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.

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.

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.

A Systematic Review of Animal Models of Disuse-Induced Bone Loss.

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.

A follistatin-based molecule increases muscle and bone mass without affecting the red blood cell count in mice.

Inhibitors of the activin receptor signaling pathway (IASPs) have become candidate therapeutics for sarcopenia and bone remodeling disorders because of their ability to increase muscle and bone mass. However, IASPs utilizing activin type IIA and IIB receptors are also potent stimulators of erythropoiesis, a feature that may restrict their usage to anemic patients because of increased risk of venous thromboembolism. Based on the endogenous TGF-ß superfamily antagonist follistatin (FST), a molecule in the IASP class, FSTΔHBS-mFc, was generated and tested in both ovariectomized and naive BALB/c and C57BL/6 mice. In ovariectomized mice, FSTΔHBS-mFc therapy dose-dependently increased cancellous bone mass up to 42% and improved bone microstructural indices. For the highest dosage of FSTΔHBS-mFc (30 mg/kg, 2 times/wk), the increase in cancellous bone mass was similar to that observed with parathyroid hormone therapy (1-34, 80 µg/kg, 5 times/wk). Musculus quadriceps femoris mass dose-dependently increased up to 21% in ovariectomized mice. In both ovariectomized and naive mice, FSTΔHBS-mFc therapy did not influence red blood cell count or hematocrit or hemoglobin levels. If the results are reproduced, a human FSTΔHBS-mFc version could be applicable in patients with musculoskeletal conditions irrespective of hematocrit status.-Lodberg, A., van der Eerden, B. C. J., Boers-Sijmons, B., Thomsen, J. S., Brüel, A., van Leeuwen, J. P. T. M., Eijken, M. A follistatin-based molecule increases muscle and bone mass without affecting the red blood cell count in mice.

Lipidoid-siRNA Nanoparticle-Mediated IL-1ß Gene Silencing for Systemic Arthritis Therapy in a Mouse Model.

Interleukin-1 beta (IL-1ß) plays a central role in the induction of rheumatoid arthritis (RA). In the present study, we demonstrated that lipidoid-polymer hybrid nanoparticle (FS14-NP) can efficiently deliver siRNA against IL-1ß (siIL-1ß) to macrophages and effectively suppress the pathogenesis of experimental arthritis induced by collagen antibody (CAIA mice). FS14-NP/siIL-1ß achieved approximately 70% and 90% gene-silencing efficiency in the RAW 264.7 cell line and intraperitoneal macrophages, respectively. Intravenous administration of FS14-NP/siRNA led to rapid accumulation of siRNA in macrophages within the arthritic joints. Furthermore, FS14-NP/siIL-1ß treatment lowered the expression of pro-inflammatory cytokines in arthritic joints and dramatically attenuated ankle swelling, bone erosion, and cartilage destruction. These results demonstrate that FS14-NP/siIL-1ß may represent an effective therapy for systemic arthritis and other inflammatory disorders.

Histomorphometric case-control study of subarticular osteophytes in patients with osteoarthritis of the hip.

OBJECTIVE: The objective of this cross-sectional case-control study was to determine the prevalence and size of marginal and subarticular osteophytes in patients with osteoarthritis (OA), and to compare these to that of a control group. DESIGN: We investigated femoral heads from 25 patients with OA following hip replacement surgery, and 25 femoral heads from a control group obtained post-mortem. The area and boundary length of the femoral head, marginal osteophytes, and subarticular osteophytes were determined with histomorphometry. Marginal osteophytes were defined histologically as bony projections at the peripheral margin of the femoral head, while subarticular osteophytes were defined as areas of bone that expanded from the normal curvature of the femoral head into the articular cartilage. RESULTS: The prevalence of OA patients with marginal- and subarticular osteophytes were 100 and 84%, respectively. Whereas the prevalence of the participants in the control group with marginal- and subarticular osteophytes were 56 and 28%, respectively. The area and boundary length of marginal osteophytes was (median (Interquartile range)) 165.3mm2 (121.4-254.0) mm2 and 75.1 mm (50.8-99.3) mm for patients with OA compared to 0 mm2 (0-0.5) mm2 and 0 mm (0-0.5) mm for the control group (P <  0.001). For the subarticular osteophytes, the area and boundary length was 1.0 mm2 (0-4.4) mm2 and 1.4 mm (0-6.5) mm for patients with OA compared to 0 mm2 (0-0.5) mm2 and 0 mm (0-0.5) mm for the control group (P <  0.001). CONCLUSION: As expected, both marginal- and subarticular osteophytes at the femoral head, were more frequent and larger in patients with OA than in the control group. However, in the control group, subarticular osteophytes were more prevalent than expected from the minor osteophytic changes at the femoral head margin, which may suggest that subarticular osteophytes are an early degenerative phenomenon that ultimately might develop into clinical osteoarthritis.

