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Despite distinct aetiologies, the end-stages of primary osteoarthritis (OA) and secondary OA are described by common radiological features. However, the morphology of the bone–cartilage unit may differ depending on the pathogenesis. In this cross-sectional study, we aimed to investigate the histological differences in the bone–cartilage unit of the femoral head between patients with primary OA and secondary OA due to rheumatoid arthritis (RA). Femoral heads were obtained from 12 patients with primary OA, six patients with secondary OA due to RA, and 12 control subjects. The femoral heads were investigated using stereological methods to ensure unbiased quantification. The volume (mean difference [95% confidence interval]) (2.1 [0.5;3.8] cm3, p = 0.016) and thickness (413 [78.9;747] µm, p = 0.029) of the articular cartilage and the thickness of the calcified cartilage (56.4 [0.4;113] µm, p = 0.017) were larger in patients with primary OA than in patients with secondary OA due to RA. Femoral head volume (1.2 [−3.6;6.1] cm3, p = 0.598), bone volume fraction (−1.1 [−2.8;5.1] cm3, p = 0.553), subchondral bone thickness (−2.5 [−212;207] µm, p = 0.980), and osteophyte area (25.3 [−53.6;104] cm2, p = 0.506) did not differ between patients. The thicker calcified cartilage in primary OA preceding the loss of articular cartilage can be attributed to endochondral ossification. Patients with secondary OA due to RA had severely thinner calcified cartilage as the pathogenesis is driven by inflammation and is characterized by a generalized and more severe loss of articular cartilage. [ABSTRACT FROM AUTHOR]

Objective: To investigate bone changes in the metacarpophalangeal (MCP) joints of anti-citrullinated peptide antibody (ACPA)-positive patients with arthralgia, but not arthritis, compared to healthy controls.Method: Using a cross-sectional study design, patients were recruited from hospitals and private care rheumatologists, and controls from a test subject website. All subjects underwent medical history interview, clinical examination, and biochemical screening including ACPA. Patients with positive ACPA, arthralgia, and no rheumatic disease were included. Controls without a history or signs of rheumatological disease or positive ACPA were included. A 2.7-cm-long region around the second and third MCP joints was evaluated using high-resolution peripheral quantitative computed tomography with a voxel size of 82 µm.Results: Twenty-nine ACPA-positive patients and 29 healthy controls were evaluated. Trabecular volumetric bone mineral density and bone volume fraction did not differ between the groups. In addition, the cortical bone was not affected in patients, as we found no difference in average cortical thickness and cortical bone area between the groups. In contrast, the trabeculae were significantly (p < 0.05) thinner in both second and third MCP heads compared with controls, whereas trabecular number and trabecular separation did not differ between the groups. No erosions were demonstrated and the number of non-specific breaks did not differ between the groups.Conclusion: Trabecular bone changes were observed in ACPA-positive patients with arthralgia compared with healthy controls. The results may reflect inflammatory up-regulated trabecular bone resorption leading to early bone loss before the onset of clinical arthritis. [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE: Parathyroid hormone (PTH) has attracted considerable interest as a bone anabolic agent. Recently, it has been suggested that PTH can also enhance bone repair after fracture and distraction osteogenesis. We analyzed bone density and strength of the newly regenerated mineralized tissue after intermittent treatment with PTH in rabbits, which undergo Haversian bone remodeling similar to that in humans. METHODS: 72 New Zealand White rabbits underwent tibial mid-diaphyseal osteotomy and the callus was distracted 1 mm/day for 10 days. The rabbits were divided into 3 groups, which received injections of PTH 25 µg/kg/day for 30 days, saline for 10 days and PTH 25 µg/kg/day for 20 days, or saline for 30 days. At the end of the study, the rabbits were killed and the bone density was evaluated with DEXA. The mechanical bone strength was determined by use of a 3-point bending test. RESULTS: In the 2 PTH-treated groups the regenerate callus ultimate load was 33% and 30% higher, absorbed energy was 100% and 65% higher, BMC was 61% and 60% higher, and callus tissue volume was 179% and 197% higher than for the control group. INTERPRETATION: We found that treatment with PTH during distraction osteogenesis resulted in substantially higher mineralized tissue volume, mineral content, and bending strength. This suggests that treatment with PTH may benefit new bone formation during distraction osteogenesis and could form a basis for clinical application of this therapy in humans. [ABSTRACT FROM AUTHOR]

Objective: To compare the number of new erosions in two metacarpophalangeal (MCP) joints over one year assessed by high-resolution peripheral quantitative computed tomography (HR-pQCT) and conventional radiography (CR). Furthermore, to estimate the diagnostic accuracy of erosive progression by CR with HR-pQCT as the reference standard., Methods: Paired sets of image data were available from patients with RA (n=310), who underwent HR-pQCT and CR, including the 2 nd and 3 rd MCP joints only of the dominant hand at baseline and at the one-year follow-up. Erosion number was determined using HR-pQCT and CR. Erosive volume was estimated from segmented HR-pQCT images, and erosion scores were obtained by the Sharp/van der Heijde method from CR. Erosive progression was defined as an increase in total erosion number or a study-specified increase in total erosive volume or total erosion score., Results: At baseline, 250 erosions were identified by CR compared to 1864 erosions by HR-pQCT. After one year, 3 new erosions were detected by CR compared to 66 new erosions by HR-pQCT. Erosive progression was identified in 40 patients using HR-pQCT and in 3 patients using CR. With HR-pQCT as reference, CR had a sensitivity of 2.5% (95% CI: 0.1-13.2%) and a specificity of 99.3% (95% CI: 97.3-99.9%) for detecting erosive progression., Conclusion: HR-pQCT identified more than 20 times the number of new erosions, and more than 10 times as many patients with erosive progression than CR. HR-pQCT is a sensitive tool for monitoring new erosions and erosive progression over one year in RA., (Copyright © 2024. Published by Elsevier Masson SAS.)

Purpose: Documentation of the wrist stabilizing effect and mechanical properties of common splinting materials is warranted to support evidence-based condition-specific recommendations for wrist immobilization. The objectives of this study were to assess the wrist stabilizing properties of volar and dorsal short-arm splints made of four different materials and evaluate the mechanical properties of the splinting materials., Methods: Dorsal and volar short arm splints made of plaster of Paris (PoP) (eight layers), Woodcast (2 mm, rigid vented), X-lite (classic, two layers) or a 3D-printed material (polypropylene) were sequentially mounted on 10 cadaveric arm specimens and fixed in a radiolucent fixture. This enabled the evaluation of maximum wrist flexion and extension relative under an orthogonal load of 42 N via radiographic images. In addition, a three-point bending test was performed on ten sheet duplicates of each of the four splinting materials., Results: When applied as a volar splint, PoP had the highest capability to resist wrist flexion and extension. However, when applied as a dorsal splint, Woodcast exhibited a lower wrist flexion and a similar wrist extension. The 3D-printed splints-both volar and dorsal-showed the highest mean wrist flexion and extension. The mechanical properties of the Woodcast, X-lite and 3D-printed splinting materials were very similar. PoP exhibited distinct properties with a stiffness of 146 (95% confidence interval [CI]: 120-173) N/mm and a deflection at F max of 0.6 (95% CI: 0.5-0.7) mm compared to ≤7.7 (95% CI: 7.4-7.9) N/mm and ≥20 (95% CI: 18-22) mm for the other materials., Conclusion: PoP displayed better wrist stabilizing properties and material stiffness than Woodcast, X-lite and 3D-printed polypropylene. When considering wrist stabilizing properties, PoP may still prove to be the preferred choice for wrist immobilization., Level of Evidence: Not applicable., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Journal of Experimental Orthopaedics published by John Wiley & Sons Ltd on behalf of European Society of Sports Traumatology, Knee Surgery and Arthroscopy.)

