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Background: Zinc finger-containing transcription factor Osterix/Specificity protein-7 (Sp7) is an essential transcription factor for osteoblast differentiation. However, its functions in differentiated osteoblasts remain unclear and the effects of osteoblast-specific Sp7 deletion on osteocytes have not been sufficiently studied. Methods: Sp7 floxneo/floxneo mice, in which Sp7 expression was 30 % of that in wild-type mice because of disturbed splicing by neo gene insertion, and osteoblast-specific knockout (Sp7 fl/fl;Col1a1-Cre) mice using 2.3-kb Col1a1 enhanced green fluorescent protein (EGFP)-Cre were examined by micro-computed tomography (micro-CT), bone histomorphometry, serum markers, and histological analyses. The expression of osteoblast and osteocyte marker genes was examined by real-time reverse transcription (RT)-PCR analysis. Osteoblastogenesis, osteoclastogenesis, and regulation of the expression of collagen type I alpha 1 chain (Col1a1) were examined in primary osteoblasts. Results: Femoral trabecular bone volume was higher in female Sp7 floxneo/floxneo and Sp7 fl/fl;Col1a1-Cre mice than in the respective controls, but not in males. Bromodeoxyuridine (BrdU)-positive osteoblastic cells were increased in male Sp7 fl/fl;Col1a1-Cre mice, and osteoblast number and the bone formation rate were increased in tibial trabecular bone in female Sp7 fl/fl;Col1a1-Cre mice, although osteoblast maturation was inhibited in female Sp7 fl/fl;Col1a1-Cre mice as shown by the increased expression of an immature osteoblast marker gene, secreted phosphoprotein 1 (Spp1), and reduced expression of a mature osteoblast marker gene, bone gamma-carboxyglutamate protein/bone gamma-carboxyglutamate protein 2 (Bglap/Bglap2). Furthermore, alkaline phosphatase activity was increased but mineralization was reduced in the culture of primary osteoblasts from Sp7 fl/fl;Col1a1-Cre mice. Therefore, the accumulated immature osteoblasts in Sp7 fl/fl;Col1a1-Cre mice was likely compensated for the inhibition of osteoblast maturation at different levels in males and females. Vertebral trabecular bone volume was lower in both male and female Sp7 fl/fl;Col1a1-Cre mice than in the controls and the osteoblast parameters and bone formation rate in females were lower in Sp7 fl/fl;Col1a1-Cre mice than in Sp7 fl/fl mice, suggesting differential regulatory mechanisms in long bones and vertebrae. The femoral cortical bone was thin and porous in Sp7 floxneo/floxneo and Sp7 fl/fl;Col1a1-Cre mice of both sexes, the number of canaliculi was reduced, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL)-positive lacunae and the osteoclasts were increased, whereas the bone formation rate was similar in Sp7 fl/fl;Col1a1-Cre and Sp7 fl/fl mice. The serum levels of total procollagen type 1 N-terminal propeptide (P1NP), a marker for bone formation, were similar, while those of tartrate-resistant acid phosphatase 5b (TRAP5b), a marker for bone resorption, were higher in Sp7 fl/fl;Col1a1-Cre mice. Osteoblasts were less cuboidal, the expression of Col1a1 and Col1a1-EGFP-Cre was lower in Sp7 fl/fl;Col1a1-Cre mice, and overexpression of Sp7 induced Col1a1 expression. Conclusions: Our studies indicated that Sp7 inhibits the proliferation of immature osteoblasts, induces osteoblast maturation and Col1a1 expression, and is required for osteocytes to acquire a sufficient number of processes for their survival, which prevents cortical porosity. The translational potential of this article: This study clarified the roles of Sp7 in differentiated osteoblasts in proliferarion, maturation, Col1a1 expression, and osteocyte process formation, which are required for targeting SP7 in the development of therapies for osteoporosis.

