Trabecular Mineral Research Articles

Morphological and Immunohistochemical Characterization of Bone Structure and Cell–Cell Communication in a Rat Osteoporosis Model

Bone remodeling is essential for maintaining bone health. The imbalance between bone formation and bone resorption leads to bone diseases such as osteoporosis. Connexin43 (Cx43) is a gap junction molecule that plays an important role in bone homeostasis. The present study investigates the morphological characteristics of bone trabeculae and the distribution of Cx43 in bone cells using osteoporotic rat models to explore the relationship between osteoporosis and bone remodeling. Female Sprague–Dawley rats were divided into three groups: sham, ovarectomy with food deprivation (OVX+diet), and ovarectomy with steroid administration (OVX+steroid) for 3 and 12 months to induce osteoporosis. The lumbar vertebrae were processed for histomorphometric and immunohistochemical evaluation of the trabeculae and the distribution of Cx43 in bone cells. The data showed a significant reduction in trabecular bone in both osteoporotic groups. After 12 months, the OVX+diet treatment resulted in reduced mineralization and an increase in unmineralized bone. The percentage of alkaline phosphatase-positive areas in the OVX+diet vertebrae was lower at 12 months compared to the sham group. A significant increase in tartrate-resistant acid phosphatase (TRAP) positive osteoclasts was observed in the OVX+diet group. Both osteoporotic groups showed a decrease in Cx43-positive osteoblasts areas. An increase in the number of osteoclasts positive for Cx43 was detected in the OVX+diet group. The changes in Cx43 distribution in bone cells, together with trabecular mineralization, suggest that Cx43 may play a role in the progression of osteoporosis and could be a valuable target to improve bone remodeling.

Pronounced cortical porosity and sex-specific patterns of increased bone and osteocyte lacunar mineralization characterize the human distal fibula with aging

The high occurrence of distal fibula fractures among older women suggests a potential link to impaired bone health. Here we used a multiscale imaging approach to investigate the microarchitecture, mineralization, and biomechanics of the human distal fibula in relation to age and sex. Micro-computed tomography was performed to analyze the local volumetric bone mineral density and various microarchitectural parameters of the trabecular and the cortical compartment. Bone mineral density distribution and osteocyte lacunar parameters were quantified using quantitative backscattered electron imaging in periosteal, endocortical, and trabecular regions. Additionally, cortical hardness and Young's modulus were assessed by nanoindentation. While cortical porosity strongly increased with age independent of sex, trabecular microarchitecture remained stable. Notably, nearly half of the specimens showed non-bony hypermineralized tissue located at the periosteum, similar to that previously detected in the femoral neck, with no consistent association with advanced age. Independent of this finding, cortical and trabecular mineralization, i.e., mean calcium content, as well as endocortical tissue hardness increased with age in males but not females. Importantly, we also observed mineralized osteocyte lacunae that increased with age specifically in females. In conclusion, our results indicate that skeletal aging of the distal fibula is signified not only by pronounced cortical porosity but also by an increase in mineralized osteocyte lacunae in females. These findings may provide an explanation for the increased occurrence of ankle fractures in older women.

Zucker Diabetic-Sprague Dawley Rats Have Impaired Peri-Implant Bone Formation, Matrix Composition, and Implant Fixation Strength.

An increasing number of patients with type 2 diabetes (T2DM) will require total joint replacement (TJR) in the next decade. T2DM patients are at increased risk for TJR failure, but the mechanisms are not well understood. The current study used the Zucker Diabetic-Sprague Dawley (ZDSD) rat model of T2DM with Sprague Dawley (SPD) controls to investigate the effects of intramedullary implant placement on osseointegration, peri-implant bone structure and matrix composition, and fixation strength at 2 and 10 weeks post-implant placement. Postoperative inflammation was assessed with circulating MCP-1 and IL-10 2 days post-implant placement. In addition to comparing the two groups, stepwise linear regression modeling was performed to determine the relative contribution of glucose, cytokines, bone formation, bone structure, and bone matrix composition on osseointegration and implant fixation strength. ZDSD rats had decreased peri-implant bone formation and reduced trabecular bone volume per total volume compared with SPD controls. The osseointegrated bone matrix of ZDSD rats had decreased mineral-to-matrix and increased crystallinity compared with SPD controls. Osseointegrated bone volume per total volume was not different between the groups, whereas implant fixation was significantly decreased in ZDSD at 2 weeks but not at 10 weeks. A combination of trabecular mineral apposition rate and postoperative MCP-1 levels explained 55.6% of the variance in osseointegration, whereas cortical thickness, osseointegration mineral apposition rate, and matrix compositional parameters explained 69.2% of the variance in implant fixation strength. The results support the growing recognition that both peri-implant structure and matrix composition affect implant fixation and suggest that postoperative inflammation may contribute to poor outcomes after TJR surgeries in T2DM patients. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Application of Nano-Calcium Phosphate Coated Porous Tantalum Scaffold in Repair of Rabbit Cartilage Defect