Morphology of the initial nephron-collecting duct connection in mice using computerized 3D tracing and electron microscopy.

Recently, the cellular origin of the connecting tubule (CNT) has been genetically characterized. The CNT is a segment between two embryonically different structures, the collecting duct originating from ureteric bud (UB), and the nephron derived from the cap mesenchyme. However, the cellular detail at the initial connection is limited. The present study demonstrated that the initial connection was composed of cells which were closely associated with the renal vesicle (RV), the initial nephron, and connected with the basal epithelium of the terminal UB tip at discrete points. The identification of the RV and UB tip was based on tracing of tubules on serial epoxy sections at mouse embryonic day 17.5. The cells at the initial connection were characterized by 1) irregularly-shaped nuclei and cells with cytoplasmic processes, 2) electron dense nuclei, 3) abundant intercellular spaces, 4) extensive cell-cell contacts with cell junctions, often zonulae adherences and occasionally focal fusion of opposing plasma membranes, and 5) numerous mitochondria, densely packed rosette-like polyribosomes, and widespread rER in the cytoplasm. Moreover, the tracing revealed that a terminal UB tip frequently connected to two nephrons at different developing stages. The UB tips, the initial connections, and the distal tubules of the S-shaped bodies did not express Na+-Cl- cotransporter, H+-ATPase, or aquaporin 2, while they were expressed in immature CNT of the capillary-loop stage nephrons throughout the kidney development. Consequently, the cells at the initial connection exhibit the morphological features suggestive of energy demanding, protein producing, and intercellular communicating. The cell morphology together with transporter development indicates that these cells serve several functions during the development of the initial connection, and that these functions are different from the cells' final functions as transportation.

No Signature of Osteocytic Osteolysis in Cortical Bone from Lactating NMRI Mice.

The roles of osteocytes in bone homeostasis have garnered increasing attention since it has been realized that osteocytes communicate with other organs. It has long been debated whether and/or to which degree osteocytes can break down the bone matrix surrounding them in a process called osteocytic osteolysis. Osteocytic osteolysis has been indicated to be induced by a number of skeletal challenges including lactation in CD1 and C57BL/6 mice, whereas immobilization-induced osteocytic osteolysis is still a matter of controversy. Motivated by the wish to understand this process better, we studied osteocyte lacunae in lactating NMRI mice, which is a widely used outbred mouse strain. Surprisingly, no trace of osteocytic osteolysis could be detected in tibial or femoral cortical bone either by 3D investigation by synchrotron nanotomography, by studies of lacunar cross-sectional areas using scanning electron microscopy, or by light microscopy. These results lead us to conclude that osteocytic osteolysis does not occur in NMRI mice as a response to lactation, in turn suggesting that osteocytic osteolysis may not play a generic role in mobilizing calcium during lactation.

Morphologic and morphometric study on microvasculature of developing mouse kidneys.

A proper morphogenesis of the renal microvasculature is crucial not only for fulfilling the renal function but also to slow down the progression of chronic kidney disease in adulthood. However, the current description of the developing microvasculature is incomplete. The present study investigated the morphogenesis and volume densities of the renal microvasculature using computer-assisted tubular tracing, immunohistochemistry for CD34, and unbiased stereology. The earliest glomerular capillaries were observed at the lower cleft of the S-shaped nephrons, as simple loops connecting the afferent and efferent arterioles. In parallel with this, the peritubular capillaries were established. Noticeably, from early nephrogenesis on, the efferent arterioles of the early-formed glomeruli ran in close proximity to their own thick ascending limbs. In addition, the ascending vasa recta arising from the arcuate or interlobular veins also ran in close proximity to the thick descending limb. Thus, the tubules and vessels formed the typical countercurrent relation in the medulla. No loop bends were observed between descending and ascending vasa recta. The volume density of the cortical and medullary peritubular capillary increased 3.3- and 2.6-fold, respectively, from 2.34 (0.13) and 7.03 (0.09)% [means (SD)] at embryonic day 14.5 (E14.5) to 7.71 (0.44) and 18.27 (1.17)% at postnatal day 40 (P40). In contrast, the volume density of glomeruli changed only slightly during kidney development, from 4.61 (0.47)% at E14.5 to 6.07 (0.2)% at P7 to 4.19 (0.47)% at P40. These results reflect that the growth and formation of the renal microvasculature closely correspond to functional development of the tubules.