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Marfan syndrome (MFS) is a connective tissue disorder caused by pathogenic mutations in FBN1. In bone, the protein fibrillin-1 is found in the extracellular matrix where it provides structural support of elastic fiber formation, stability for basement membrane, and regulates the bioavailability of growth factors. Individuals with MFS exhibit a range of skeletal complications including low bone mineral density and long bone overgrowth. However, it remains unknown if the bone phenotype is caused by alteration of fibrillin-1's structural function or distortion of its interactions with bone cells. To assess the structural effects of the fibrillin-1 mutation, we characterized bone curvature, microarchitecture, composition, porosity, and mechanical behavior in the Fbn1 C1041G/+ mouse model of MFS. Tibiae of 10, 26, and 52-week-old female Fbn1 C1041G/+ and littermate control (LC) mice were analyzed. Mechanical behavior was assessed via in vivo strain gauging, finite element analysis, ex vivo three-point bending, and nanoindentation. Tibial bone morphology and curvature were assessed with micro computed tomography (µCT). Bone composition was measured with Fourier transform infrared (FTIR) imaging. Vascular and osteocyte lacunar porosity were assessed by synchrotron computed tomography. Fbn1 C1041G/+ mice exhibited long bone overgrowth and osteopenia consistent with the MFS phenotype. Trabecular thickness was lower in Fbn1 C1041G/+ mice but cortical bone microarchitecture was similar in Fbn1 C1041G/+ and LC mice. Whole bone curvature was straighter below the tibio-fibular junction in the medial-lateral direction and more curved above in LC compared to Fbn1 C1041G/+ mice. The bone matrix crystallinity was 4 % lower in Fbn1 C1041G/+ mice compared to LC, implying that mineral platelets in LCs have greater crystal size and perfection than Fbn1 C1041G/+ mice. Structural and mechanical properties were similar between genotypes. Cortical diaphyseal lacunar porosity was lower in Fbn1 C1041G/+ mice compared to LC; this was a result of the average volume of an individual osteocyte lacunae being smaller. These data provide valuable insights into the bone phenotype and its contribution to fracture risk in this commonly used mouse model of MFS.

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To clarify the cellular mechanism of cortical porosity induced by intermittent parathyroid hormone (PTH) administration, we examined the femoral cortical bone of mice that received 40 µg/kg/day (four times a day) human PTH (hPTH) (1-34). The PTH-driven cortical porosity initiated from the metaphyseal region and chronologically expanded toward the diaphysis. Alkaline phosphatase (ALP)-positive osteoblasts in the control mice covered the cortical surface, and endomucin-positive blood vessels were distant from these osteoblasts. In PTH-administered mice, endomucin-reactive blood vessels with TRAP-positive penetrated the ALP-positive osteoblast layer, invading the cortical bone. Statistically, the distance between endomucin-positive blood vessels and the cortical bone surface abated after PTH administration. Transmission electron microscopic observation demonstrated that vascular endothelial cells often pass through the flattened osteoblast layer and accompanied osteoclasts in the deep region of the cortical bone. The cell layers covering mature osteoblasts thickened with PTH administration and exhibited ALP, α-smooth muscle actin (αSMA), vascular cell adhesion molecule-1 (VCAM1), and receptor activator of NF-κB ligand (RANKL). Within these cell layers, osteoclasts were found near endomucin-reactive blood vessels. In PTH-administered femora, osteocytes secreted Dkk1, a Wnt inhibitor that affects angiogenesis, and blood vessels exhibited plasmalemma vesicle-associated protein, an angiogenic molecule. In summary, endomucin-positive blood vessels, when accompanied by osteoclasts in the ALP/αSMA/VCAM1/RANKL-reactive osteoblastic cell layers, invade the cortical bone, potentially due to the action of osteocyte-derived molecules such as DKK1.