Calcium coated scaffolds and their role in repair of bone defects have been widely studied in the last decades. This study aimed to evaluate the effect of bone morphogenetic protein-7 (BMP-7) loaded nano-calcium phosphate (CaP) coated porous tantalum scaffolds (PTS) on regenerative and reparative potency of cartilage defects in rabbits. We used New Zealand white rabbit cartilage defect models. The subjects were randomly grouped into 4: (1) a control group (no treatment), (2) a PTS group, (3) a CaP/PTS group, and (4) a CaP/PTS/BMP-7 groups. The characterization and in vitro biocompatibility of each scaffold were evaluated. Following scaffold implantation, we evaluated the gross observation score, Micro-CT observation parameters, and the mRNA of target genes. No visible difference was detected in water contact angle (WCA) and cell proliferation activity after co-culture among the PTS, CaP/PTS, and CaP/PTS/BMP-7 scaffold groups (P >0.05). Gross observation scores, trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular mineral density (TMD), bone volume fraction (BV/TV), COL2A1, SOX-9, Aggrecan, and RUNX-2 showed an obvious increase among the PTS, CaP/PTS, and CaP/PTS/BMP-7 groups to those with no treatment (P <0.05). Also, the PPARγ showed an obvious decline (P <0.05). Gross observation scores, Tb.Th, Tb.N, TMD, BV/TV, COL2A1, SOX-9, Aggrecan, and RUNX-2 increased and PPARγ decreased in CaP/PTS/BMP-7 group to PTS and CaP/PTS groups (P <0.05). Our findings indicate that PT scaffolds enhance the reparative potency of cartilage defects in New Zealand rabbits promoted the repair of cartilage defects in rabbits. This effect is boosted among those treated with the BMP-7 loaded nano-CaP coated PTS, and this group showed improved regenerative and reparative potency.

Mineralized Cartilage and Bone-Like Tissues in Chondrichthyans Offer Potential Insights Into the Evolution and Development of Mineralized Tissues in the Vertebrate Endoskeleton.

The impregnation of biominerals into the extracellular matrix of living organisms, a process termed biomineralization, gives rise to diverse mineralized (or calcified) tissues in vertebrates. Preservation of mineralized tissues in the fossil record has provided insights into the evolutionary history of vertebrates and their skeletons. However, current understanding of the vertebrate skeleton and of the processes underlying its formation is biased towards biomedical models such as the tetrapods mouse and chick. Chondrichthyans (sharks, skates, rays, and chimaeras) and osteichthyans are the only vertebrate groups with extant (living) representatives that have a mineralized skeleton, but the basal phylogenetic position of chondrichthyans could potentially offer unique insights into skeletal evolution. For example, bone is a vertebrate novelty, but the internal supporting skeleton (endoskeleton) of extant chondrichthyans is commonly described as lacking bone. The molecular and developmental basis for this assertion is yet to be tested. Subperichondral tissues in the endoskeleton of some chondrichthyans display mineralization patterns and histological and molecular features of bone, thereby challenging the notion that extant chondrichthyans lack endoskeletal bone. Additionally, the chondrichthyan endoskeleton demonstrates some unique features and others that are potentially homologous with other vertebrates, including a polygonal mineralization pattern, a trabecular mineralization pattern, and an unconstricted perichordal sheath. Because of the basal phylogenetic position of chondrichthyans among all other extant vertebrates with a mineralized skeleton, developmental and molecular studies of chondrichthyans are critical to flesh out the evolution of vertebrate skeletal tissues, but only a handful of such studies have been carried out to date. This review discusses morphological and molecular features of chondrichthyan endoskeletal tissues and cell types, ultimately emphasizing how comparative embryology and transcriptomics can reveal homology of mineralized skeletal tissues (and their cell types) between chondrichthyans and other vertebrates.

Comparison of Trabecular Bone Score-Adjusted FRAX™ and FRAX™-Bone Mineral Density in Postmenopausal Women with Clinical Vertebral Fractures: A Cross-Sectional, Analytic Study

