintaining GNAS methylation.
PARATHYROID HORMONE; PSEUDOHYPOPARATHYROIDISM IB; GNAS LOCUS; METHYLATION; AUTOSOMAL RECESSIVE
Alkaline phosphatase (ALP) plays an essential role in the regulation of tissue mineralization, and its activity is highly heritable. Guided by genetic associations discovered in a murine model, we hypothesized a role for rare coding variants in determining serum ALP level and bone mineral density (BMD) in humans. We sequenced the coding regions of the ALP gene (ALPL) in men with low and normal serum ALP activity levels. Single-nucleotide ALPL variants, including 19 rare nonsynonymous variants (minor allele frequency <1%), were much more frequent among the low ALP group (33.8%) than the normal group (1.4%, p = 1 × 10−11). Within the low ALP group, men with a rare, nonsynonymous variant had 11.2% lower mean serum ALP (p = 3.9 × 10−4), 6.7% lower BMD (p = 0.03), and 11.1% higher serum phosphate (p = 0.002) than those without. In contrast, common nonsynonymous variants had no association with serum ALP, phosphate, or BMD. Multiple rare ALPL coding variants are present in the general population, and nonsynonymous coding variants may be responsible for heritable differences in mineralization and thus BMD.
OSTEOPOROSIS; HEREDITY; GENETICS; ALKALINE PHOSPHATASE
Dickkopf-related protein 1 (DKK1) is essential to maintain skeletal homeostasis as an inhibitor of Wnt signaling and osteogenic differentiation. The purpose of this study was to investigate the molecular mechanisms underlying the developmental stage–specific regulation of the DKK1 protein level. We performed a series of studies including luciferase reporter assays, micro-RNA microarray, site-specific mutations, and gain- and loss-of-function analyses. We found that the DKK1 protein level was regulated via DKK1 3′ UTR by miRNA control, which was restricted to osteoblast-lineage cells. As a result of decreased DKK1 protein level by miR-335-5p, Wnt signaling was enhanced, as indicated by elevated GSK-3β phosphorylation and increased β-catenin transcriptional activity. The effects of miR-335-5p were reversed by anti-miR-335-5p treatment, which downregulated endogenous miR-335-5p. In vivo studies showed high expression levels of miR-335-5p in osteoblasts and hypertrophic chondrocytes of mouse embryos, indicating a pivotal role of miR-335-5p in regulating bone development. In conclusion, miR-335-5p activates Wnt signaling and promotes osteogenic differentiation by down-regulating DKK1. This cell- and development-specific regulation is essential and mandatory for the initiation and progression of osteogenic differentiation. miR-335-5p proves to be a potential and useful targeting molecule for promoting bone formation and regeneration.
MIR-335-5P; OSTEOGENIC DIFFERENTIATION; DKK1; WNT; BONE FORMATION
We have demonstrated previously that higher birth weight is associated with greater peak and later-life bone mineral content and that maternal body build, diet, and lifestyle influence prenatal bone mineral accrual. To examine prenatal influences on bone health further, we related ultrasound measures of fetal growth to childhood bone size and density. We derived Z-scores for fetal femur length and abdominal circumference and conditional growth velocity from 19 to 34 weeks’ gestation from ultrasound measurements in participants in the Southampton Women’s Survey. A total of 380 of the offspring underwent dual-energy X-ray absorptiometry (DXA) at age 4 years [whole body minus head bone area (BA), bone mineral content (BMC), areal bone mineral density (aBMD), and estimated volumetric BMD (vBMD)]. Volumetric bone mineral density was estimated using BMC adjusted for BA, height, and weight. A higher velocity of 19- to 34-week fetal femur growth was strongly associated with greater childhood skeletal size (BA: r = 0.30, p < .0001) but not with volumetric density (vBMD: r = 0.03, p = .51). Conversely, a higher velocity of 19- to 34-week fetal abdominal growth was associated with greater childhood volumetric density (vBMD: r = 0.15, p = .004) but not with skeletal size (BA: r = 0.06, p = .21). Both fetal measurements were positively associated with BMC and aBMD, indices influenced by both size and density. The velocity of fetal femur length growth from 19 to 34 weeks’ gestation predicted childhood skeletal size at age 4 years, whereas the velocity of abdominal growth (a measure of liver volume and adiposity) predicted volumetric density. These results suggest a discordance between influences on skeletal size and volumetric density.
EPIDEMIOLOGY; OSTEOPOROSIS; PROGRAMMING; DEVELOPMENTAL ORIGINS
Transient receptor potential cation channel, subfamily V, member 6 (TRPV6) is an apical membrane calcium (Ca) channel in the small intestine proposed to be essential for vitamin D regulated intestinal Ca absorption. Recent studies have challenged the proposed role for TRPV6 in Ca absorption. We directly tested intestinal TRPV6 function in Ca and bone metabolism in wild-type (WT) and vitamin D receptor knockout (VDRKO) mice. Transgenic mice (TG) were made with intestinal epithelium-specific expression of a 3X flag-tagged human TRPV6 protein. TG and VDRKO mice were crossed to make TG-VDRKO mice. Ca and bone metabolism was examined in WT, TG, VDRKO, and TG-VDRKO mice. TG mice developed hypercalcemia and soft tissue calcification on a chow diet. In TG mice fed a 0.25% Ca diet, Ca absorption was >3 fold higher and femur bone mineral density (BMD) was 26% higher than WT. Renal CYP27B1 mRNA and intestinal expression of the natural mouse TRPV6 gene were reduced to <10% of WT but small intestine calbindin-D9k expression was elevated >15X in TG mice. TG-VDRKO mice had high Ca absorption that prevented the low serum Ca, high renal CYP27B1 mRNA, and low BMD and abnormal bone microarchitecture seen in VDRKO mice. In addition, small intestinal calbindin D9K mRNA and protein levels were elevated in TG-VDRKO. Transgenic TRPV6 expression in intestine is sufficient to increase Ca absorption and bone density, even in VDRKO mice. VDR independent up-regulation of intestinal calbindin D9k in TG-VDRKO suggests this protein may buffer intracellular Ca during Ca absorption.