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BACKGROUND: Cortical thickness and porosity are two main determinants of cortical bone strength. Thus, mapping variations in these parameters across the full width of the distal end of the clavicle may be helpful for better understanding the basis of distal clavicle fractures and for selecting optimal surgical treatment. METHODS: Distal ends of 11 clavicles (6 men, 5 women; age: 81.9 ± 15.1 years) were scanned by micro-computed tomography at 10-µm resolution. We first analyzed cortical thickness and porosity of each 500-µm-wide area across the superior surface of distal clavicle at the level of conoid tubercle in an antero-posterior direction. This level was chosen for detailed evaluation because previous studies have demonstrated its superior microarchitecture relative to the rest of the distal clavicle. Subsequently, we divided the full width of distal clavicle to three subregions (anterior, middle, and posterior) and analyzed cortical porosity, pore diameter, pore separation, and cortical thickness. RESULTS: We found the largest number of low-thickness and high-porosity areas in the anterior subregion. Cortical porosity, pore diameter, pore separation, and cortical thickness varied significantly among the three subregions (p < 0.001 p = 0.016, p = 0.001, p < 0.001, respectively). Cortex of the anterior subregion was more porous than that of the middle subregion (p < 0.001) and more porous and thinner than that of the posterior subregion (p < 0.001, p = 0.030, respectively). Interaction of site and sex revealed higher porosity of the anterior subregion in women (p < 0.001). The anterior subregion had larger pores than the middle subregion (p = 0.019), whereas the middle subregion had greater pore separation compared with the anterior (p = 0.002) and posterior subregions (p = 0.006). In general, compared with men, women had thinner (p < 0.001) and more porous cortex (p = 0.03) with larger cortical pores (p < 0.001). CONCLUSIONS: Due to high cortical porosity and low thickness, the anterior conoid subregion exhibits poor bone microarchitecture, particularly in women, which may be considered in clinical practice. LEVELS OF EVIDENCE: Level IV.

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INTRODUCTION: Patients with chronic kidney disease (CKD) are at an alarming risk of fracture compared to age and sex-matched non-CKD individuals. Clinical and preclinical data highlight two key factors in CKD-induced skeletal fragility: cortical porosity and reduced matrix-level properties including bone hydration. Thus, strategies are needed to address these concerns to improve mechanical properties and ultimately lower fracture risk in CKD. We sought to evaluate the singular and combined effects of mechanical and pharmacological interventions on modulating porosity, bone hydration, and mechanical properties in CKD. METHODS: Sixteen-week-old male C57BL/6J mice underwent a 10-week CKD induction period via a 0.2 % adenine-laced casein-based diet (n = 48) or remained as non-CKD littermate controls (Con, n = 48). Following disease induction (26 weeks of age), n = 7 CKD and n = 7 Con were sacrificed (baseline cohort) to confirm a steady-state CKD state was achieved prior to the initiation of treatment. At 27 weeks of age, all remaining mice underwent right tibial loading to a maximum tensile strain of 2050 µÆ 3× a week for five weeks with the contralateral limb as a non-loaded control. Half of the mice (equal number CKD and Con) received subcutaneous injections of 0.5 mg/kg raloxifene (RAL) 5× a week, and the other half remained untreated (UN). Mice were sacrificed at 31 weeks of age. Serum biochemistries were performed, and bi-lateral tibiae were assessed for microarchitecture, whole bone and tissue level mechanical properties, and composition including bone hydration. RESULTS: Regardless of intervention, BUN and PTH were higher in CKD animals throughout the study. In CKD, the combined effects of loading and RAL were quantified as lower cortical porosity and improved mechanical, material, and compositional properties, including higher matrix-bound water. Loading was generally responsible for positive impacts in cortical geometry and structural mechanical properties, while RAL treatment improved some trabecular outcomes and material-level mechanical properties and was responsible for improvements in several compositional parameters. While control animals responded positively to loading, their bones were less impacted by the RAL treatment, showing no deformation, toughness, or bound water improvements which were all evident in CKD. Serum PTH levels were negatively correlated with matrix-bound water. DISCUSSION: An effective treatment program to improve fracture risk in CKD ideally focuses on the cortical bone and considers both cortical porosity and matrix properties. Loading-induced bone formation and mechanical improvements were observed across groups, and in the CKD cohort, this included lower cortical porosity. This study highlights that RAL treatment superimposed on active bone formation may be ideal for reducing skeletal complications in CKD by forming new bone with enhanced matrix properties.