Background: Ethnicity differentially modulate fracture risk prediction using FRAX™-bone mineral density (BMD). Objective: To compare FRAX™-BMD and trabecular bone score (TBS)-adjusted FRAX™ in the 10-year probability of fracture among Thai postmenopausal woman (PMW) with vertebral fracture (VF). Materials and Methods: The present study was a cross-sectional study conducted by retrospective review of medical records of PMW with VF older than 45 years undergoing lumbar and hip DXA scan. The authors excluded the PMW having been treated with metal implant at the spine, 3 or more lumbar VF, or cancer spreading to vertebral spine. The authors assessed the difference in means of normally distributed data by dependent sample t-test, the correlation between TBS and LS BMD by Pearson correlation, and the difference in proportions of PMW who met intervention threshold (IT) before and after TBS-adjusted FRAX™ by McNemar’s test. A p<0.05 was considered statistically significant. Results: Of the 119 patients, the mean age was 70.8±8.1 years. The mean 10-year probability of a hip fracture by TBS-adjusted FRAX™ was significantly higher than that by FRAX™-BMD with the mean difference of 0.44% (95% CI 0.13 to 0.76). The higher difference in means, using TBSadjusted FRAX™, was even significantly greater in the 10-year probability of major osteoporotic fractures. For either the IT of major osteoporotic fracture or hip fracture, TBS-adjusted FRAX™ resulted in four added PMWs who needed treatment, but without statistical significance. Conclusion: TBS-adjusted FRAX™ had a higher 10-year probability of fracture than FRAX™-BMD. Keywords: Trabecular bone score; FRAX™; Bone mineral density; Postmenopausal women; Osteoporosis

Erythropoietin Eliminates Increased Bone Marrow Adiposity and Alters Bone Features in Obese Mice

Bone marrow (BM) adipose tissue (BMAT) and BM adipocytes are developmentally distinct from other fat depots and differ from white and brown adipose tissue adipocytes in their lipid composition. BMAT also negatively regulates hematopoiesis. Obesity results in BMAT expansion, which increases bone resorbing osteoclasts and is associated with reduced osteoblast bone forming potential leading to increased risk of fractures. Factors that could limit the expansion of BMAT and decrease the negative effects on bones and hematopoiesis could be useful in restoring bone and BM homeostasis.Erythropoietin (EPO) is essential for erythroid differentiation. In animal models of diet induced obesity, EPO lowers inflammation, reduces white fat mass and promotes glucose tolerance. Here, we investigated the EPO effect on BMAT, bones and distinct BM cell populations under obesogenic conditions. Young C57BL6/J female mice (6 weeks) were placed on normal chow diet (ND) or on 60% kcal high fat diet (HFD) for 4 weeks and were treated with EPO 1200 IU/kg or saline daily for 10 more days on HFD. This course of EPO treatment increased hematocrit without affecting whole body fat mass. For bone analysis, femurs were harvested and analyzed using micro-computed tomography (micro-Ct), and hematoxylin and eosin staining to identify bone and marrow features. HFD decreased trabecular number and increased trabecular spacing, but increased the cortical bone volume, suggesting the adaptation of the bones to withstand the increased body mass in HFD fed mice. EPO treatment reduced trabecular mineral density, trabecular number and spacing, and bone volume/tissue volume under both ND and HFD conditions. However, with EPO treatment, cortical bone volume decreased only in ND fed mice but not in HFD fed mice and cortical bone mineral density increased in HFD fed mice. Consistent with these selective EPO associated changes in bone loss were decreased trabecular osteocytes and decreased trabecular lining osteoblasts in ND fed mice and analogous trends in HFD fed mice, while EPO treatment reduced osteocytes in the cortical bones and osteoblasts in the periosteal region of cortical bones in ND fed mice but not in HFD fed mice. Histology sections showed that EPO treatment markedly reduced BMAT in ND and HFD fed mice, and inhibited the increase in BMAT with HFD feeding. External fat depots did not exhibit any decrease in fat mass following the ten day EPO treatment, suggesting that BMAT is more sensitive and has a quicker response to EPO treatment in both ND and HFD fed mice.Contrary to published reports that link EPO stimulated bone loss to increased osteoclast number and activity, we did not find that EPO increased osteoclasts detected by tartarate resistant acid phosphatase (TRAP) staining. Although femur sections showed an increase in osteoclasts with HFD feeding, EPO treatment reduced osteoclasts in HFD fed mice and had no significant effect in ND fed mice. These data suggest that bone loss, especially trabecular bone, accompanying EPO stimulated increase in hematocrit is not due to increase osteoclast activity but rather is due in part to decreased osteoblasts and osteocytes. Flow cytometry analysis of BM cell populations demonstrated that EPO treatment in ND fed mice decreased hematopoietic cells by 40% and increased erythroid precursor cells by 70%. Although EPO stimulated increase in hematocrit was comparable in ND and HFD fed mice, corresponding changes in % hematopoietic cells and % erythroid precursor cells in BM cell populations were not evident in HFD fed mice. This suggests that in obese mice, EPO treatment abrogates the suppression of hematopoiesis exerted by the expanding BMAT and results in the increased hematopoietic cell proliferation concomitant with EPO stimulated erythropoiesis.In summary, EPO treatment decreases trabecular bone in ND and HFD fed mice, decreases cortical bone volume in ND fed mice, but increases cortical bone mineral density in HFD fed mice. Concomitant with EPO stimulated increased hematocrit is a decrease in BMAT and abrogation of BMAT expansion in diet-induced obese mice. Changes in BMAT are prior to detectable changes in fat mass in external fat depots, suggesting increased sensitivity of BMAT to EPO treatment compared with white adipose tissue. These changes exemplify EPO response in non-hematopoietic tissue of bone and BMAT that accompany bone marrow stimulated erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.