Denosumab is a monoclonal anti-RANKL antibody that inhibits bone resorption, increases bone mass, and reduces fracture risk. Denosumab discontinuation causes an extensive wave of rebound resorption, but the cellular mechanisms remain poorly characterized. We utilized in situ hybridization (ISH) as a direct approach to identify the cells that activate osteoclastogenesis through the RANKL/OPG pathway. ISH was performed across species, skeletal sites, and following recombinant OPG (OPG:Fc) and parathyroid hormone 1-34 (PTH) treatment of mice. OPG:Fc treatment in mice induced an increased expression of RANKL mRNA mainly in trabecular, but not endocortical bone surface cells. Additionally, a decreased expression of OPG mRNA was detected in bone surface cells and osteocytes of both compartments. A similar but more pronounced effect on RANKL and OPG expression was seen one hour after PTH treatment. These findings suggest that bone surface cells and osteocytes conjointly regulate the activation of osteoclastogenesis, and that OPG:Fc treatment induces a local accumulation of osteoclastogenic activation sites, ready to recruit and activate osteoclasts upon treatment discontinuation. Analysis of publicly available single-cell RNA sequencing (scRNAseq) data from murine bone marrow stromal cells revealed that Tnfsf11 + cells expressed high levels of Mmp13, Limch1, and Wif1, confirming their osteoprogenitor status. ISH confirmed co-expression of Mmp13 and Tnfsf11 in bone surface cells of both vehicle- and OPG:Fc-treated mice. Under physiological conditions of human/mouse bone, RANKL is expressed mainly by osteoprogenitors proximate to the osteoclasts, while OPG is expressed mainly by osteocytes and bone-forming osteoblasts., (© 2024. The Author(s).)

Insulin-like growth factor (IGF) signaling is required for proper growth and skeletal development in vertebrates. Consequently, its dysregulation may lead to abnormalities of growth or skeletal structures. IGF is involved in the regulation of cell proliferation and differentiation of chondrocytes. However, the availability of bioactive IGF may be controlled by antagonizing IGF binding proteins (IGFBPs) in the circulation and tissues. As the metalloproteinase PAPP-A specifically cleaves members of the IGFBP family, we hypothesized that PAPP-A activity liberates bioactive IGF in cartilage. In PAPP-A knockout mice, the femur length was reduced and the mice showed a disorganized columnar organization of growth plate chondrocytes. Similarly, zebrafish lacking pappaa showed reduced length of Meckel's cartilage and disorganized chondrocytes, reminiscent of the mouse knockout phenotype. Expression of chondrocyte differentiation markers (sox9a, ihha, and col10a1) was markedly affected in Meckel's cartilage of pappaa knockout zebrafish, indicating that differentiation of chondrocytes was compromised. Additionally, the zebrafish pappaa knockout phenotype was mimicked by pharmacological inhibition of IGF signaling, and it could be rescued by treatment with exogenous recombinant IGF-I. In conclusion, our data suggests that IGF activity in the growing cartilage, and hence IGF signaling in chondrocytes, requires the presence of PAPP-A. The absence of PAPP-A causes aberrant chondrocyte organization and compromised growth in both mice and zebrafish., (© 2024. The Author(s).)

Nephron loop-vessel countercurrent arrangement in the medulla provides the structural basis for the formation of concentrated urine. To date, the morphogenesis of it and relevant water and solutes transportation has not been fully elucidated. In this study, with immunohistochemistry for aquaporins (AQP) and Na-K-2Cl co-transporter (NKCC2), as well as 3D visualization, we noticed in embryonic day 14.5 kidneys that the countercurrent arrangement of two pairs of loop-vessel was established as soon as the loop and vessel both extended into the medulla. One pair happened between descending limb and ascending vasa recta, the other occurred between thick ascending limb and descending vasa recta. Meanwhile, the immunohistochemical results showed that the limb and vessel expressing AQP-1 such as descending thick and thin limb and descending vasa recta was always accompanied with AQP-1 negative ascending vasa recta or capillaries and thick ascending limb, respectively. Moreover, the thick ascending limb expressing NKCC2 closely contacted with descending vasa recta without expressing NKCC2. As kidney developed, an increasing number of loop-vessels in countercurrent arrangement extended into the interstitium of the medulla. In addition, we observed that the AQP-2 positive ureteric bud and their branches were separated from those pairs of tubule-vessels by a relatively large and thin-walled veins or capillaries. Thus, the present study reveals that the loop-vessel countercurrent arrangement is formed at the early stage of nephrogenesis, which facilitates the efficient transportation of water and electrolytes to maintain the medullary osmolality and to form a concentrated urine., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objectives: To investigate the diagnostic accuracy of high-resolution peripheral quantitative computed tomography (HR-pQCT) to assess erosive progression during one year compared to conventional radiography (CR) in rheumatoid arthritis (RA)., Methods: This prospective study included 359 patients with RA (disease duration ≥ 5 years) between March 2018 and October 2020. HR-pQCT and CR were obtained at inclusion and after one year. Erosive assessment was performed at two metacarpophalangeal joints of the dominant hand using HR-pQCT and progression was defined as an increase in erosion number ≥ 1 or an increase in erosive volume > least significant change. CR of hands, wrists, and feet were evaluated using Sharp/van der Heijde scores and erosive progression was defined as a 1.1-point increase in erosion score according to the smallest detectable change., Results: In paired analyses (n = 310), erosive progression was identified in 30 patients using CR and in 40 patients using HR-pQCT. In the 40 patients with erosive progression on HR-pQCT, progression was not identified by CR in 33 patients. Adding HR-pQCT to CR doubled the proportion of patients identified with progression from 30 (10%) to 63 (20%) patients. Using CR as the reference, the sensitivity (% (95% CI)) of HR-pQCT for identifying erosive progression was 23.3 (9.9-42.3) and the specificity was 88.2 (83.8-91.7)., Conclusion: A substantial proportion of patients with erosive progression are overlooked using CR only to monitor erosive progression. Adding high-resolution peripheral CT to CR doubles the proportion of patients, who may benefit from individualised therapy targeting erosive progression in RA., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Ten percent of the female population suffers from congenital abnormalities of the vagina, uterus, or oviducts, with severe consequences for reproductive and psychological health. Yet, the underlying causes of most of these malformations remain largely unknown. ADGRA3 (GPR125) is involved in WNT signaling and planar cell polarity, mechanisms vital to female reproductive tract development. Although ADGRA3 is a well-established spermatogonial stem cell marker, its role within the female urogenital system remains unclear., Results: In this study, we found Adgra3 to be expressed throughout the murine female urogenital system, with higher expression pre-puberty than after sexual maturation. We generated a global Adgra3 -/- mouse line and observed imperforate vagina in 44% of Adgra3 -/- females, resulting in distension of the reproductive tract and infertility. Ovarian morphology, plasma estradiol, ovarian Cyp19a1, and vaginal estrogen receptor α (Esr1) expression were unaffected. However, compared to controls, a significantly lower bone mineral density was found in Adgra3 -/- mice. Whereas vaginal opening in mice is an estrogen-dependent process, 17β-estradiol treatment failed to induce vaginal canalization in Adgra3 -/- mice. Furthermore, a marked reduction in vaginal and ovarian progesterone receptor expression was observed concomitant with an upregulation of apoptotic regulators Bcl2, Bid, and Bmf in adult Adgra3 -/- females with a closed vagina., Conclusions: Our collective results shed new insights into the complex mechanisms by which the adhesion receptor ADGRA3 regulates distal vaginal tissue remodeling during vaginal canalization via altered sex hormone responsiveness and balance in apoptotic regulators. This highlights the potential of ADGRA3 as a target in diagnostic screening and/or therapy for obstructive vaginal malformations in humans., (© 2024. The Author(s).)