Neuronal Cell Adhesion Molecule 1 Regulates Leptin Sensitivity and Bone Mass.

The central nervous system is widely known to exert control over our systemic physiology via several mechanisms including the regulation of skeletal metabolism. Neuronal circuits within the hypothalamus have been shown to impact bone mass via leptin-dependent and independent mechanisms; however, the full extent to which the brain controls bone homeostasis is not known. We previously identified cell adhesion molecule1 (Cadm1) as a regulator of body weight and energy homeostasis via its expression in multiple regions of the brain. Here, we show that loss of Cadm1 expression in excitatory neurons results in increased leptin sensitivity in addition to a concomitant reduction in bone mass. Femoral length, bone mineral content, diaphyseal cross-sectional area, and bone strength were all lower in Cadm1-deficient animals. Conversely, inducing expression of Cadm1 in excitatory neurons decreased leptin sensitivity and increased femoral length, bone mineral content, and diaphyseal cross-sectional area. Together, these results illustrate an essential role for this synaptic protein in the neuronal regulation of skeletal bone metabolism.

Long-Term High-Dose Resveratrol Supplementation Reduces Bone Mass and Fracture Strength in Rats.

Resveratrol (RSV) is a natural polyphenolic compound. A recent study suggests a positive effect on BMD in men; however, the underlying changes in microstructure and strength remain unknown. We aimed to investigate the effects of RSV on the skeleton in hindlimb-immobilized and non-immobilized rats. Seventy-two female Wistar rats were divided into six groups. Two baseline (BSL) groups underwent short-term diet intervention for 4 weeks before sacrifice [phytoestrogen-deficient diet (PD) (BSL + PD) or RSV diet (600 mg/kg body weight/day) (BSL + RSV)]. Four groups were injected in the right hindlimb with botulinum toxin (BTX) (immobilized) or saline (non-immobilized), and fed either PD diet or RSV diet 4 weeks pre-injection and 6 weeks post-injection before sacrifice (BTX + PD, BTX + RSV, PD, and RSV, respectively). DXA, µCT, dynamic histomorphometry, and mechanical tests were performed. Short-term RSV treatment did not affect bone parameters, whereas long-term RSV exposure had a consistent negative impact on non-immobilized rats (RSV vs. PD); whole femoral aBMD (p = 0.01) and distal femoral metaphyseal Tb.N (p = 0.01), Tb.Sp (p = 0.02), and BV/TV (p = 0.07). At the femoral mid-diaphysis, RSV increased periosteal resorption (p = 0.01) and increased endosteal formation (p = 0.02), while mineralization was unaffected. In addition, RSV reduced femoral mid-diaphyseal three-point bending strength (p = 0.03) and stiffness (p = 0.04). BTX-induced immobilization resulted in significant bone loss and reduced bone strength; however, RSV supplementation was unable to prevent this. In conclusion, long-term high-dose RSV reduced bone mass and fracture strength and did not prevent immobilization-induced bone loss in rats.

Disuse osteopenia induced by botulinum toxin is similar in skeletally mature young and aged female C57BL/6J mice.

Osteopenia and osteoporosis predominately occur in the fully grown skeleton. However, it is unknown whether disuse osteopenia in skeletally mature, but growing, mice resembles that of fully grown mice. Twenty-four 16-week-old (young) and eighteen 44-week-old (aged) female C57BL/6J mice were investigated. Twelve young and nine aged mice were injected with botulinum toxin in one hind limb; the remaining mice served as controls. The mice were euthanized after 3 weeks of disuse. The femora were scanned by micro-computed tomography (µCT) and bone strength was determined by mechanically testing the femoral mid-diaphysis and neck. At the distal femoral metaphysis, the loss of trabecular bone volume fraction (BV/TV) differed between the young and aged mice. However, at the distal femoral epiphysis, no age-dependent differences were observed. Thinning of the trabeculae was not affected by the age of the mice at either the distal femoral metaphysis or the epiphysis. Furthermore, the aged mice lost more bone strength at the femoral mid-diaphysis, but not at the femoral neck, compared to the young mice. In general, the bone loss induced by botulinum toxin did not differ substantially between young and aged mice. Therefore, the loss of bone in young mice resembles that of aged mice, even though they are not fully grown.

Zoledronic acid prevents disuse osteopenia and augments gene expression of osteoclastic differentiation markers in mice.