Short-term glucocorticoid excess blunts abaloparatide-induced increase in femoral bone mass and strength in mice

Glucocorticoids (GCs), such as prednisolone, are widely used to treat inflammatory diseases. Continuously long-term or high dose treatment with GCs is one of the most common causes of secondary osteoporosis and is associated with sarcopenia and increased risk of debilitating osteoporotic fragility fractures. Abaloparatide (ABL) is a potent parathyroid hormone-related peptide analog, which can increase bone mineral density (aBMD), improve trabecular microarchitecture, and increase bone strength. The present study aimed to investigate whether GC excess blunts the osteoanabolic effect of ABL. Sixty 12–13-week-old female RjOrl:SWISS mice were allocated to the following groups: Baseline, Control, ABL, GC, and GC + ABL. ABL was administered as subcutaneous injections (100 μg/kg), while GC was delivered by subcutaneous implantation of a 60-days slow-release prednisolone-pellet (10 mg). The study lasted four weeks. GC induced a substantial reduction in muscle mass, trabecular mineral apposition rate (MAR) and bone formation rate (BFR/BS), and endocortical MAR compared with Control, but did not alter the trabecular microarchitecture or bone strength. In mice not receiving GC, ABL increased aBMD, bone mineral content (BMC), cortical and trabecular microarchitecture, mineralizing surface (MS/BS), MAR, BFR/BS, and bone strength compared with Control. However, when administered concomitantly with GC, the osteoanabolic effect of ABL on BMC, cortical morphology, and cortical bone strength was blunted. In conclusion, at cortical bone sites, the osteoanabolic effect of ABL is generally blunted by short-term GC excess.

Osteoprotective effect of bread enriched with protein, dietary fiber, calcium, iron and iodine in hypoestrogen-induced osteoporosis among rats

The search for new strategies for the prevention and control of osteoporosis is an urgent task. Functional foodstuffs and their components are of particular interest in this regard. The aim was to study the effect of bread enriched with protein, dietary fiber, calcium, iron and iodine on the state of the bone tissue of rats in a model of postmenopausal osteoporosis. Material and methods. The experiment was performed on sexually mature female Wistar rats divided into groups: K - control (sham-operated rats, not ovariectomized); О30 - osteoporosis model (animals were sacrificed 30 days after ovariectomy); groups О120 and О120+ - a model of osteoporosis (rats were sacrificed 120 days after ovariectomy). All animals were fed a standard vivary diet. For rats of the О120+ group, from the 40th to the 120th day, enriched bread was included in the diet in an amount of 6 g per 100 g of body weight per day. The bread was fortified with protein (whey protein, blood plasma proteins from farm animals), dietary fiber, calcium (eggshell), iron (purified hemoglobin) and iodized whey protein. Animals of groups K and О120 received unfortified bread in the same amount. Blood levels of total calcium (by colorimetric method), gonadotropins, testosterone, and estradiol (by enzyme-linked immunosorbent assay) were analyzed. Microtomographic evaluation of the architecture and mineral density of the trabecular part of the femur and lumbar vertebrae was performed. Histomorphological analysis of the uterus and femur of animals was performed. Results and discussion. In animals of the О120+ group, in comparison with the О120 sample, there was a decrease in blood testosterone and a marked compensatory release of follicle-stimulating hormone, while no changes were detected in the concentration of estradiol and the state of the uterus atrophied against the background of ovariectomy. There was an increase in the trabecular mineral density of the femur and lumbar vertebrae. The proportion of bone trabeculae in the total volume of the femoral metaphysis (BV/TV) in animals of the О120+ sample was 12.5±0.66% compared to 10.4±0.52% in the О120 group. The values of the structural model index (SMI) reflecting the loss of bone strength and the trabecularity coefficient (TbPf) in О120+ rats (1.44±0.07 and 5.96±0.29 1/mm) were significantly lower than these parameters in the О120 group (1.74±0.08; 9.13±0.46 1/mm, р<0.05). The micro-architectural structure of the femur in the О120+ group of rats was close to that of the О30 sample, which serves as a model of the early stage of osteoporosis (SMI 1.42±0.07; TbPf 5.55±0.28 1/mm). The percentage of bone resorption perimeter and the number of osteoclasts in the О120+ femoral trabeculae were lower than in the О120 group. In the О120+ group, active osteoblasts were observed in a significant part of the resorption cavities. Cell differentiation more was observed in the osteogenic direction than in the adipogenic direction. Conclusion. Bread enriched with protein, fiber, calcium, iron and iodine, effectively weakens osteoporosis induced by ovariectomy in rats. Its inclusion in the diet may be beneficial for the prevention and treatment of systemic postmenopausal osteoporosis.