Background and Purpose: Cementless arthroplasty fixation relies on early bone ingrowth and may be poor in patients with low proximal tibial bone density or abnormal bone turnover. We aimed first to describe the baseline bone properties in patients undergoing medial unicompartmental knee replacement (UKR), and second to investigate its association with cemented and cementless tibial component migration until 2 years., Methods: A subset investigation of 2 patient groups from a 3-armed randomized controlled trial was conducted. There were 26 cemented and 25 cementless medial UKRs with twin-pegged femoral components. Volumetric bone mineral density (vBMD) and microstructure of the excised medial tibial plateau were ascertained with µCT. Bone turnover was estimated using dynamic histomorphometry (eroded surface/bone surface = ES/BS, osteoid surface/bone surface = OS/BS, mineralizing surface/bone surface = MS/BS). Tibial component migration in 4 feature points was followed for 2 years with radiostereometry., Results: At the 2-year follow-up, the cementless tibial components migrated 0.38 mm (95% confidence interval [CI] 0.14-0.62) total translation more than the cemented components at the posterior feature point. The greatest migration in the cementless group was subsidence at the posterior feature point of 0.66 mm (CI 0.48-0.84) until 6 weeks, and from 3 months the components were stable. Cemented tibial components subsided very little. Between 1- and 2-year follow-ups, no cementless but 4 cemented tibial components revealed continuous migration. OS/BS was half of the ES/BS. No µCT or histomorphometric parameters showed any clinically relevant correlation with tibial component migration at the posterior feature point for either cemented or cementless UKR at 6 weeks' or 2 years' follow-up after adjustment for age, BMI, and sex., Conclusion: Preoperative vBMD, bone turnover, and microstructure were not associated with postoperative tibial component migration of cemented and cementless medial UKR.

Liquid plays an important role in bone that has a complex 3D hierarchical pore structure. However, liquid (water) is difficult to discern from e.g. an organic matrix by X-ray imaging. Therefore, we use a correlative approach using both high resolution X-ray and neutron imaging. Human femoral bone with liquid adsorbed into some of the pores was imaged with both the Neutron Microscope at the ICON beamline, SINQ at PSI, and by lab-based μCT using 2.7 μm voxel size. Segmentation of the two datasets showed that, even though the liquid was clearly distinguishable in the neutron data and not in the X-ray data, it remained challenging to segment it from bone due to overlaps of peaks in the gray level histograms. In consequence, segmentations from X-ray and neutron data varied significantly. To address this issue, the segmented X-ray porosities was overlaid on the neutron data, making it possible to localize the liquid in the vascular porosities of the bone sample and use the neutron attenuation to identify it as H 2 O. The contrast in the neutron images was lowered slightly between the bone and the liquid compared to the bone and the air. This correlative study shows that the complementary use of X-rays and neutrons is very favorable, since H 2 O is very distinct in the neutron data, while D 2 O, H 2 O, and organic matter can barely be distinguished from air in the X-ray data., Competing Interests: Declaration of competing interest None., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Although failure to establish a vascular network has been associated with many skeletal disorders, little is known about what drives development of vasculature in the intracortical bone compartments. Here, we show that intracortical bone resorption events are coordinated with development of the vasculature. We investigated the prevalence of vascular structures at different remodeling stages as well as their 3D organization using proximal femoral cortical bone from 5 girls and 6 boys (aged 6-15 years). A 2D analysis revealed that non-quiescent intracortical pores contained more vascular structures than quiescent pores (p < 0.0001). Type 2 pores, i.e., remodeling of existing pores, had a higher density of vascular structures than type 1 pores, i.e., de novo created pores (p < 0.05). Furthermore, pores at the eroded-formative remodeling stage, had more vascular structures than pores at any other remodeling stage (p < 0.05). A 3D reconstruction of an intracortical remodeling event showed that osteoclasts in the advancing tip of the cutting cone as well as preosteoclasts in the lumen expressed vascular endothelial growth factor-A (VEGFA), while VEGFA-receptors 1 and 2 mainly were expressed in endothelial cells in the adjacent vasculature. Consequently, we propose that the progression of the vascular network in intracortical remodeling events is driven by osteoclasts expressing VEGFA. Moreover, the vasculature is continuously reconfigured according to the demands of the remodeling events at the surrounding bone surfaces., Competing Interests: Declaration of competing interest The following research grants have been received: However, none of our founders had any role in conceptualization, analysis, or interpretation of the study and I can, as last author of the submitted manuscript declare that none of the authors have any personal financial interest to declare in connection this manuscript., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The use of a phase-retrieval technique for propagation-based phase-contrast neutron imaging with a polychromatic beam is demonstrated. This enables imaging of samples with low absorption contrast and/or improving the signal-to-noise ratio to facilitate e.g. time-resolved measurements. A metal sample, designed to be close to a phase pure object, and a bone sample with canals partially filled with D 2 O were used for demonstrating the technique. These samples were imaged with a polychromatic neutron beam followed by phase retrieval. For both samples the signal-to-noise ratios were significantly improved and, in the case of the bone sample, the phase retrieval allowed for separation of bone and D 2 O, which is important for example for in situ flow experiments. The use of deuteration contrast avoids the use of chemical contrast enhancement and makes neutron imaging an interesting complementary method to X-ray imaging of bone., (© Maja Østergaard et al. 2023.)