OBJECTIVES: Disuse is characterized by a rapid and profound bone resorption. Zoledronic acid (Zol) inhibits osteoclastic bone resorption. The aim of the study was to prevent disuse osteopenia with Zol and investigate gene expression markers of osteoclastic differentiation. METHODS: Disuse osteopenia was induced by injecting botulinum toxin (BTX) into the right hind limb of 16-week-old C57BL/6J female mice. Zol (100 µg/kg) was injected s.c. once at study start. The immobilized bones were investigated with DEXA, microCT, mechanical testing, dynamic bone histomorphometry, and RT-qPCR. RESULTS: The BTX-injections resulted in a loss of cortical and trabecular bone as well as mechanical strength compared to intact baseline and control mice. Treatment with Zol prevented the loss of bone and mechanical strength. Interestingly, treatment with Zol resulted in a higher expression of Nfatc1 and Dcstamp, which are markers osteoclastic differentiation. CONCLUSIONS: Zol effectively prevented BTX-induced disuse osteopenia. Furthermore, gene expression markers of osteoclastic differentiation were increased in Zol treated immobilized mice, indicating that Zol only affect mature bone resorbing osteoclasts in vivo. However, the current findings are preliminary and calls for further studies.

Nephron morphometry in mice and rats using tomographic microscopy.

The aim was to quantify the glomerular capillary surface area, the segmental tubular radius, length, and area of single nephrons in mouse and rat kidneys. Multiple 2.5-µm-thick serial Epon sections were obtained from three mouse and three rat kidneys for three-dimensional reconstruction of the nephron tubules. Micrographs were aligned for each kidney, and 359 nephrons were traced and their segments localized. Thirty mouse and thirty rat nephrons were selected for further investigation. The luminal radius of each segment was determined by two methods. The luminal surface area was estimated from the radius and length of each segment. High-resolution micrographs were recorded for five rat glomeruli, and the capillary surface area determined. The capillary volume and surface area were corrected for glomerular shrinkage. A positive correlation was found between glomerular capillary area and proximal tubule area. The thickest part of the nephron, i.e., the proximal tubule, was followed by the thinnest part of the nephron, i.e., the descending thin limb, and the diameters of the seven identified nephron segments share the same rank in the two species. The radius and length measurements from mouse and rat nephrons generally share the same pattern; rat tubular radius-to-mouse tubular radius ratio ≈ 1.47, and rat tubular length-to-mouse tubular length ratio ≈ 2.29, suggesting relatively longer tubules in the rat. The detailed tables of mouse and rat glomerular capillary area and segmental radius, length, and area values may be used to enhance understanding of the associated physiology, including existing steady-state models of the urine-concentrating mechanism.

Simulation of heterogeneous molecular delivery in tumours using µCT reconstructions and MRI validation.

PRIMARY OBJECTIVE: Utilizing the detailed vascular network obtained from micro-computed tomography (µCT) to establish a mathematical model of the temporal molecular distribution within a murine C3H mammary carcinoma. PROCEDURES: Female CDF1 mice with a C3H mammary carcinoma on the right rear foot were used in this study. Dynamic information for each tumour was achieved by Dynamic Contrast Enhanced-Magnetic Resonance Imaging (DCE-MRI) on a 16.4 T system. Detailed morphologic information on the tumour vasculature was obtained by ex vivo µCT and compared to CD34 immunohistochemical staining of tissue sections. The reconstructed vascular network served as origin for the diffusion (described by the apparent diffusion coefficient) within the tumour (the restricted volume described by the interstitial volume fraction derived from DCE-MRI). The resulting partial differential equation was solved using Finite-Element and a combined mathematical graph describing molecular distribution within the tumour was obtained. RESULTS: The established molecular distribution model predicted a heterogeneous distribution throughout the tumour related to the layout of the vascular network. Central tumour section concentration-time curves estimated from the established molecular distribution model were compared with physical measurements obtained by DCE-MRI of the same tumours and showed excellent correlation. CONCLUSIONS: A mathematical model describing temporal molecular distribution based on detailed vascular network structures was established and compared to DCE-MRI. The improved morphological insight will enhance future studies of heterogeneous tumours.

Systemic Treatment with Strontium Ranelate Does Not Influence the Healing of Femoral Mid-shaft Defects in Rats.

