Effects of Raloxifene and tibial loading on bone mass and mechanics in male and female mice.

Purpose: Raloxifene (RAL) is a selective estrogen receptor modulator (SERM) that has previously been shown to cause acellular benefits to bone tissue. Due to these improvements, RAL was combined with targeted tibial loading to assess if RAL treatment during periods of active bone formation would allow for further mechanical enhancements.Methods: Structural, mechanical, and microstructural effects were assessed in bone from C57BL/6 mice that were treated with RAL (0.5 mg/kg), tibial loading, or both for 6 weeks, beginning at 10 weeks of age.Results:Ex vivo microcomputed tomography (CT) images indicated RAL and loading work together to improve bone mass and architecture, especially within the cancellous region of males. Increases in cancellous bone volume fraction were heavily driven by increases in trabecular thickness, though there were some effects on trabecular spacing and number. In the cortical regions, RAL and loading both increased cross-sectional area, cortical area, and cortical thickness. Whole-bone mechanical testing primarily indicated the effects of loading. Further characterization through Raman spectroscopy and nanoindentation showed load-based changes in mineralization and micromechanics, while both loading and RAL caused changes in the secondary collagen structure. In contrast to males, in females, there were large load-based effects in the cancellous and cortical regions, resulting in increased whole-bone mechanical properties. RAL had less of an effect on cancellous and cortical architecture, though some effects were still present.Conclusion: RAL and loading work together to impact bone architecture and mechanical integrity, leading to greater improvements than either treatment individually.

The impact of advanced glycation end products on bone properties in chronic kidney disease.

PURPOSE OF REVIEW: Chronic kidney disease (CKD) affects over 15% of Americans and results in an increased risk of skeletal fractures and fracture-related mortality. However, there remain great challenges in estimating fracture risk in CKD patients, as conventional metrics such as bone density assess bone quantity without accounting for the material quality of the bone tissue. The purpose of this review is to highlight the detrimental effects of advanced glycation end products (AGEs) on the structural and mechanical properties of bone, and to demonstrate the importance of including bone quality when assessing fracture risk in CKD patients. RECENT FINDINGS: Increased oxidative stress and inflammation drive the production of AGEs in CKD patients that form nonenzymatic crosslinks between type I collagen fibrils in the bone matrix. Nonenzymatic crosslinks stiffen and embrittle the bone, reducing its ability to absorb energy and resist fracture. Clinical measurement of AGEs is typically indirect and fails to distinguish the identity and properties of the various AGEs. SUMMARY: Accounting for the impact of AGEs on the skeleton in CKD patients may improve our estimation of overall bone quality, fracture risk, and treatments to improve both bone quantity and quality by reducing AGEs in patients with CKD merit investigation in order to improve our understanding of the etiology of increased fracture risk.

Six months of voluntary alcohol consumption in male cynomolgus macaques reduces intracortical bone porosity without altering mineralization or mechanical properties.

Chronic heavy alcohol consumption is a risk factor for low trauma bone fracture. Using a non-human primate model of voluntary alcohol consumption, we investigated the effects of 6 months of ethanol intake on cortical bone in cynomolgus macaques (Macaca fascicularis). Young adult (6.4 ± 0.1 years old, mean ± SE) male cynomolgus macaques (n = 17) were subjected to a 4-month graded ethanol induction period, followed by voluntary self-administration of water or ethanol (4 % w/v) for 22 h/d, 7 d/wk. for 6 months. Control animals (n = 6) consumed an isocaloric maltose-dextrin solution. Tibial response was evaluated using densitometry, microcomputed tomography, histomorphometry, biomechanical testing, and Raman spectroscopy. Global bone response was evaluated using biochemical markers of bone turnover. Monkeys in the ethanol group consumed an average of 2.3 ± 0.2 g/kg/d ethanol resulting in a blood ethanol concentration of 90 ± 12 mg/dl in longitudinal samples taken 7 h after the daily session began. Ethanol consumption had no effect on tibia length, mass, density, mechanical properties, or mineralization (p > 0.642). However, compared to controls, ethanol intake resulted in a dose-dependent reduction in intracortical bone porosity (Spearman rank correlation = -0.770; p < 0.0001) and compared to baseline, a strong tendency (p = 0.058) for lower plasma CTX, a biochemical marker of global bone resorption. These findings are important because suppressed cortical bone remodeling can result in a decrease in bone quality. In conclusion, intracortical bone porosity was reduced to subnormal values 6 months following initiation of voluntary ethanol consumption but other measures of tibia architecture, mineralization, or mechanics were not altered.