The stomatopod is a fascinating animal that uses its weaponized appendage dactyl clubs for breaking mollusc shells. Dactyl clubs are a well studied example of biomineralized hierarchical structures. Most research has focused on the regions close to the action, namely the impact region and surface composed of chitin and apatite crystallites. Further away from the site of impact, the club has lower mineralization and more amorphous phases; these areas have not been as actively studied as their highly mineralized counterparts. This work focuses on the side of the club, in what is known as the periodic and striated regions. A combination of laboratory micro-computed tomography, synchrotron X-ray diffraction mapping and synchrotron X-ray fluorescence mapping has shown that the mineral in this region undergoes the transition from an amorphous to a crystalline phase in some, but not all, clubs. This means that this side region can be mineralized by either an amorphous phase, calcite crystallites or a mixture of both. It was found that when larger calcite crystallites form, they are organized (textured) with respect to the chitin present in this biocomposite. This suggests that chitin may serve as a template for crystallization when the side of the club is fully mineralized. Further, calcite crystallites were found to form as early as 1 week after moulting of the club. This suggests that the side of the club is designed with a significant safety margin that allows for a variety of phases, i.e. the club can function independently of whether the side region has a crystalline or amorphous mineral phase., (open access.)

The adhesion receptor ADGRA3 (GPR125) is a known spermatogonial stem cell marker, but its impact on male reproduction and fertility has not been examined. Using a mouse model lacking Adgra3 (Adgra3 -/- ), we show that 55% of the male mice are infertile from puberty despite having normal spermatogenesis and epididymal sperm count. Instead, male mice lacking Adgra3 exhibited decreased estrogen receptor alpha expression and transient dilation of the epididymis. Combined with an increased estradiol production, this indicates a post-pubertal hormonal imbalance and fluid retention. Dye injection revealed a blockage between the ejaculatory duct and the urethra, which is rare in mice suffering from infertility, thereby mimicking the etiologies of obstructive azoospermia found in human male infertility. To summarize, male reproductive tract development is dependent on ADGRA3 function that in concert with estrogen signaling may influence fluid handling during sperm maturation and storage., (© 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Background: Bone fractures are common conditions of the musculoskeletal system. Several animal models of bone fractures have been established to help elucidate the complex process of bone healing. In the last decades, drill-hole bone defects have emerged as a method to study bone healing. Animal models of drill-hole defects are easy to standardize and do not require external fixation of the bone. However, current studies of drill-hole bone defects lack detailed descriptions of techniques and interstudy standardization., Objective: This systematic review aims to present a detailed description of the different methods used to induce drill-hole bone defects in long bones of laboratory animals and to provide a comprehensive overview of their methodology and potential for investigation of bone healing., Methods: A systematic search of PubMed and Embase will be performed of abstracts containing variations of the following four keywords: "long bone," "drill-hole," "regeneration," and "animal model." Abstract screening and full-text screening will be performed independently by 2 reviewers, and data will be extracted to a predesigned extraction protocol. The primary outcome of the included studies is the technique used to create the drill-hole bone defect, and secondary outcomes are any measurements or analyses of bone defect and regeneration. A narrative synthesis will be used to present the primary outcome, while information on secondary outcomes will be displayed graphically. The study protocol follows the PRISMA-P (Preferred Reporting Items for Systematic Review and Meta-analysis Protocols) guidelines., Results: Abstract and full-text screening is ongoing and is expected to be completed by October 2022. Data extraction will commence immediately after, and the manuscript is expected to be completed by December 2023. The systematic review will follow the PRISMA statement., Conclusions: The strength of this systematic review is that it provides a comprehensive methodological overview of the different drill-hole methods and their advantages and disadvantages. This will assist researchers in choosing which model to use when studying different aspects of bone healing., Trial Registration: International Prospective Register of Systematic Reviews CRD42020213076; https://tinyurl.com/bp56wdwe., International Registered Report Identifier (irrid): PRR1-10.2196/34887., (©Frederik Duch Bromer, Mikkel Bo Brent, Jesper Skovhus Thomsen, Annemarie Brüel. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 18.07.2022.)

Prolonged disuse and substantial mechanical unloading are particularly damaging to skeletal integrity. Preclinical studies in rodents and clinical studies have highlighted the need for potent bone anabolic drugs to counteract disuse-induced osteopenia. The aim of present study was to compare the efficacy of romosozumab (Scl-Ab) and abaloparatide (ABL), alone or in combination, to prevent botulinum toxin (BTX) induced bone loss in a rat model. Eighty female Wistar rats were divided into the following six groups: 1. Baseline (n = 12); 2. Control (Ctrl) (n = 12); 3. BTX (n = 12); 4. BTX + Scl-Ab (n = 16); 5. BTX + ABL (n = 12); and 6. BTX + Scl-Ab + ABL (n = 16). Disuse was achieved by injecting 4 IU BTX into the hind limb musculature at study start. Scl-Ab (25 mg/kg) was injected s.c. twice weekly, while ABL (80 μg/kg) was injected s.c. five days a week for four weeks. Hind limb disuse dramatically decreased muscle mass and skeletal integrity and deteriorated the cortical morphology and trabecular microstructure. Treatment with Scl-Ab alone prevented most of the adverse cortical and trabecular effects of disuse, while ABL monotherapy mainly attenuated the disuse-induced loss of femoral areal bone mineral density (aBMD). Moreover, the combination of Scl-Ab and ABL not only counteracted most of the negative skeletal effects of unloading, but also increased aBMD (+10% and +20%), epiphyseal trabecular bone volume fraction (BV/TV) (+25% and +73%), and metaphyseal bone strength (+18% and +30%) significantly above that of Scl-Ab or ABL monotherapy, respectively. The potent and additive osteoanabolic effect of Scl-Ab and ABL, when given in combination, is highly intriguing and underlines that an osteoanabolic bone gain can be maximized by utilizing two pharmaceuticals targeting different cellular signaling pathways. From a clinical perspective, a combination treatment may be warranted in patients where the osteoanabolic effect of either monotherapy is not sufficient, or if a dose-reduction is required due to adverse effects., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Bone tissue is continuously remodeled. In trabecular bone, each remodeling transaction forms a microscopic bone structural unit (BSU), also known as a hemiosteon or a trabecular packet, which is bonded to existing tissue by osteopontin-rich cement lines. The size and shape of the BSUs are determined by the size and shape of the resorption cavity, and whether the cavity is potentially over- or under-filled by the subsequent bone formation. The present study focuses on the recently formed trabecular BSUs, and how their 2D size and shape changes with age and trabecular microstructure. The study was performed using osteopontin-immunostained frontal sections of L2 vertebrae from 8 young (aged 18.5-37.6 years) and 8 old (aged 69.1-96.4 years) control females, which underwent microcomputed tomography (μCT) imaging prior to sectioning. The contour of 4230 BSU profiles (181-385 per vertebra) within 1024 trabecular profiles were outlined, and their 2D width, length, area, and shape were assessed. Of these BSUs, 22 (0.5%) were generated by modeling-based bone formation (i.e. without prior resorption), while 99.5% were generated by remodeling-based bone formation (i.e. with prior resorption). The distributions of BSU profile width, length, and area were significantly smaller in the old versus young females (p < 0.005), and the median profile width, length, and area were negative correlated with age (p < 0.018). Importantly, these BSU profile size parameters were more strongly correlated with trabecular bone volume (BV/TV, p < 0.002) and structure model index (SMI, p < 0.008) assessed by μCT, than age. Moreover, the 2D BSU size parameters were positively correlated to the area of the individual trabecular profiles (p < 0.0001), which were significantly smaller in the old versus young females (p < 0.024). The BSU shape parameters (aspect ratio, circularity, and solidity) were not correlated with age, BV/TV, or SMI. Collectively, the study supports the notion that not only the BSU profile width, but also its length and area, are more influenced by the age-related bone loss and shift from plates to rods (SMI), than age itself. This implies that BSU profile size is mainly driven by changes in the trabecular microstructure, which affect the size of the resorption cavity that the BSU refills., (Copyright © 2022. Published by Elsevier Inc.)