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Skeletal fragility and high fracture rates are common in CKD. A key component of bone loss in CKD with secondary hyperparathyroidism is high bone turnover and cortical bone deterioration through both cortical porosity and cortical thinning. We hypothesized that RANKL drives high bone resorption within cortical bone leading to the development of cortical porosity in CKD (study 1) and that systemic inhibition of RANKL would mitigate the skeletal phenotype of CKD (study 2). In study 1, we assessed the skeletal properties of male and female Dmp1-cre RANKLfl/fl (cKO) and control genotype (Ranklfl/fl; Con) mice after 10 wk of adenine-induced CKD (AD; 0.2% dietary adenine). All AD mice regardless of sex or genotype had elevated blood urea nitrogen and high PTH. Con AD mice in both sexes had cortical porosity and lower cortical thickness as well as high osteoclast-covered trabecular surfaces and higher bone formation rate. cKO mice had preserved cortical bone microarchitecture despite high circulating PTH as well as no CKD-induced increases in osteoclasts. In study 2, male mice with established AD CKD were either given a single injection of an anti-RANKL antibody (5 mg/kg) 8 wk post-induction of CKD or subjected to 3×/wk dosing with risedronate (1.2 µg/kg) for 4 wk. Anti-RANKL treatment significantly reduced bone formation rate as well as osteoclast surfaces at both trabecular and cortical pore surfaces; risedronate treatment had little effect on these bone parameters. In conclusion, these studies demonstrate that bone-specific RANKL is critical for the development of high bone formation/high osteoclasts and cortical bone loss in CKD with high PTH. Additionally, systemic anti-RANKL ligand therapy in established CKD may help prevent the propagation of cortical bone loss via suppression of bone turnover.

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Preclinical models (typically ovariectomized rats and genetically altered mice) have underpinned much of what we know about skeletal biology. They have been pivotal for developing therapies for osteoporosis and monogenic skeletal conditions, including osteogenesis imperfecta, achondroplasia, hypophosphatasia, and craniodysplasias. Further therapeutic advances, particularly to improve cortical strength, require improved understanding and more rigorous use and reporting. We describe here how trabecular and cortical bone structure develop, are maintained, and degenerate with aging in mice, rats, and humans, and how cortical bone structure is changed in some preclinical models of endocrine conditions (eg, postmenopausal osteoporosis, chronic kidney disease, hyperparathyroidism, diabetes). We provide examples of preclinical models used to identify and test current therapies for osteoporosis, and discuss common concerns raised when comparing rodent preclinical models to the human skeleton. We focus especially on cortical bone, because it differs between small and larger mammals in its organizational structure. We discuss mechanisms common to mouse and human controlling cortical bone strength and structure, including recent examples revealing genetic contributors to cortical porosity and osteocyte network configurations during growth, maturity, and aging. We conclude with guidelines for clear reporting on mouse models with a goal for better consistency in the use and interpretation of these models.

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Age-related changes in human trabecular bone and cortical bone are known to vary. Although the porosity of cortical bone has been suggested to increase the risk of bone fracture, most of the currently available instruments for osteoporosis testing target trabecular bone. In this study, we evaluated cortical bone density using clinical computed tomography (CT) and compared the reliability of the cortical bone density index (CDI) with that of a polished male femoral bone from the same region. CDI images revealed that the porous area of cortical bone was extended in low CDI values. Moreover, this method was used to semi-quantitatively evaluate the cortical bones of the diaphysis of male femur specimens (n = 46). We found that there was a significant relationship (r = 0.70, p < 0.01) between the value of the cortical index (the ratio of cortical bone area to the cross-sectional area of the femoral diaphysis) and the average of CDI in the low signal area. Our findings suggest that the smaller the cortical bone occupancy, the more areas of consequential bone density loss were present. This may be the first step toward using clinical CT to assess cortical bone density.

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To elucidate the effect of evocalcet, a new oral calcimimetic to bone of secondary hyperparathyroidism (SHPT) with chronic kidney disease (CKD), the rats were 5/6 nephrectomized and fed on a high-phosphate diet. The treated rats were then divided into vehicle groups and evocalcet administered groups. The rats in the vehicle groups exhibited increased levels of serum PTH and inorganic phosphate (Pi) levels, high bone turnover, and severe cortical porosity, mimicking SHPT (CKD-SHPT rats). The cortical bone of the CKD-SHPT rats showed broad demineralization around the osteocytes, suppression of Phex/small integrin-binding ligand N-linked glycoprotein-mediated mineralization in the periphery of the osteocytic lacunae, and increased levels of osteocytic cell death, all of which were considered as the first steps of cortical porosity. In contrast, evocalcet ameliorated the increased serum PTH levels, the enlarged osteocytic lacunae, and the cortical porosity of the CKD-SHPT rats. Osteocytes of CKD-SHPT rats strongly expressed PTH receptor and Pit1/Pit2, which sense extracellular Pi, indicating that PTH and Pi affected these osteocytes. Cell death of cultured osteocytes increased in a Pi concentration-dependent manner, and PTH administration rapidly elevated Pit1 expression and enhanced osteocytic death, indicating the possibility that the highly concentrated serum PTH and Pi cause severe perilacunar osteolysis and osteocytic cell death. It is likely therefore that evocalcet not only decreases serum PTH but also reduces the exacerbation combined with PTH and Pi to the demineralization of osteocytic lacunae and osteocytic cell death, thereby protecting cortical porosity in CKD-SHPT rats.

