High incidence of imperforate vagina in ADGRA3-deficient mice.

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, 17ß-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.

Preoperative proximal tibial bone density, bone microarchitecture, and bone turnover are not associated with postoperative tibial component migration in cemented and cementless medial unicompartmental knee replacements: secondary analyses from a randomized controlled trial.

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.

Transitory Activation and Improved Transition from Erosion to Formation within Intracortical Bone Remodeling in Hypoparathyroid Patients Treated with rhPTH(1-84).

In hypoparathyroidism, lack of parathyroid hormone (PTH) leads to low calcium levels and decreased bone remodeling. Treatment with recombinant human PTH (rhPTH) may normalize bone turnover. This study aimed to investigate whether rhPTH(1-84) continued to activate intracortical bone remodeling after 30 months and promoted the transition from erosion to formation and whether this effect was transitory when rhPTH(1-84) was discontinued. Cortical histomorphometry was performed on 60 bone biopsies from patients (aged 31 to 78 years) with chronic hypoparathyroidism randomized to either 100 µg rhPTH(1-84) a day (n = 21) (PTH) or similar placebo (n = 21) (PLB) for 6 months as add-on to conventional therapy. This was followed by an open-label extension, where patients extended their rhPTH(1-84) (PTH) (n = 5), continued conventional treatment (CON) (n = 5), or withdrew from rhPTH(1-84) and resumed conventional therapy (PTHw) for an additional 24 months (n = 8). Bone biopsies were collected at months 6 (n = 42) and 30 (n = 18). After 6 and 30 months, the overall cortical microarchitecture (cortical porosity, thickness, pore density, and mean pore diameter) in the PTH group did not differ from that of the PLB/CON and PTHw groups. Still, the PTH group had a significantly and persistently higher percentage of pores undergoing remodeling than the PLB/CON groups. A significantly higher percentage of these pores was undergoing bone formation in the PTH compared with the PLB/CON groups, whereas the percentage of pores with erosion only was not different. This resulted in a shift in the ratio between formative and eroded pores, reflecting a faster transition from erosion to formation in the PTH-treated patients. In the rhPTH(1-84) withdrawal group PTHw, the latter effects of PTH were completely reversed in comparison to those of the PLB/CON groups. In conclusion, rhPTH(1-84) replacement therapy in hypoparathyroidism patients promotes intracortical remodeling and its transition from erosion to formation without affecting the overall cortical microstructure. The effect persists for at least 30 months and is reversible when treatment is withdrawn. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Trabecular bone deterioration in a postmenopausal female suffering multiple spontaneous vertebral fractures due to a delayed denosumab injection - A post-treatment re-initiation bone biopsy-based case study.

Background: Denosumab, is a potent anti-resorptive that, increases bone mineral density, and reduces fracture risk in osteoporotic patients. However, several case studies have reported multiple vertebral fractures in patients discontinuing denosumab. Case presentation: This case report describes a 64-year-old female with postmenopausal osteoporosis treated with denosumab, who had her 11th injection delayed by 4 months. The patient suffered eight spontaneous vertebral fractures. After consent, an iliac crest bone biopsy was obtained following re-initiation of the denosumab treatment and analyzed by micro-computed tomography and histomorphometry. Results: micro-computed tomography analysis revealed a low trabecular bone volume of 10 %, a low trabecular thickness of 97 µm, a low trabecular spacing of 546 µm, a high trabecular number of 1.8/mm, and a high structure model index of 2.2, suggesting trabecular thinning and loss of trabecular plates. Histomorphometric trabecular bone analysis revealed an eroded perimeter per bone perimeter of 33 % and an osteoid perimeter per bone perimeter of 62 %. Importantly, 88 % of the osteoid perimeter was immediately above an eroded-scalloped cement line with no sign of mineralization, and often with no clear bone-forming osteoblasts on the surface. Moreover, only 5 % of the bone perimeter was mineralizing, reflecting that only 8 % of the osteoid perimeter underwent mineralization, resulting in a mineralization lag time of 545 days. Taken together, this indicates limited bone formation and delayed mineralization. Conclusion: We present a case report of multiple vertebral fractures after denosumab discontinuation with histomorphometric evidence that denosumab discontinuation leads to extensive trabecular bone resorption followed by a limited bone formation and delayed mineralization if the denosumab treatment is reinitiated. This highlights the importance of developing optimal discontinuation strategies for patients that are to discontinue treatment.

Correlative study of liquid in human bone by 3D neutron microscopy and lab-based X-ray µCT.

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 H2O. 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 H2O is very distinct in the neutron data, while D2O, H2O, and organic matter can barely be distinguished from air in the X-ray data.

Polychromatic neutron phase-contrast imaging of weakly absorbing samples enabled by phase retrieval.

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 D2O 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 D2O, 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.

Local coordination between intracortical bone remodeling and vascular development in human juvenile bone.

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.

Flexible design in the stomatopod dactyl club.

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.

Drill-Hole Bone Defects in Animal Models of Bone Healing: Protocol for a Systematic Review.

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.

2D size of trabecular bone structure units (BSU) correlate more strongly with 3D architectural parameters than age in human vertebrae.

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.

Anti-sclerostin antibodies and abaloparatide have additive effects when used as a countermeasure against disuse osteopenia in female rats.

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.

Hypomorphic expression of parathyroid Bmal1 disrupts the internal parathyroid circadian clock and increases parathyroid cell proliferation in response to uremia.

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;Bmal1flox/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;Bmal1flox/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.

The Programmed Death-1 Pathway Counter-Regulates Inflammation-Induced Osteoclast Activity in Clinical and Experimental Settings.

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.

Effect of Acetazolamide and Zoledronate on Simulated High Altitude-Induced Bone Loss.

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.

The Effect of Bone Marrow Stimulation for Cartilage Repair on the Subchondral Bone Plate.

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/mm3 vs. 21 1/mm3, 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.

Hypobaric hypoxia deteriorates bone mass and strength in mice.

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.

Opportunities for biomineralization research using multiscale computed X-ray tomography as exemplified by bone imaging.

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.

Artificial intelligence-assisted identification and quantification of osteoclasts.

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.

Ultrastructural identification of developing proximal tubules based on three-dimensional reconstruction.

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.

Mesenchymal Stem Cell Extracellular Vesicles as Adjuvant to Bone Marrow Stimulation in Chondral Defect Repair in a Minipig Model.

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/cm3 vs. 838 mg HA/cm3, 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.