The molecular circadian clock is an evolutionary adaptation to anticipate recurring changes in the environment and to coordinate variations in activity, metabolism and hormone secretion. Parathyroid hyperplasia in uremia is a significant clinical challenge. Here, we examined changes in the transcriptome of the murine parathyroid gland over 24 hours and found a rhythmic expression of parathyroid signature genes, such as Casr, Vdr, Fgfr1 and Gcm2. Overall, 1455 genes corresponding to 6.9% of all expressed genes had significant circadian rhythmicity. Biological pathway analysis indicated that the circadian clock system is essential for the regulation of parathyroid cell function. To study this, a novel mouse strain with parathyroid gland-specific knockdown of the core clock gene Bmal1 (PTHcre;Bmal1 flox/flox ) was created. Dampening of the parathyroid circadian clock rhythmicity was found in these knockdown mice, resulting in abrogated rhythmicity of regulators of parathyroid cell proliferation such as Sp1, Mafb, Gcm2 and Gata3, indicating circadian clock regulation of these genes. Furthermore, the knockdown resulted in downregulation of genes involved in mitochondrial function and synthesis of ATP. When superimposed by uremia, these PTHcre;Bmal1 flox/flox mice had an increased parathyroid cell proliferative response, compared to wild type mice. Thus, our findings indicate a role of the internal parathyroid circadian clock in the development of parathyroid gland hyperplasia in uremia., (Copyright © 2022 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Objective: The programmed death-1 (PD-1) pathway is essential for maintaining self-tolerance and plays an important role in autoimmunity, including rheumatoid arthritis (RA). Here, we investigated how membrane-bound and soluble (s)PD-1 influence bone homeostasis during chronic inflammation, exemplified in RA., Methods: Bone mineral density and bone microstructure were examined in PD-1 and PD-L1 knockout (KO) mice and compared with wild-type (WT) mice. Receptor activator of nuclear factor kappa-B ligand (RANKL) was measured in serum, and the expression examined on activated bone marrow cells. Osteoclast formation was examined in cells from murine spleen and bone marrow and from human synovial fluid cells. sPD-1 was measured in chronic and early (e)RA patients and correlated to markers of disease activity and radiographic scores., Results: PD-1 and PD-L1 KO mice showed signs of osteoporosis. This was supported by a significantly reduced trabecular bone volume fraction and deteriorated microstructure, as well as increased osteoclast formation and an increased RANKL/OPG ratio. The recombinant form of sPD-1 decreased osteoclast formation in vitro , but was closely associated with disease activity markers in eRA patients. Sustained elevated sPD-1 levels indicated ongoing inflammation and were associated with increased radiographic progression., Conclusion: The PD-1 pathway is closely associated with bone homeostasis, and lacking members of this pathway causes a deteriorated bone structure. The immunological balance in the microenvironment determines how the PD-1 pathway regulates osteoclast formation. In eRA patients, sPD-1 may serve as a biomarker, reflecting residual but clinically silent disease activity and radiographic progression., Competing Interests: GF and AS have patents/pending royalties on the PD-1 pathway from Roche, Merck, Bristol-Myers-Squibb, EMD-Serono, Boehringer-Ingelheim, AstraZeneca, Dako, and Novartis. GF has equity in Nextpoint, Triursus, iTeos, and Xios. GF has served on advisory boards for Roche, Bristol-Myers-Squibb, Xios, Origimed, iTeos, and Nextpoint. AS has served on advisory boards for Novartis, Surface Oncology, Elstar, SQZ Biotechnologies, Adaptimmune, Elpiscience, and Monopteros. AS has received research funding from Novartis, Roche, UCB, Ipsen, Merck, and Quark. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Greisen, Kragstrup, Thomsen, Hørslev-Pedersen, Hetland, Stengaard-Pedersen, Østergaard, Ørnbjerg, Junker, Sharpe, Freeman, Hvid, Moestrup, Hauge and Deleuran.)

Biominerals typically have complex hierarchical structures traversing many length scales. This makes their structural characterization complicated, since it requires 3D techniques that can probe full specimens at down to nanometer-resolution, a combination that is difficult - if not impossible - to achieve simultaneously. One challenging example is bone, a mineralized tissue with a highly complex architecture that is replete with a network of cells. X-ray computed tomography techniques enable multiscale structural characterization through the combination of various equipment and emerge as promising tools for characterizing biominerals. Using bone as an example, we discuss how combining different X-ray imaging instruments allow characterizing bone structures from the nano- to the organ-scale. In particular, we compare and contrast human and rodent bone, emphasize the importance of the osteocyte lacuno-canalicular network in bone, and finally illustrate how combining synchrotron X-ray imaging with laboratory instrumentation for computed tomography is especially helpful for multiscale characterization of biominerals., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Exposure to hypobaric hypoxia at high altitude puts mountaineers at risk of acute mountain sickness. The carbonic anhydrase inhibitor acetazolamide is used to accelerate acclimatization, when it is not feasible to make a controlled and slow ascend. Studies in rodents have suggested that exposure to hypobaric hypoxia deteriorates bone integrity and reduces bone strength. The study investigated the effect of treatment with acetazolamide and the bisphosphonate, zoledronate, on the skeletal effects of exposure to hypobaric hypoxia. Eighty 16-week-old female RjOrl : SWISS mice were divided into five groups: 1. Baseline; 2. Normobaric; 3. Hypobaric hypoxia; 4. Hypobaric hypoxia + acetazolamide, and 5. Hypobaric hypoxia + zoledronate. Acetazolamide was administered in the drinking water (62 mg/kg/day) for four weeks, and zoledronate (100 μg/kg) was administered as a single subcutaneous injection at study start. Exposure to hypobaric hypoxia significantly increased lung wet weight and decreased femoral cortical thickness. Trabecular bone was spared from the detrimental effects of hypobaric hypoxia, although a trend towards reduced bone volume fraction was found at the L4 vertebral body. Treatment with acetazolamide did not have any negative skeletal effects, but could not mitigate the altitude-induced bone loss. Zoledronate was able to prevent the altitude-induced reduction in cortical thickness. In conclusion, simulated high altitude affected primarily cortical bone, whereas trabecular bone was spared. Only treatment with zoledronate prevented the altitude-induced cortical bone loss. The study provides preclinical support for future studies of zoledronate as a potential pharmacological countermeasure for altitude-related bone loss., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Brent, Simonsen, Thomsen and Brüel.)