Large osteocyte lacunae in iliac crest infantile bone are not associated with impaired mineral distribution or signs of osteocytic osteolysis.

The enlargement of osteocyte lacunae via osteocytic osteolysis was previously detected in situations of increased calcium demand (e.g., lactation, vitamin D deficiency). However, it is unclear whether similar processes occur also in the growing infantile skeleton and how this is linked to the mineral distribution within the bone matrix. Human iliac crest biopsies of 30 subjects (0-6months, n=14; 2-8years, n=6 and 18-25years, n=10) were acquired. Bone microarchitecture was assessed by micro-CT, while cellular bone histomorphometry was performed on undecalcified histological sections. Quantitative backscattered electron imaging (qBEI) was conducted to determine the bone mineral density distribution (BMDD) as well as osteocyte lacunar size and density. We additionally evaluated cathepsin K positive osteocytes using immunohistochemistry. Infantile bone was characterized by various signs of ongoing bone development such as higher bone (re)modeling, lower cortical and trabecular thickness compared to young adults. Importantly, a significantly higher osteocyte lacunar density and increased lacunar area were detected. Large osteocyte lacunae were associated with a more heterogeneous bone mineral density distribution of the trabecular bone matrix due to the presence of hypermineralized cartilage remnants, whereas the mean mineralization (i.e., CaMean) was not different in infantile bone. Absence of cathepsin K expression in osteocyte lacunae indicated nonexistent osteocytic osteolysis. Taken together, we demonstrated that the overall mineralization distribution in infantile bone is not altered compared to young adults besides high trabecular mineralization heterogeneity. Our study also provides important reference values for bone microstructure, BMDD and osteocyte characteristics in infants, children and young adults. Infantile bone displays large osteocyte lacunae indicating a developmental phenomenon rather than osteocytic osteolysis. Larger osteocytes may have superior mechanosensory abilities to enable bone adaption during growth.

The long-term residual effects of low-magnitude mechanical stimulation therapy on skeletal health

BackgroundLow-magnitude mechanical stimulation (LMMS) may improve skeletal health. The objective of this research was to investigate the long-term residual effects of LMMS on bone health. 10-week old female mice were given LMMS for 8 weeks; SHAM did not receive LMMS. Some groups remained on study for an additional 8 or 16 weeks post treatment (N = 17).ResultsEpiphyseal trabecular mineralizing surface to bone surface ratio (MS/BS) and bone formation rate (BFR/BS) were significantly greater in the LMMS group compared to the SHAM group at 8 weeks by 92 and 128% respectively. Mineral apposition rate (MAR) was significantly greater in the LMMS group 16 weeks post treatment by 14%.Metaphyseal trabecular bone mineral density (BMD) increased by 18%, bone volume tissue volume ratio (BV/TV) increased by 37%, and trabecular thickness (Tb.Th.) increased by 10% with LMMS at 8 weeks post treatment. Significant effects 16 weeks post treatment were maintained for BV/TV and Tb.Th. The middle-cortical region bone volume (BV) increased by 4% and cortical thickness increased by 3% with 8-week LMMS.ConclusionsLMMS improves bone morphological parameters immediately after and in some cases long-term post LMMS. Results from this work will be helpful in developing treatment strategies to increase bone health in younger individuals.

Secondary alterations in bone mineralisation and trabecular thickening occur after long-term estrogen deficiency in ovariectomised rat tibiae, which do not coincide with initial rapid bone loss.

Recent studies have shown that osteoporosis is not simply a disease of bone loss and microarchitectural degradation but that important changes in tissue composition also occur. Such changes may be a secondary response to early bone loss, but the time sequence of changes in bone mineral distribution is not fully understood. The objective of this study was to quantify the temporal effects of estrogen deficiency on trabecular mineral distribution in the tibia of ovariectomised (OVX) rats. Weekly in vivo micro-CT scans and morphometric and bone mineral density distribution analyses of the proximal tibia were conducted for the first 4 weeks of estrogen deficiency and then at 8, 14 and 34 weeks. Here we report that although trabecular bone volume and architecture are significantly deteriorated within the first 4 weeks of estrogen deficiency, there is no change in the distribution of bone mineral within trabeculae during this initial period. The rate of bone loss in OVX animals dramatically reduced between week 4 and week 14, which coincided with the initiation of increases in trabecular thickness and mineralisation in the OVX group. Together this study reveals for the first time that alterations in bone mineralisation and trabecular thickening arise secondary to the initial rapid bone loss. We propose that these secondary mineralisation changes act to reinforce the trabecular network in an attempt to compensate for the increased loading that ensues after severe bone loss. This study provides an insight into temporal changes in bone mineral distribution in estrogen deficiency.