Calcimimetics Alter Periosteal and Perilacunar Bone Matrix Composition and Material Properties in Early Chronic Kidney Disease.

Chronic kidney disease (CKD) affects 15% of Americans and greatly increases fracture risk due to elevated parathyroid hormone, cortical porosity, and reduced bone material quality. Calcimimetic drugs are used to lower parathyroid hormone (PTH) in CKD patients, but their impact on bone matrix properties remains unknown. We hypothesized that tissue-level bone quality is altered in early CKD and that calcimimetic treatment will prevent these alterations. To test this hypothesis, we treated Cy/+ rats, a model of spontaneous and progressive CKD-mineral and bone disorder (CKD-MBD), with KP-2326, a preclinical analogue of etelcalcetide, early in the CKD disease course. To measure tissue-level bone matrix composition and material properties, we performed colocalized Raman spectroscopy and nanoindentation on new periosteal bone and perilacunar bone using hydrated femur sections. We found that CKD and KP treatment lowered mineral type B carbonate substitution whereas KP treatment increased mineral crystallinity in new periosteal bone. Reduced elastic modulus was lower in CKD but was not different in KP-treated rats versus CTRL. In perilacunar bone, KP treatment lowered type B carbonate substitution, increased crystallinity, and increased mineral-to-matrix ratio in a spatially dependent manner. KP treatment also increased reduced elastic modulus and hardness in a spatially dependent manner. Taken together, these data suggest that KP treatment improves material properties on the tissue level through a combination of lowering carbonate substitution, increasing mineral crystallinity, and increasing relative mineralization of the bone early in CKD. As a result, the mechanical properties were improved, and in some regions, were the same as control animals. Therefore, calcimimetics may help prevent CKD-induced bone deterioration by improving bone quality in new periosteal bone and in bone tissue near osteocyte lacunae. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

A novel murine model of combined insulin-dependent diabetes and chronic kidney disease has greater skeletal detriments than either disease individually.

Diabetes and chronic kidney disease (CKD) consistently rank among the top ten conditions in prevalence and mortality in the United States. Insulin-dependent diabetes (IDD) and CKD each increase the risk of skeletal fractures and fracture-related mortality. However, it remains unknown whether these conditions have interactive end-organ effects on the skeleton. We hypothesized that combining IDD and CKD in mice would cause structural and mechanical bone alterations that are more deleterious compared to the single disease states. Female C57BL6/J mice were divided into four groups: 1) N = 12 Control (CTRL), 2) N = 10 Streptozotocin-induced IDD (STZ), 3) N = 10 Adenine diet-induced CKD (AD), and 4) N = 9 Combination (STZ+AD). STZ administration resulted in significantly higher blood glucose, HbA1c (p < 0.0001), and glucose intolerance (p < 0.0001). AD resulted in higher blood urea nitrogen (p = 0.0002) while AD, but not STZ+AD mice, had high serum parathyroid hormone (p < 0.0001) and phosphorus (p = 0.0005). STZ lowered bone turnover (p = 0.001). Trabecular bone volume was lowered by STZ (p < 0.0001) and increased by AD (p = 0.003). Tissue mineral density was lowered by STZ (p < 0.0001) and AD (p = 0.02) in trabecular bone but only lowered by STZ in cortical bone (p = 0.002). Cortical porosity of the proximal tibia was increased by AD, moment of inertia was lower in both disease groups, and most cortical properties were lower in all groups vs CTRL. Ultimate force, stiffness, toughness, and total displacement/strain were lowered by STZ and AD. Fracture toughness was lower by AD (p = 0.003). Importantly, Cohen's D indicated that STZ+AD most strongly lowered bone turnover and mechanical properties. Taken together, structural and material-level bone properties are altered by STZ and AD while their combination resulted in greater detriments, indicating that improving bone health in the combined disease state may require novel interventions.

Non-Additive Effects of Combined NOX1/4 Inhibition and Calcimimetic Treatment on a Rat Model of Chronic Kidney Disease-Mineral and Bone Disorder (CKD-MBD).