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Increased cortical porosity is associated with a heightened risk of skeletal fragility due to bone loss and structural decay in adults. However, few studies have examined the etiology of cortical porosity in infants and children. This study examines whether age-related changes in femoral growth and locomotor development influence femoral midshaft cortical porosity in a sample of 48 individuals (fetal to 3.99 years) from the 10th-13th century cemetery of St. Étienne de Toulouse, France. Histological sections were prepared and imaged using light microscopy. Midshaft geometric variables such as total area, cortical area, and pore area were calculated using BoneJ. Increased porosity and cortical trabecularization were found to be significantly associated with age, being almost exclusively present in individuals aged 0.5-1.99 years. At approximately 6 months of age infants typically begin engaging in regular femoral loading and experience an acceleration in growth. The observed increase in midshaft porosity and trabecularization, therefore, likely results from the reorganization and redistribution of cortical bone, stimulated by increased growth velocity and the onset of weight-bearing activities. The reduction in cortical porosity and trabecularization in individuals aged 2.0-3.99 years indicates that children are approaching some sort of homeostasis as growth velocity slows and their femora adapt to consistent loading. Understanding what expected skeletal development looks like is necessary when conducting bioarcheological studies and this study provides evidence for a pattern of transient midshaft porosity during infancy and early childhood.

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Mechanical unloading causes rapid loss of bone structure and strength, which gradually recovers after resuming normal loading. However, it is not well established how this adaptation to unloading and reloading changes with age. Clinically, elderly patients are more prone to musculoskeletal injury and longer periods of bedrest, therefore it is important to understand how periods of disuse will affect overall skeletal health of aged subjects. Bone also undergoes an age-related decrease in osteocyte density, which may impair mechanoresponsiveness. In this study, we examined bone adaptation during unloading and subsequent reloading in mice. Specifically, we examined the differences in bone adaptation between young mice (3-month-old), old mice (18-month-old), and transgenic mice that exhibit diminished osteocyte density at a young age (3-month-old BCL-2 transgenic mice). Mice underwent 14 days of hindlimb unloading followed by up to 14 days of reloading. We analyzed trabecular and cortical bone structure in the femur, mechanical properties of the femoral cortical diaphysis, osteocyte density and cell death in cortical bone, and serum levels of inflammatory cytokines. We found that young mice lost ~10% cortical bone volume and 27-42% trabecular bone volume during unloading and early reloading, with modest recovery of metaphyseal trabecular bone and near total recovery of epiphyseal trabecular bone, but no recovery of cortical bone after 14 days of reloading. Old mice lost 12-14% cortical bone volume and 35-50% trabecular bone volume during unloading and early reloading but had diminished recovery of trabecular bone during reloading and no recovery of cortical bone. In BCL-2 transgenic mice, no cortical bone loss was observed during unloading or reloading, but 28-31% trabecular bone loss occurred during unloading and early reloading, with little to no recovery during reloading. No significant differences in circulating inflammatory cytokine levels were observed due to unloading and reloading in any of the experimental groups. These results illustrate important differences in bone adaptation in older and osteocyte deficient mice, suggesting a possible period of vulnerability in skeletal health in older subjects during and following a period of disuse that may affect skeletal health in elderly patients.