Strontium ranelate (SrR) has both bone anabolic and anti-resorption properties and has therefore the potential to increase the healing of bone defects. The aim of the present study was to investigate the effect of systemic treatment with SrR during the healing of cortical bone defects in rats. In addition, the vertebral bodies were examined in order to elucidate the effect of short-term treatment with SrR on intact trabecular bone. Sixty 16-week-old female Wistar rats were randomized into four groups. A cylindrical defect was drilled through the anterior cortex of the mid-femoral diaphysis in both hind limbs. Two of the groups were treated with SrR (900 mg/kg b.w.) mixed into the food and two groups served as controls. The animals were euthanized after either 3 or 8 weeks of treatment. Healing of the defects was analyzed with µCT, mechanical testing, and stereology. Treatment with SrR resulted in increased thickness of the defects after 3 weeks of treatment, whereas no effect on bone volume fraction (BV/TV), mechanical properties (maximum strength and maximum stiffness), periosteal callus volume, or osteoclast-covered bone surfaces (Oc.S/BS) after either 3 or 8 weeks of treatment was found. Furthermore, SrR increased the bone material density (ρ) of the vertebral bodies, and tended to increase BV/TV after 8 weeks of treatment (p = 0.087). The mechanical properties of the vertebral bodies were not influenced by SrR treatment. In conclusion, 3 weeks of treatment with SrR increased the thickness of the healing mid-femoral cortical bone defects in rats, but did not influence BV/TV, mechanical properties, periosteal callus volume, or Oc.S/BS after either 3 or 8 weeks. Furthermore, SrR had no effect on the microstructure and mechanical properties of the vertebral bodies.

Osteocyte lacunar properties in rat cortical bone: Differences between lamellar and central bone.

Recently, the roles of osteocytes in bone maintenance have gained increasing attention. Osteocytes reside in lacunae that are interconnected by canaliculi resulting in a vast cellular network within the mineralized bone matrix. As the structure of the lacuno-canalicular network is highly connected to osteocyte function, osteocyte lacunar properties such as volume, shape, orientation, and density are now frequently reported in studies investigating osteocyte activity. Despite this increasing interest in lacunar morphometrics, many studies show a large spread in such values, suggesting a large inter-species but also inter-site variation in lacunar properties. Here, osteocyte lacunae in rat cortical bone have been studied using synchrotron radiation micro computed tomography (SR µCT) and backscattered electron (BE) microscopy. Quantitative lacunar geometric characteristics are reported based on the synchrotron radiation data, differentiating between circumferential lamellar bone and a central, more disordered bone type. From these studies, no significant differences were found in lacunar volumes between lamellar and central bone, whereas significant differences in lacunar orientation, shape and density values were observed. The 3D nature of the SR µCT data sets furthermore revealed that lacunae in central bone, which appear to be poorly aligned in transverse 2D cross sections, are in fact highly aligned along the bone long axis. These results demonstrate the importance of using 3D methods to investigate anisotropic biological materials such as bone and that the appropriate choice of subregions for high resolution imaging is not trivial.

Vertical Trabeculae are Thinned More Than Horizontal Trabeculae in Skeletal-Unloaded Rats.

Skeletal unloading results in a rapid thinning of the trabecular bone network, but it is unknown whether vertical and horizontal trabeculae are equally affected. Therefore, the purpose of the present study was to investigate whether horizontal and vertical trabeculae were thinned similarly during skeletal unloading in rats. Fifty-seven 16-week-old female Wistar rats were randomized into six groups: baseline; control 4 weeks; botulinum toxin A (BTX) 4 weeks; control 8 weeks; BTX 8 weeks; and two BTX injections 8 weeks (BTX + BTX8). The BTX animals were injected in the right hind limb with 4 IU BTX at the start of the study, while the BTX + BTX8 were also injected with 2 IU BTX after 4 weeks. The animals were killed after 0, 4, or 8 weeks. The distal femoral metaphyses were µCT scanned, and the strengths of the femoral necks, mid-diaphyses, and distal femoral metaphyses were ascertained. Disuse resulted in a significant loss of BV/TV, thinning of the trabeculae, and decrease in the degree of anisotropy, and in a significant reduced bone strength after both 4 and 8 weeks. The ratio of horizontal to vertical trabecular thickness (Tb.Th.horz/Tb.Th.vert) and the ratio of horizontal to vertical bone volume (BV.horz/BV.vert) were significantly higher in BTX animals than in control animals. In addition, the horizontal and vertical trabecular thickness probability density functions were more similar in BTX animals than in control animals. In conclusion, skeletal unloading decreased BV/TV, Tb.Th, the degree of anisotropy, and mechanical strength, while BV.horz/BV.vert and Tb.Th.horz/Tb.Th.vert were increased. This indicates that the more loaded vertical trabeculae are pronouncedly more thinned than the less loaded supporting horizontal trabeculae during unloading.