Chronic kidney disease-mineral and bone disorder (CKD-MBD) increases cardiovascular calcification and skeletal fragility in part by increasing systemic oxidative stress and disrupting mineral homeostasis through secondary hyperparathyroidism. We hypothesized that treatments to reduce reactive oxygen species formation and reduce parathyroid hormone (PTH) levels would have additive beneficial effects to prevent cardiovascular calcification and deleterious bone architecture and mechanics before end-stage kidney disease. To test this hypothesis, we treated a naturally progressive model of CKD-MBD, the Cy/+ rat, beginning early in CKD with the NADPH oxidase (NOX1/4) inhibitor GKT-137831 (GKT), the preclinical analogue of the calcimimetic etelcalcetide, KP-2326 (KP), and their combination. The results demonstrated that CKD animals had elevated blood urea nitrogen, PTH, fibroblast growth factor 23 (FGF23), and phosphorus. Treatment with KP reduced PTH levels compared with CKD animals, whereas GKT treatment increased C-terminal FGF23 levels without altering intact FGF23. GKT treatment alone reduced aortic calcification and NOX4 expression but did not alter the oxidative stress marker 8-OHdG in the serum or aorta. KP treatment reduced aortic 8-OHdG and inhibited the ability for GKT to reduce aortic calcification. Treatments did not alter heart calcification or left ventricular mass. In the skeleton, CKD animals had reduced trabecular bone volume fraction and trabecular number with increased trabecular spacing that were not improved with either treatment. The cortical bone was not altered by CKD or by treatments at this early stage of CKD. These results suggest that GKT reduces aortic calcification while KP reduces aortic oxidative stress and reduces PTH, but the combination was not additive. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Segregating the effects of ferric citrate-mediated iron utilization and FGF23 in a mouse model of CKD.

Ferric citrate (FC) is an approved therapy for chronic kidney disease (CKD) patients as a phosphate (Pi) binder for dialysis-dependent CKD, and for iron deficiency anemia (IDA) in non-dialysis CKD. Elevated Pi and IDA both lead to increased FGF23, however, the roles of iron and FGF23 during CKD remain unclear. To this end, iron and Pi metabolism were tested in a mouse model of CKD (0.2% adenine) ± 0.5% FC for 6 weeks, with and without osteocyte deletion of Fgf23 (flox-Fgf23/Dmp1-Cre). Intact FGF23 (iFGF23) increased in all CKD mice but was lower in Cre+ mice with or without FC, thus the Dmp1-Cre effectively reduced FGF23. Cre+ mice fed AD-only had higher serum Pi than Cre- pre- and post-diet, and the Cre+ mice had higher BUN regardless of FC treatment. Total serum iron was higher in all mice receiving FC, and liver Tfrc, Bmp6, and hepcidin mRNAs were increased regardless of genotype; liver IL-6 showed decreased mRNA in FC-fed mice. The renal 1,25-dihydroxyvitamin D (1,25D) anabolic enzyme Cyp27b1 had higher mRNA and the catabolic Cyp24a1 showed lower mRNA in FC-fed mice. Finally, mice with loss of FGF23 had higher bone cortical porosity, whereas Raman spectroscopy showed no changes in matrix mineral parameters. Thus, FC- and FGF23-dependent and -independent actions were identified in CKD; loss of FGF23 was associated with higher serum Pi and BUN, demonstrating that FGF23 was protective of mineral metabolism. In contrast, FC maintained serum iron and corrected inflammation mediators, potentially providing ancillary benefit.

Control of Bone Matrix Properties by Osteocytes.

Osteocytes make up 90-95% of the cellular content of bone and form a rich dendritic network with a vastly greater surface area than either osteoblasts or osteoclasts. Osteocytes are well positioned to play a role in bone homeostasis by interacting directly with the matrix; however, the ability for these cells to modify bone matrix remains incompletely understood. With techniques for examining the nano- and microstructure of bone matrix components including hydroxyapatite and type I collagen becoming more widespread, there is great potential to uncover novel roles for the osteocyte in maintaining bone quality. In this review, we begin with an overview of osteocyte biology and the lacunar-canalicular system. Next, we describe recent findings from in vitro models of osteocytes, focusing on the transitions in cellular phenotype as they mature. Finally, we describe historical and current research on matrix alteration by osteocytes in vivo, focusing on the exciting potential for osteocytes to directly form, degrade, and modify the mineral and collagen in their surrounding matrix.