Bone-like features in skate suggest a novel elasmobranch synapomorphy and deep homology of trabecular mineralization patterns

Bone is a defining characteristic of the vertebrate skeleton, and while chondrichthyans (sharks, skates, and other cartilaginous fishes) are vertebrates, they are hypothesized to have lost the ability to make bone during their evolution. Multiple descriptions of a bone-like tissue in neural arches of vertebrae in various shark species (selachians), however, challenge this hypothesis. Here, we extend this argument by analyzing vertebrae of two members of the batoids (the little skate Leucoraja erinacea and Eaton’s skate Bathyraja eatonii), the sister group to selachians within elasmobranchs. Micro-CT images showed a bone-like mineralization pattern in neural arches of each skate species, and histological analyses confirmed that this bone-like tissue surrounded a cartilage core, exactly as described in sharks. Another mineralization pattern identified in skate vertebrae was distinct from the polygonal tesseral and areolar patterns that classically are associated with the chondrichthyan endoskeleton. Many regions of the vertebrae, including the neural spine and transverse processes, showed this perichondral mineralization pattern, termed here trabecular tesseral. Other than the cartilage core of the neural arch, all mineralized tissues in skate vertebrae had flattened cells surrounded by matrix with bone-like histology. Analyses of quantitative microstructural parameters revealed that, compared to rat vertebrae, the bone-like mineralization pattern in the neural arches of skate vertebrae was more similar to compact bone than trabecular bone. In contrast, the thickness of the trabecular tesseral pattern was more similar to trabecular bone than compact bone of rat vertebrae. In conclusion, a bone-like tissue in neural arches of skate vertebrae appears to be a novel elasmobranch synapomorphy. We propose that the trabecular tesseral mineralization pattern in the skate might have deep homology to the mineralization pattern utilized in trabecular bone. Statement of SignificanceMineralization patterns of skeletal tissues have not been investigated thoroughly in all vertebrate clades. Despite their designation as ‘cartilaginous fish’, chondrichthyans clearly evolved from ancestral vertebrates that made bone. The consensus that chondrichthyans lost the ability to make bone during their evolution, however, is challenged by reports of bone and bone-like tissues in the neural arches of vertebrae in extant sharks (selachians). Here, we provide evidence from micro-CT imaging and histological analyses to support our hypothesis that a bone-like tissue is present in the neural arches of batoids (the sister group to selachians within elasmobranchs). These results argue strongly that the neural arch bone-like tissue is a previously unknown synapomorphy of elasmobranchs. In addition to the bone-like mineralization pattern identified in the neural arches, micro-CT images also showed a novel mineralization pattern which we described as trabecular tesseral. Quantitative microstructural features shared between trabecular tesseral pattern and trabecular bone (from homologous rat vertebrae) suggest that both patterns might derive from an ancestral gene network driving trabecular mineralization (i.e., deep homology).

The effect of parity and lactation on postmenopausal osteoporosis in the Korean National Health and Nutrition Examination Survey (KNHANES)

Introduction: Maternal skeletal loss during pregnancy is minimal despite the fetal demand for calcium. However, in case of some pregnancies with risk factors, the maternal skeleton undergoes more progressive bone loss due to excessive bone resorption. Loss of trabecular mineral content resulted by lactation is expected to be subsequently restored, but only a few studies in Korea have investigated the effects of pregnancy and lactation on postmenopausal osteoporosis.

The Bone Sparing Effects of 2-Methoxyestradiol Are Mediated via Estrogen Receptor-α in Male Mice.

2-Methoxyestradiol (2ME2), a metabolite of 17β-estradiol (E2), exerts bone sparing effects in animal models. We hypothesized that the underlying mechanism is back conversion of 2ME2 to E2, which subsequently acts via estrogen receptor (ER)α. We measured serum E2 levels in orchidectomized wild-type (WT) mice treated with 2ME2 66.6 μg/d or placebo. In placebo-treated animals, E2 was below the detection limit. In 2ME2-treated mice, the serum E2 level was 4.97 ± 0.68 pg/mL. This corresponds to the level found in diesterus in cycling female mice. Next, we investigated bone parameters in orchidectomized WT and ERα knockout mice treated with 2ME2 or placebo for 35 days. 2ME2 (6.66 μg/d) preserved trabecular and cortical bone in WT mice. Trabecular volumetric-bone mineral density was 64 ± 20%, and trabecular bone volume/total volume was 60 ± 20% higher in the metaphyseal region of the femur in the 2ME2 group, compared with placebo (P < .01). Both trabecular number and trabecular thickness were increased (P < .01). Cortical bone mineral content in the diaphyseal region of the femur was 31 ± 3% higher in the 2ME2 group, compared with placebo (P < .001). This was due to larger cortical area (P < .001). Three-point bending showed an increased bone strength in WT 2ME2-treated animals compared with placebo (maximum load [Fmax] +19±5% in the 2ME2 group, P < .05). Importantly, no bone parameter was affected by 2ME2 treatment in ERα knockout mice. In conclusion, 2ME2 treatment of orchidectomized mice results in increased serum E2. ERα mediates the bone sparing effects of 2ME2. The likely mediator of this effect is E2 resulting from back conversion of 2ME2.