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Introduction: High-resolution peripheral quantitative computed tomography (HR-pQCT) is a powerful tool that has revolutionized 3D longitudinal assessment of bone microarchitecture. However, cortical porosity, a common characteristic of cortical bone loss, is still often determined by static evaluation of overall porosity at one timepoint. Therefore, we sought to 1) describe a technique to evaluate individual cortical pore dynamics in aging females over one year using HR-pQCT imaging and 2) determine whether formation and expansion of pores would exceed contraction and infilling of pores. Methods: HR-pQCT (60.7 µm resolution) images were acquired one year apart at the distal tibia and distal radius in seven female volunteers (60-72 years of age). Baseline and one-year images were registered at each bone site and a custom software was used to quantify dynamic activity of individual cortical pores using the following categories: developed, infilled, expanded, contracted, and static. Results: Over the one-year period, cortical pores actively developed, contracted, expanded, and infilled. More pores expanded and developed vs. infilled or contracted leading to increased pore area in both tibial and radial sites (p = 0.0034 and p = 0.0474, respectively). Closed pores in the tibia, those that were not connected to the endosteal or periosteal surfaces, were the most dynamic of any pores type (open/closed) at either bone site. Conclusion: This study demonstrates an approach to longitudinally track individual cortical pore activity in tibial and radial sites. These data expand conventional parameters for assessing cortical porosity and show increased porosity in one year of aging is caused by newly developed pores and expansion of existing pores.

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Cortical bone remodeling is carried out by basic multicellular units (BMUs), which couple resorption to formation. Although fluorochrome labeling has facilitated study of BMU formative parameters since the 1960s, some resorptive parameters, including the longitudinal erosion rate (LER), have remained beyond reach of direct measurement. Indeed, our only insights into this spatiotemporal parameter of BMU behavior come from classical studies that indirectly inferred LER. Here, we demonstrate a 4D in vivo method to directly measure LER through in-line phase contrast synchrotron imaging. The tibias of rabbits (n = 15) dosed daily with parathyroid hormone were first imaged in vivo (synchrotron micro-CT; day 15) and then ex vivo 14 days later (conventional micro-CT; day 29). Mean LER assessed by landmarking the co-registered scans was 23.69 ± 1.73 µm/d. This novel approach holds great promise for the direct study of the spatiotemporal coordination of bone remodeling, its role in diseases such as osteoporosis, as well as related treatments. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

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Cortical porosity develops in chronic kidney disease (CKD) and increases with progressing disease. Cortical porosity is likely a prominent contributor to skeletal fragility/fracture. The degree to which cortical porosity occurs throughout the skeleton is not fully known. In this study, we assessed cortical bone porosity via micro-computed tomography at multiple skeletal sites in rats with progressive chronic kidney disease. We hypothesized that cortical porosity would occur in long bones throughout the body, but to a lesser degree in flat bones and irregular bones. Porosity was measured, using micro-CT, at 17 different skeletal sites in 6 male rats with CKD. Varying degrees of porosity were seen throughout the skeleton with higher porosity in flat and irregular bone (i.e. parietal bone, mandible) vs. long bones (p = 0.01) and in non-weightbearing bones vs. weightbearing bones (p = 0.01). Porosity was also higher in proximal sites vs. distal sites in long bones (p < 0.01 in all comparisons). There was large heterogeneity in porosity within skeletal sites across rats and within the same rat across skeletal sites. Correlations showed cortical porosity of the proximal tibia was positively associated with porosity at the other sites with the strongest correlation to the parietal bone and the weakest to the ulna. Overall, our data demonstrates varying and significant cortical bone porosity across the skeleton of animals with chronic kidney disease. These data point to careful selection of skeletal sites to assess porosity in pre-clinical studies and the potential for fractures at multiple skeletal sites in patients with CKD.

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Individuals with diabetes mellitus type 2 (T2DM) have approximately 30% increased risk of hip fracture; however, the main cause of the elevated fracture risk in those subjects remains unclear. Moreover, micromechanical and microarchitectural properties of the superolateral femoral neck-the common fracture-initiating site-are still unknown. We collected proximal femora of 16 men (eight with T2DM and eight controls; age: 61 ± 10 years) at autopsy. After performing post-mortem bone densitometry (DXA), the superolateral neck was excised and scanned with microcomputed tomography (microCT). We also conducted Vickers microindentation testing. T2DM and control subjects did not differ in age (p = 0.605), body mass index (p = 0.114), and femoral neck bone mineral density (BMD) (p = 0.841). Cortical porosity (Ct.Po) was higher and cortical thickness (Ct.Th) was lower in T2DM (p = 0.044, p = 0.007, respectively). Of trabecular microarchitectural parameters, only structure model index (p = 0.022) was significantly different between T2DM subjects and controls. Control group showed higher cortical (p = 0.002) and trabecular bone microhardness (p = 0.005). Increased Ct.Po and decreased Ct.Th in T2DM subjects increase the propensity to femoral neck fracture. Apart from the deteriorated cortical microarchitecture, decreased cortical and trabecular microhardness suggests altered bone composition of the superolateral femoral neck cortex and trabeculae in T2DM. Significantly deteriorated cortical microarchitecture of the superolateral femoral neck is not recognized by standard DXA measurement of the femoral neck.