Purpose: To investigate the effect of bone-marrow stimulation (BMS) on subchondral bone plate morphology and remodeling compared to untreated subchondral bone in a validated minipig model., Methods: Three Göttingen minipigs received BMS with drilling as treatment for two chondral defects in each knee. The animals were euthanized after six months. Follow-up consisted of a histological semiquantitative evaluation using a novel subchondral bone scoring system and micro computed tomography (µCT) of the BMS subchondral bone. The histological and microstructural properties of the BMS-treated subchondral bone were compared to that of the adjacent healthy subchondral bone., Results: The µCT analysis showed that subchondral bone treated with BMS had significantly higher connectivity density compared to adjacent untreated subchondral bone (26 1/mm 3 vs. 21 1/mm 3 , P = 0.048). This was the only microstructural parameter showing a significant difference. The histological semiquantitative score differed significantly between the subchondral bone treated with BMS and the adjacent untreated subchondral (8.0 vs. 10 P = < 0.001). Surface irregularities were seen in 43% and bone overgrowth in 27% of the histological sections. Only sparse formation of bone cysts was detected (1%)., Conclusions: BMS with drilling does not cause extensive changes to the subchondral bone microarchitecture. Furthermore, the morphology of BMS subchondral bone resembled that of untreated subchondral bone with almost no formation of bone cyst, but some surface irregularities and bone overgrowth.

Mountaineers at high altitude are at increased risk of acute mountain sickness as well as high altitude pulmonary and cerebral edema. A densitometric study in mountaineers has suggested that expeditions at high altitude decrease bone mineral density. Surprisingly, the in vivo skeletal effects of hypobaric hypoxia are largely unknown, and have not been studied using advanced contemporary methods to assess bone microstructure. Eighty-four 22-week-old female mice were divided into seven groups with 12 mice in each group: 1. Baseline; 2. Normobaric, 4 weeks; 3. Hypobaric hypoxia, 4 weeks; 4. Normobaric, 8 weeks; 5. Hypobaric hypoxia, 8 weeks; 6. Normobaric, 12 weeks; and 7. Hypobaric hypoxia, 12 weeks. Hypobaric hypoxia mice were housed in hypobaric chambers at an ambient pressure of 500 mbar (5500 m altitude), while normobaric mice were housed at sea level atmospheric pressure for 4, 8, or 12 weeks, respectively. Hypobaric hypoxia had a profound impact on femoral cortical bone and L4 trabecular bone, while the effect on femoral trabecular bone was less pronounced. Hypobaric hypoxia reduced the bone strength of the femoral mid-diaphysis and L4 at all time-points. At femoral cortical bone, hypobaric hypoxia reduced bone formation through fewer mineralizing surfaces and lower bone formation rate after 2 weeks. In addition, bone strength decreased, and C-terminal telopeptide of type I collagen (CTX-I) increased independently of the duration of exposure to simulated high altitude. At L4, hypobaric hypoxia resulted in a substantial reduction in bone volume fraction, trabecular thickness, and trabecular number after 4 weeks of exposure. Hypobaric hypoxia reduced bone strength and femoral bone mass, while femoral trabecular bone was much less affected, indicating the skeletal response to hypobaric hypoxia differ between cortical and trabecular bone. These findings provide initial preclinical support for future clinical studies in mountaineers to assess bone status and bone strength after exposure to prolonged high altitude exposure., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: This study evaluated the effects of mesenchymal stem cell-extracellular vesicles (MSC-EVs) on chondrocyte proliferation in vitro and on cartilage repair in vivo following bone marrow stimulation (BMS) of focal chondral defects of the knee., Methods: Six adult Göttingen minipigs received 2 chondral defects in each knee. The pigs were randomized to treatment with either BMS combined with MSC-EVs or BMS combined with phosphate-buffered saline (PBS). Intraarticular injections MSC-EVs or PBS were performed immediately after closure of the surgical incisions, and at 2 and 4 weeks postoperatively. Repair was evaluated after 6 months with gross examination, histology, histomorphometry, immunohistochemistry, and micro-computed tomography (µCT) analysis of the trabecular bone beneath the defect., Results: Defects treated with MSC-EVs had more bone in the cartilage defect area than the PBS-treated defects (7.9% vs. 1.5%, P = 0.02). Less than 1% of the repair tissue in both groups was hyaline cartilage. International Cartilage and Joint Preservation Society II histological scoring showed that defects treated with MSC-EVs scored lower on "matrix staining" (20.8 vs. 50.0, P = 0.03), "cell morphology" (35.4 vs. 53.8, P = 0.04), and "overall assessment" (30.8 vs. 52.9, P = 0.03). Consistently, defects treated with MSC-EVs had lower collagen II and higher collagen I areal deposition. Defects treated with MSC-EVs had subchondral bone with significantly higher tissue mineral densities than PBS-treated defects (860 mg HA/cm 3 vs. 838 mg HA/cm 3 , P = 0.02)., Conclusion: Intraarticular injections of MSC-EVs in conjunction with BMS led to osseous ingrowth that impaired optimal cartilage repair, while enhancing subchondral bone healing.