TRPV6; calbindin D9k; vitamin D; intestine; absorption
To better understand the biomechanical mechanisms underlying the association between hyperkyphosis of the thoracic spine and risk of vertebral fracture and other degenerative spinal pathology, we used a previously validated musculoskeletal model of the spine to determine how thoracic kyphosis angle and spinal posture affect vertebral compressive loading. We simulated an age-related increase in thoracic kyphosis (T1-T12 Cobb angle 50° to 75°) during two different activities (relaxed standing and standing with 5 kg weights in the hands) and three different posture conditions: 1) an increase in thoracic kyphosis with no postural adjustment (uncompensated posture), 2) an increase in thoracic kyphosis with a concomitant increase in pelvic tilt that maintains a stable center of mass and horizontal eye gaze (compensated posture), and 3) an increase in thoracic kyphosis with a concomitant increase in lumbar lordosis that also maintains a stable center of mass and horizontal eye gaze (congruent posture). For all posture conditions, compressive loading increased with increasing thoracic kyphosis, with loading increasing more in the thoracolumbar and lumbar regions than in the mid-thoracic region. Loading increased the most for the uncompensated posture, followed by the compensated posture, with the congruent posture almost completely mitigating any increases in loading with increased thoracic kyphosis. These findings indicate that thoracic kyphosis and spinal posture both influence vertebral loading during daily activities, implying that thoracic kyphosis measurements alone are not sufficient to characterize the impact of spinal curvature on vertebral loading.
Kyphosis; Spinal Loading; Posture; Biomechanical Model; Vertebral Fracture
Though osteonecrosis of the jaw (ONJ) is temporally-associated with the use of nitrogen-containing bisphosphonates (N-BPs), a cause/effect relationship has not yet been established. We hypothesize that ONJ is a two-stage process in which: a) risk factors initiate pathologic processes in the oral cavity that lead to a supranormal rate of hard tissue necrosis, and b) powerful anti-resorptives reduce the rate of removal of necrotic bone sufficiently to allow its net accumulation in the jaw. To test this hypothesis, we used the rice rat model of periodontitis. At age 28 days, rats (n=15/group) were placed on a high sucrose and casein diet to exacerbate the development of periodontitis. Animals were injected SC biweekly with vehicle or alendronate (ALN, 15μg/kg), or IV once monthly with vehicle, a low dose (LD), or a high dose (HD) of zoledronic acid (ZOL) and sacrificed after 6, 12, 18, and 24 wks. Mandibles and maxillae were analyzed to determine the effects on the: a) progression of periodontitis, b) integrity of alveolar bone, c) status of bone resorption and formation, d) vascularity, and e) osteocyte viability. We found that only HD-ZOL induced ONJ-like lesions in mandibles of rice rats after 18 and 24 wks of treatment. These lesions were characterized by areas of exposed necrotic alveolar bone, osteolysis, a honey comb-like appearance of the alveolar bone, presence of bacterial colonies, and periodontal tissue destruction. In addition, inhibition of bone formation, a paradoxical abolition of the antiresorptive effect of only HD-ZOL, increased osteocyte necrosis/apoptosis, and decreased blood vessel number were found after 18 and/or 24 wks. Our study suggests that only HD-ZOL exacerbates the inflammatory response and periodontal tissue damage in rice rats, inducing bone lesions that resemble ONJ.
ONJ; Zoledronic acid; alendronate; periodontal disease; rice rats
The mechanisms of age-related vertebral fragility remain unclear, but may be related to the degree of “structural redundancy” of the vertebra, that is, its ability to safely redistribute stress internally after local trabecular failure from an isolated mechanical overload. To better understand this issue, we performed biomechanical testing and nonlinear micro-CT-based finite element analysis on 12 elderly human thoracic ninth vertebral bodies (ages 76.9 ± 10.8 years). After experimentally overloading the vertebrae to measure strength, we used the nonlinear finite element analysis to estimate the amount of failed tissue and understand failure mechanisms. We found that the amount of failed tissue per unit bone mass decreased with decreasing bone volume fraction (r2 = 0.66, p < 0.01). Thus, for the weak vertebrae with low bone volume fraction, overall failure of the vertebra occurred after failure of just a tiny proportion of the bone tissue (< 5%). This small proportion of failed tissue had two sources: the existence of fewer vertically oriented load paths to which load could be redistributed from failed trabeculae; and the vulnerability of the trabeculae in these few load paths to undergo bending-type failure mechanisms, which further weaken the bone. Taken together, these characteristics suggest that diminished structural redundancy may be an important aspect of age-related vertebral fragility: vertebrae with low bone volume fraction are highly susceptible to collapse since so few trabeculae are available for load redistribution if the external loads cause any trabeculae to fail.
bone strength; finite element analysis; osteoporosis; vertebra; biomechanics