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GS, the stimulatory heterotrimeric G protein, is an essential regulator of osteogenesis and bone turnover. To determine if increasing GαS in osteoblasts alters bone responses to hyperparathyroidism, we used a transgenic mouse line overexpressing GαS in osteoblasts (GS-Tg mice). Primary osteoblasts from GS-Tg mice showed increased basal and parathyroid hormone (PTH)-stimulated cAMP and greater responses to PTH than cells from WT mice. Skeletal responses to 2-week continuous PTH administration (cPTH) in female mice resulted in trabecular bone loss in WT mice but 74% and 34% increase in trabecular bone mass in long bones and vertebrae, respectively, in GS-Tg mice. Vertebral biomechanical strength was compromised by cPTH treatment in WT mice but not in GS-Tg. Increased peritrabecular fibrosis was greatly increased by cPTH in Gs-Tg compared to WT mice and corresponded with greater increases in Wnt pathway proteins in trabecular bone. Cortical bone responded negatively to cPTH in WT and Gs-Tg mice with large increases in porosity, decreased cortical thickness and compromised biomechanical properties. These results demonstrate that hyperparathyroidism can increase trabecular bone when GS expression and cAMP stimulation in osteoblasts are increased but this is not the case in cortical bone where increased GS expression exacerbates cortical bone loss.

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Recent advances in imaging of bone microstructure have led to a growing recognition of the role of cortical microstructure in osteoporosis. It is now accepted that the assessment of the microstructure of cortical porosity is essential to assess bone mechanical competence and predict fracture risk. Cortical porosity affects the propagation of ultrasound waves because pores act as ultrasound scatterers. Scattering by the porosity is an opportunity that should be leveraged to extract quantitative information about cortical microstructure. Scattering by the pores affects a number of ultrasound parameters that should be quantified, including attenuation, backscatter coefficient, ultrasound diffusivity, and their frequency dependence. Measuring these ultrasound parameters and developing models that describe their dependence upon parameters of cortical microstructure is the key to solve inverse problems that will allow the quantitative assessment of cortical porosity and ultimately will improve the non-invasive ultrasound-based evaluation of bone mechanical competence and fracture risk. In this chapter, we present recent advances in measuring and modeling those parameters in cortical bone.

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PURPOSE: Patients with chronic kidney disease (CKD) have high risk of fracture in part due to cortical bone deterioration. The goal of this study was to assess the impact of two different bisphosphonates and dosing regimens on cortical microstructure (porosity, thickness, area) and bone mechanical properties in animal models of CKD. METHODS: In experiment 1, Male Cy/+ (CKD) rats were treated with either a single dose or ten fractionated doses of zoledronate at 18 weeks of age. Fractionated animals received 1/10th of single dose given weekly for 10 weeks, with study endpoint at 28 weeks of age. In experiment 2, male C57Bl/6 J mice were given dietary adenine (0.2%) to induce CKD. Bisphosphonate treated groups were given either a single dose of zoledronate or weekly risedronate injections for 4 weeks. Cortical microstructure was assessed via µCT and mechanical parameters evaluated by monotonic bending tests. RESULTS: Exp 1: CKD rats had higher blood urea nitrogen (BUN) and parathyroid hormone (PTH) compared to NL littermate controls. Single dose zoledronate had significantly higher cortical porosity in CKD S.Zol (2.29%) compared to NL control (0.04%) and untreated CKD (0.14%) (p = 0.004). Exp 2: All adenine groups had significantly higher BUN and PTH compared to control mice. Mice treated with single dose zoledronate (Ad + Zol) had the highest porosity (~6%), which was significantly higher compared to either Ad or Ad + Ris (~3%; p < 0.0001) and control mice had the lowest cortical porosity (0.35%). In both experiments, mechanics were minimally affected by any bisphosphonate dosing regimen. CONCLUSION: A single dose of zoledronate leads to higher cortical porosity compared to more frequent dosing of bisphosphonates (fractionated zoledronate or risedronate). Bisphosphonate treatment demonstrated limited effectiveness in preventing cortical bone microstructure deterioration with mechanical parameters remaining compromised due to CKD and/or secondary hyperparathyroidism irrespective of bisphosphonate treatment.