Bone metabolism during pregnancy

During pregnancy, mineral concentrations, of calcium and phosphorus in particular, are maintained at a high level in fetal blood so that the developing skeleton may accrete adequate mineral content. The placenta actively transports minerals for this purpose. Maternal intestinal absorption increases in order to meet the fetal demand for calcium, which is only partly dependent on calcitriol. Mineral regulation is essentially dependent on parathyroid hormone (PTH) and PTH-related protein (PTHrP). The calcium-sensing receptor (CaSR) regulates PTH and PTHrP production. If calcium intake is insufficient, the maternal skeleton will undergo resorption due to PTHrP. After birth, a switch from fetal to neonatal homeostasis occurs through increase in PTH and calcitriol, and developmental adaptation of the kidneys and intestines with bone turnover contributing additional mineral to the circulation. Calcium absorption becomes progressively active and dependent on calcitriol. The postnatal skeleton can transiently present with osteoposis but adequate mineral diet usually allows full restoration. Cases of primary osteoporosis must be identified. Loss of trabecular mineral content occurs during lactation in order to provide calcium to the newborn. This programmed bone loss is dependent on a “brain-breast-bone” circuit. The physiological bone resorption during reproduction does not normally cause fractures or persistent osteoporosis. Women who experience fracture are likely to have other causes of bone loss.

Altered distributions of bone tissue mineral and collagen properties in women with fragility fractures

Heterogeneity of bone tissue properties is emerging as a potential indicator of altered bone quality in pathologic tissue. The objective of this study was to compare the distributions of tissue properties in women with and without histories of fragility fractures using Fourier transform infrared (FTIR) imaging. We extended a prior study that examined the relationship of the mean FTIR properties to fracture risk by analyzing in detail the widths and the tails of the distributions of FTIR properties in biopsies from fracture and non-fracture cohorts. The mineral and matrix properties of cortical and trabecular iliac crest tissue were compared in biopsies from women with a history of fragility fracture (+Fx; n=21, age: mean 54±SD 15y) and with no history of fragility fracture (−Fx; n=12, age: 57±5y). A subset of the patients included in the −Fx group were taking estrogen-plus-progestin hormone replacement therapy (HRT) (−Fx+HRT n=8, age: 58±5y) and were analyzed separately from patients with no history of HRT (−Fx−HRT n=4, age: 56±7y). When the FTIR parameter mean values were examined by treatment group, the trabecular tissue of −Fx−HRT patients had a lower mineral:matrix ratio (M:M) and collagen maturity (XLR) than that of −Fx+HRT patients (−22% M:M, −18% XLR) and +Fx patients (−17% M:M, −18% XLR). Across multiple FTIR parameters, tissue from the −Fx−HRT group had smaller low-tail (5th percentile) values than that from the −Fx+HRT or +Fx groups. In trabecular collagen maturity and crystallinity (XST), the −Fx−HRT group had smaller low-tail values than those in the –Fx+HRT group (−16% XLR, −5% XST) and the +Fx group (−17% XLR, −7% XST). The relatively low values of trabecular mineral:matrix ratio and collagen maturity and smaller low-tail values of collagen maturity and crystallinity observed in the −Fx−HRT group are characteristic of younger tissue. Taken together, our data suggest that the presence of newly formed tissue that includes small/imperfect crystals and immature crosslinks, as well as moderately mature tissue, is an important characteristic of healthy, fracture-resistant bone. Finally, the larger mean and low-tail values of mineral:matrix ratio and collagen maturity noted in our −Fx+HRT vs. −Fx−HRT biopsies are consistent with greater tissue age and greater BMD arising from decreased osteoclastic resorption in HRT-treated patients.

Decreased bone cortical density at the forearm in subjects with subclinical peripheral arterial disease.