Background: The cellular mechanisms involved in the development of proximal tubules are not only associated with morphogenesis in fetal life, but also with restoration of damaged tubules in adulthood. Knowledge about morphological features of cell differentiation and proliferation along the developing tubule is insufficient, which hinders identification of the cellular origin., Objectives: This study aimed to investigate ultrastructures of the proximal tubule at different stages of nephrogenesis., Methods: Electron microscopy was used and guided by computer-assisted tubular tracing to identify the cellular structures., Results: Renal vesicles and S-shaped bodies revealed more proliferative features, such as densely-packed fusiform-shaped cells with numerous protein-producing organelles than membrane specializations typical for mature tubules. At the capillary-loop stage the proximal tubules demonstrated all characteristics of the mature tubules, but not as developed, including shorter but densely packed microvilli, fewer lateral processes with cell-cell contacts, lower basal membrane infoldings, and lower mitochondrial volume density. However, they exhibited an elaborated endocytic system above the nucleus, indicating a membrane transport is being established. Abundant free- and endoplasmic reticulum-adhered ribosomes and Golgi complexes reflected active protein synthesis for cell growth and proliferation. Interestingly, electron dense cells were occasionally intermixed with electron lucent cells characterized by various organelles in less cytosol and a larger nucleus with abundant euchromatin, which is a feature of active proliferation., Conclusions: These ultrastructures indicate that the morphogenesis of the developing proximal tubule corresponds to the gradually established physiological activities. The two different cellular electron densities may suggest distinctive differentiation of the cells along the tubule., (© 2021 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

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.

Bromer, Frederik Duch, Mikkel Bo Brent, Michael Pedersen, Jesper Skovhus Thomsen, Annemarie Brüel, and Casper Bindzus Foldager. The effect of normobaric intermittent hypoxia therapy on bone in normal and disuse osteopenic mice. High Alt Med Biol . 22: 225-234, 2021. Background: Systemic intermittent hypoxia therapy (IHT) has been shown to elicit beneficial effects on multiple physiological systems. However, only few studies have investigated the effect of long-term normobaric IHT on bone mass and mechanical and microstructural properties. The aim of the present study was to examine the effect of IHT on bone in both healthy and osteopenic mice. Materials and Methods: Thirty mice were stratified into four groups: Ctrl, Ctrl+IHT, Botox, and Botox+IHT. Osteopenia was induced by injecting Botox into the right hindlimb of the mice causing paralysis and disuse. IHT animals were placed in a normobaric hypoxia-chamber (10% oxygen) for 1 hour twice daily 5 days/week. Animals were sacrificed after 21 days, and DEXA, micro-computed tomography, and mechanical testing were performed on the femora. Results: As expected, Botox resulted in a significant reduction of bone mineral content (-23.4%), area bone mineral density (-19.1%), femoral neck strength (F max : -54.7%), bone volume fraction (bone volume/tissue volume: -41.8%), and trabecular thickness (-32.4%). IHT had no measurable effect on the bone properties in either healthy or osteopenic mice. Conclusion: The study confirmed that Botox led to loss of bone mass, deterioration of trabecular microstructure, and loss of bone strength. These changes were not influenced by IHT. Notably, IHT had no detrimental effect on bone in either healthy or osteopenic mice. This indicates that IHT of ailments outside of the skeletal system may be administered without causing harm to the bone.

Objective: Several different animal models are used to study disuse-induced bone loss. This systematic review aims to give a comprehensive overview of the animal models of disuse-induced bone loss and provide a detailed narrative synthesis of each unique animal model., Methods: PubMed and Embase were systematically searched for animal models of disuse from inception to November 30, 2019. In addition, Google Scholar and personal file archives were searched for relevant publications not indexed in PubMed or Embase. Two reviewers independently reviewed titles and abstracts for full-text inclusion. Data were extracted using a predefined extraction scheme to ensure standardization., Results: 1964 titles and abstracts were screened of which 653 full-text articles were included. The most common animal species used to model disuse were rats (59%) and mice (30%). Males (53%) where used in the majority of the studies and genetically modified animals accounted for 7%. Twelve different methods to induce disuse were identified. The most frequently used methods were hindlimb unloading (44%), neurectomy (15%), bandages and orthoses (15%), and botulinum toxin (9%). The median time of disuse was 21 days (quartiles: 14 days, 36 days) and the median number of animals per group subjected to disuse was 10 (quartiles: 7, 14). Random group allocation was reported in 43% of the studies. Fewer than 5% of the studies justified the number of animals per group by a sample size calculation to ensure adequate statistical power., Conclusion: Multiple animal models of disuse-induced bone loss exist, and several species of animals have successfully been studied. The complexity of disuse-induced bone loss warrants rigid research study designs. This systematic review emphasized the need for standardization of animal disuse research and reporting.

Three bone anabolic pharmaceuticals are currently approved for treatment of osteoporosis, teriparatide (PTH (1-34)), the parathyroid hormone-related protein analog abaloparatide (ABL), and romosozumab. The present study compared the effect of intermittent PTH (1-34) and ABL on bone tissue directly mole-to-mole in female mice. Forty-seven C57BL/6 mice were randomly allocated to the following groups: Baseline ( n = 11), Control (Ctrl) ( n = 12), PTH ( n = 12), and ABL ( n = 12). The mice were injected s.c. with PTH (100 µg/kg), ABL (96 µg/kg), or saline (Ctrl) five days a week for three weeks. To assess the effect of PTH and ABL, the hindlimb bones were analyzed with DXA, µCT, mechanical testing, dynamic bone histomorphometry, and histological quantification of bone cells. In addition, serum calcium concentration was determined. PTH and ABL significantly increased femoral areal bone mineral density (aBMD) (borderline significant p = 0.06 for PTH), femoral mid-diaphyseal bone strength, femoral metaphyseal and epiphyseal and vertebral bone volume fraction (BV/TV), connectivity density, volumetric bone mineral density (vBMD), and bone formation rate (BFR/BS) compared to Ctrl. In addition, ABL also significantly increased mid-diaphyseal cortical thickness and bone area compared to Ctrl. Neither PTH nor ABL significantly increased bone strength at the femoral neck. In conclusion, abaloparatide and PTH have similar bone anabolic properties when compared directly mole-to-mole in mice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2021 Brent, Stoltenborg, Brüel and Thomsen.)