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Roux-en Y gastric bypass (RYGB) surgery is an effective treatment for obesity; however, it may negatively impact skeletal health by increasing fracture risk. This increase may be the result not only of decreased bone mineral density but also of changes in bone microstructure, for example, increased cortical porosity. Increased tibial and radial cortical porosity of patients undergoing RYGB surgery has been observed as early as 6 months postoperatively; however, local microstructural changes and associated biological mechanisms driving this increase remain unclear. To provide insight, we studied the spatial distribution of cortical porosity in 42 women and men (aged 46 ± 12 years) after RYGB surgery. Distal tibias and radii were evaluated with high-resolution peripheral quantitative computed tomography (HR-pQCT) preoperatively and at 12 months postoperatively. Laminar analysis was used to determine cortical pore number and size within the endosteal, midcortical, and periosteal layers of the cortex. Paired t tests were used to compare baseline versus follow-up porosity parameters in each layer. Mixed models were used to compare longitudinal changes in laminar analysis outcomes between layers. We found that the midcortical (0.927 ± 0.607 mm-2 to 1.069 ± 0.654 mm-2 , p = 0.004; 0.439 ± 0.293 mm-2 to 0.509 ± 0.343 mm-2 , p = 0.03) and periosteal (0.642 ± 0.412 mm-2 to 0.843 ± 0.452 mm-2 , p < 0.0001; 0.171 ± 0.101 mm-2 to 0.230 ± 0.160 mm-2 , p = 0.003) layers underwent the greatest increases in porosity over the 12-month period at the distal tibia and radius, respectively. The endosteal layer, which had the greatest porosity at baseline, did not undergo significant porosity increase over the same period (1.234 ± 0.402 mm-2 to 1.259 ± 0.413 mm-2 , p = 0.49; 0.584 ± 0.290 mm-2 to 0.620 ± 0.299 mm-2 , p = 0.35) at the distal tibia and radius, respectively. An alternative baseline-mapping approach for endosteal boundary definition confirmed that cortical bone loss was not primarily endosteal. These findings indicate that increases in cortical porosity happen in regions distant from the endosteal surface, suggesting that the underlying mechanism driving the increase in cortical porosity is not merely endosteal trabecularization. © 2022 American Society for Bone and Mineral Research (ASBMR).

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PURPOSE: Chronic kidney disease (CKD) and aging are each independently associated with higher fracture risk. Although CKD is highly prevalent in the aging population, the interaction between these two conditions with respect to bone structure and mechanics is not well understood. The purpose of this study was to examine cortical porosity and mechanical properties in skeletally mature young and aging mice with CKD. METHODS: CKD was induced by feeding 16-week and 78-week male mice 0.2% adenine (AD) for six weeks followed by two weeks of maintenance on a control diet for a total study duration of eight weeks of CKD; control (CON) animals of each age were fed a standard diet. Serum biochemistries, µCT imaging, and mechanical properties via four-point bending were assessed at the endpoint. RESULTS: Phosphorus, parathyroid hormone, and blood urea nitrogen were elevated in both ages of AD mice compared to age-matched CON; aging AD mice had PTH and BUN values higher than all other groups. Femoral cortical porosity was more than four-fold higher in aging AD mice compared to young AD mice and more than two-fold higher compared to age-matched controls. Structural and estimated material mechanical properties were both lower in aging mice, but there were no significant interactions between AD treatment and age. CONCLUSION: These data show an interaction between CKD and aging that produces a more severe biochemical and cortical bone phenotype. This highlights the importance of studying mechanisms and potential interventions in both young and aged animals to translate to a broader spectrum of CKD patients.

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