The association between peripheral arterial disease (PAD) and low bone mass is controversial. In our study, peripheral quantitative computed tomography shows a reduction of cortical but not trabecular, bone mineral density (BMD) at the forearm, in patients with subclinical PAD. Some controversy exists regarding the association between peripheral arterial disease (PAD) and low bone mass. Previous studies have evaluated bone mineral density (BMD) in patients with subclinical PAD, with mixed results. Inconsistency of data may depend on the fact that most studies measured areal bone mineral density (aBMD) by Dual-energy-x ray absorptiometry (DXA). Because DXA cannot distinguish between cortical and trabecular compartments, we reasoned that a study aimed to establish whether these compartments were differentially affected by PAD status could give more information on the nature of this association. In this cross-sectional study, we used peripheral quantitative computed tomography (pQCT) to examine volumetric cortical and trabecular mineral density at the radius (vBMD) in a cohort of subjects with subclinical PAD as defined by ABI ≤0.90 and compared them with healthy subjects with no evidence of PAD. Patients with subclinical PAD had significantly reduced cortical density (1101.0 ± 45.4 vs 1156.2 ± 51.3 mg/cm(3), p < 0.001) and cortical area (75.0 ± 20.9 vs 99.9 ± 18.2 mm(2), p < 0.001) than healthy subjects. Trabecular density (178.1 ± 47.9 vs 165.8 ± 29.6 mg/cm(3)) was not significantly different in the two groups. Subclinical PAD induces a selective bone loss at the radius compartment, not identified by standard DXA, which seems to occur primarily at the cortical level.

SPR4-peptide alters bone metabolism of normal and HYP mice

ContextASARM-peptides are substrates and ligands for PHEX, the gene responsible for X-linked hypophosphatemic rickets (HYP). PHEX binds to the DMP1-ASARM-motif to form a trimeric-complex with α5β3-integrin on the osteocyte surface and this suppresses FGF23 expression. ASARM-peptide disruption of this complex increases FGF23 expression. We used a 4.2kDa peptide (SPR4) that binds to ASARM-peptide and ASARM-motif to study DMP1–PHEX interactions and to assess SPR4 for treating inherited hypophosphatemic rickets. DesignSubcutaneously transplanted osmotic pumps were used to infuse SPR4-peptide or vehicle into wild-type mice (WT) and HYP-mice for 4weeks. ResultsAsymmetrically distributed mineralization defects occurred with WT-SPR4 femurs. Specifically, SPR4 induced negative effects on trabecular bone and increased bone volume and mineralization in cortical-bone. Markedly increased sclerostin and reduced active β-catenin occurred with HYP mice. SPR4-infusion suppressed sclerostin and increased active β-catenin in WT and HYP mice and improved HYP-mice trabecular mineralization defects but not cortical mineralization defects. ConclusionsSPR4-peptide has bimodal activity and acts by: (1) preventing DMP1 binding to PHEX and (2) sequestering an inhibitor of DMP1–PHEX binding, ASARM-peptide. In PHEX defective HYP-mice the second pathway predominates. Although SPR4-peptide improved trabecular calcification defects, decreased sclerostin and increased active β-catenin it did not correct HYP-mice cortical mineralization defects on a normal phosphate diet. Thus, for inherited hypophosphatemic rickets patients on a normal phosphate diet, SPR4-peptide is not a useful therapeutic.

Anti-sclerostin antibody treatment in a rat model of progressive renal osteodystrophy.

Chronic kidney disease (CKD) is associated with abnormalities in bone quantity and quality, leading to increased fractures. Recent studies suggest abnormalities of Wnt signaling in animal models of CKD and elevated sclerostin levels in patients with CKD. The goal of this study was to evaluate the effectiveness of anti-sclerostin antibody treatment in an animal model of progressive CKD with low and high parathyroid hormone (PTH) levels. Cy/+ male rats (CKD) were treated without or with calcium in the drinking water at 25 weeks of age to stratify the animals into high PTH and low PTH groups, respectively, by 30 weeks. Animals were then treated with anti-sclerostin antibody at 100 mg/kg i.v. weekly for 5 doses, a single 20-µg/kg subcutaneous dose of zoledronic acid, or no treatment, and were then euthanized at 35 weeks. As a positive control, the efficacy of anti-sclerostin antibody treatment was also evaluated in normal littermates. The results demonstrated that the CKD animals with high PTH had lower calcium, higher phosphorus, and lower FGF23 compared to the CKD animals with low PTH. Treatment with anti-sclerostin antibody had no effect on any of the biochemistries, whereas zoledronic acid lowered dkk-1 levels. The anti-sclerostin antibody increased trabecular bone volume/total volume (BV/TV) and trabecular mineralization surface in animals with low PTH, but not in animals with high PTH. Neither anti-sclerostin antibody nor zoledronic acid improved biomechanical properties in the animals. Cortical porosity was severe in high-PTH animals and was unaffected by either treatment. In contrast, in normal animals treated with anti-sclerostin antibody, there was an improvement in bone volume, cortical geometry, and biomechanical properties. In summary, this is the first study to test the efficacy of anti-sclerostin antibody treatment on animals with advanced CKD. We found efficacy in improving bone properties only when the PTH levels were low.