Proteinuria is a well-established marker and predictor of kidney disease. The receptors megalin and cubilin reabsorb filtered proteins and thereby proteinuria is avoided. It is unknown if all segments of the proximal tubule are involved in clearing the filtrate or if there exists a reserve capacity in case of increased glomerular protein filtration. To determine this, we performed serial sectioning of rat kidney and used stereology to quantify the endolysosomal system of the three segments of cortical and juxtamedullary nephrons by electron microscopy. Immunohistochemistry was applied to analyze the adaptor protein Dab2, which assists in megalin mediated endocytosis, megalin, and endocytic uptake of two endogenous megalin ligands; retinol binding protein and β2-microglobulin at exact tubular positions. Proteinuric rats (puromycin-treated) and mice (podocin knock-out) were analyzed to clarify the response of the tubule to increased protein filtration. We found that the endolysosomal system was most prominent in segment 1 and 2, whereas segment 3 was less developed. The depth of ligand uptake varied among nephrons, but it descended into segment 2 although uptake was lower than in segment 1 and it was never observed in segment 3. This was supported by prominent expression of Dab2 in segment 1 and 2. When protein filtration increased, segment 3 was included in the reabsorption process in proteinuric animals. Thus, segment 1 and 2 are responsible for clearing the filtrate for protein during normal physiological conditions, but the tubule exhibits plasticity and is able to include segment 3 under proteinuric stress., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Superoxide dismutase 3 (SOD3) is an extracellular protein with the capacity to convert superoxide into hydrogen peroxide, an important secondary messenger in redox regulation. To investigate the utility of zebrafish in functional studies of SOD3 and its relevance for redox regulation, we have characterized the zebrafish orthologues; Sod3a and Sod3b. Our analyses show that both recombinant Sod3a and Sod3b express SOD activity, however, only Sod3b is able to bind heparin. Furthermore, RT-PCR analyses reveal that sod3a and sod3b are expressed in zebrafish embryos and are present primarily in separate organs in adult zebrafish, suggesting distinct functions in vivo. Surprisingly, both RT-PCR and whole mount in situ hybridization showed specific expression of sod3b in skeletal tissue. To further investigate this observation, we compared femoral bone obtained from wild-type and SOD3 -/- mice to determine whether a functional difference was apparent in healthy adult mice. Here we report, that bone from SOD3 -/- mice is less mineralized and characterized by significant reduction of cortical and trabecular thickness in addition to reduced mechanical strength. These analyses show that SOD3 plays a hitherto unappreciated role in bone development and homeostasis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Quantification of osteoclasts to assess bone resorption is a time-consuming and tedious process. Since the inception of bone histomorphometry and manual counting of osteoclasts using bright-field microscopy, several approaches have been proposed to accelerate the counting process using both free and commercially available software. However, most of the present alternatives depend on manual or semi-automatic color segmentation and do not take advantage of artificial intelligence (AI). The present study directly compare estimates of osteoclast-covered surfaces (Oc.S/BS) obtained by the conventional manual method using a bright-field microscope to that obtained by a new AI-assisted method. We present a detailed step-by-step guide for the AI-based method. Tibiae from Wistar rats were either enzymatically stained for TRAP or immunostained for cathepsin K to identify osteoclasts. We found that estimation of Oc.S/BS by the new AI-assisted method was considerably less time-consuming, while still providing similar results to the conventional manual method. In addition, the retrainable AI-module used in the present study allows for fully automated overnight batch processing of multiple annotated sections.•Bone histomorphometry•AI-assisted osteoclast identification•TRAP and cathepsin K., (© 2021 The Author(s). Published by Elsevier B.V.)

Objective: Calcified cartilage is suggested to be involved in the pathogenesis of osteoarthritis (OA) by facilitating endochondral ossification at the bone-cartilage unit. Therefore, the objective was to quantify the volume and surface area of the calcified cartilage in the femoral head in OA patients and healthy subjects., Materials and Methods: We used design-based stereological principles, i.e., systematic uniform random sampling and vertical uniform random sections of the entire femoral head. We investigated the articular and calcified cartilage and femoral head surface area and volume, excluding fovea capitis and marginal osteophytes, in 20 patients with OA and 15 healthy subjects., Results: The volume of the calcified cartilage was significantly larger for the patients with OA compared with the healthy subjects (mean difference [95% CI]) (284 [110,457] mm 3 , p = 0.002). The upper and lower surface area of the calcified cartilage, i.e. the tidemark and cement line, were both significantly larger for OA patients compared with the healthy subjects (17.8 [8.4,27.3] cm 2 , p < 0.001) and (38.7 [20.8,56.7] cm 2 , p = 0.002), respectively. The volume of the calcified cartilage and the volume of the femoral head were significantly correlated for the patients with OA (Spearman's ρ = 0.51, p = 0.021), but not for the healthy subjects (ρ = 0.41, p = 0.123)., Conclusions: Patients with OA had a larger femoral head surface area and more calcified cartilage compared to healthy subjects. The volume of the calcified cartilage correlated positively with the volume of the femoral head for patients with OA, but not for healthy subjects. This strongly supports the existing view that bone growth in OA is associated with endochondral ossification., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Damage of the lower motor neuron cell bodies or their axons results in reduced or abolished voluntary movement accompanied by a substantial loss of bone and muscle mass. Intermittent parathyroid hormone 1-34 (PTH) (teriparatide) is one of the most potent bone-anabolic treatment regimens. ActRIIA-mFc is an activin type IIA decoy receptor that increases bone mass mediated by inhibition of the activin receptor signaling pathway. We investigated whether PTH or ActRIIA-mFc alone or in combination could prevent loss of bone and muscle mass induced by injecting botulinum toxin A (BTX) into the right hind limb in mice. Seventy-two 16-week-old female C57BL/6 mice were allocated to the following groups: Baseline, Control, BTX, BTX + ActRIIA-mFc (10 mg/kg), BTX + PTH (100 μg/kg), and BTX + ActRIIA-mFc + PTH. The mice were sacrificed after three weeks of disuse and treatment. In contrast to monotherapy with PTH, ActRIIA-mFc alone or in combination with PTH was able partly or completely to prevent disuse-induced loss of whole femoral bone mass, trabecular thickness, and bone strength. Moreover, an additive effect of ActRIIA-mFc and PTH on areal bone mineral density and trabecular bone volume was found. In summary, ActRIIA-mFc and PTH in combination were more effective in preventing disuse-induced bone loss and deterioration of trabecular micro-architecture than either treatment alone., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Obesity is associated with increased plasma glycerol levels. The coordinated regulation of glycerol channels in adipose tissue (AQP7) and the liver (AQP9) has been suggested as an important contributor to the pathophysiology of type-2-diabetes mellitus, as it would provide glycerol for hepatic synthesis of glucose and triglycerides. The regulation of AQP7 and AQP9 is influenced by sex. This study investigates the effect of a high-fat diet (HFD) on glycerol metabolism in mice and the influence of sex and GLP-1-receptor agonist treatment. Female and male C57BL/6JRj mice were fed either a control diet or a HFD for 12 or 24 weeks. Liraglutide was administered (1 mg/kg/day) to a subset of female mice. After 12 weeks of HFD, females had gained less weight than males. In adipose tissue, only females demonstrated an increased abundance of AQP7, whereas only males demonstrated a significant increase in glycerol kinase abundance and adipocyte size. 24 weeks of HFD resulted in a more comparable effect on weight gain and adipose tissue in females and males. HFD resulted in marked hepatic steatosis in males only and had no significant effect on the hepatic abundance of AQP9. Liraglutide treatment generally attenuated the effects of HFD on glycerol metabolism. In conclusion, no coordinated upregulation of glycerol channels in adipose tissue and liver was observed in response to HFD. The effect of HFD on glycerol metabolism is sex-specific in mice, and we propose that the increased AQP7 abundance in female adipose tissue could contribute to their less severe response to HFD., (Copyright © 2020 Iena, Jul, Vegger, Lodberg, Thomsen, Brüel and Lebeck.)