The prevention of fragility fractures in bone-pathologic fractures resulting from daily activity and mostly occurring in the elderly population-has been a long-term clinical quest. Recent research indicating that falls in the elderly might be the consequence of fracture rather than its cause has raised fundamental questions about the origin of fragility fractures. Is day-to-day cyclic loading, instead of a single-load event such as a fall, the main cause of progressively growing fractures? Are fragility fractures predominantly affected by bone quality rather than bone mass, which is the clinical indicator of fracture risk? Do osteocytes actively participate in the bone repair process? In this Perspective, we discuss the central role of cyclic fatigue in bone fragility fracture.

Osteocytes locally remodel their surrounding tissue through perilacunar canalicular remodeling (PLR). During lactation, osteocytes remove minerals to satisfy the metabolic demand, resulting in increased lacunar volume, quantifiable with synchrotron X-ray radiation micro-tomography (SRµCT). Although the effects of lactation on PLR are well-studied, it remains unclear whether PLR occurs uniformly throughout the bone and what mechanisms prevent PLR from undermining bone quality. We used SRµCT imaging to conduct an in-depth spatial analysis of the impact of lactation and osteocyte-intrinsic MMP13 deletion on PLR in murine bone. We found larger lacunae undergoing PLR are located near canals in the mid-cortex or endosteum. We show lactation-induced hypomineralization occurs 14 µm away from lacunar edges, past a hypermineralized barrier. Our findings reveal that osteocyte-intrinsic MMP13 is crucial for lactation-induced PLR near lacunae in the mid-cortex but not for whole-bone resorption. This research highlights the spatial control of PLR on mineral distribution during lactation.

Bone fragility is the product of defects in bone mass and bone quality, both of which show sex-specific differences. Despite this, the cellular and molecular mechanisms underpinning the sexually dimorphic control of bone quality remain unclear, limiting our ability to effectively prevent fractures, especially in postmenopausal osteoporosis. Recently, using male mice, we found that systemic or osteocyte-intrinsic inhibition of TGFβ signaling, achieved using the 9.6-kb DMP1 promoter-driven Cre recombinase (TβRII^ocy-/- mice), suppresses osteocyte perilacunar/canalicular remodeling (PLR) and compromises bone quality. Because systemic TGFβ inhibition more robustly increases bone mass in female than male mice, we postulated that sex-specific differences in bone quality could likewise result, in part, from dimorphic regulation of PLR by TGFβ. Moreover, because lactation induces PLR, we examined the effect of TGFβ inhibition on the female skeleton during lactation. In contrast to males, female mice that possess an osteocyte-intrinsic defect in TGFβ signaling were protected from TGFβ-dependent defects in PLR and bone quality. The expression of requisite PLR enzymes, the lacunocanalicular network (LCN), and the flexural strength of female TβRII^ocy-/- bone was intact. With lactation, however, bone loss and induction in PLR and osteocytic parathyroid hormone type I receptor (PTHR1) expression, were suppressed in TβRII^ocy-/- bone, relative to the control littermates. Indeed, differential control of PTHR1 expression, by TGFβ and other factors, may contribute to dimorphism in PLR regulation in male and female TβRII^ocy-/- mice. These findings provide key insights into the sex-based differences in osteocyte PLR that underlie bone quality and highlight TGFβ signaling as a crucial regulator of lactation-induced PLR. © 2020 American Society for Bone and Mineral Research.

All levels of the unique hierarchical structure of bone, consisting of collagen and hydroxyapatite crystals at the nanoscale to osteon/lamellae structures at the microscale, contribute to its characteristic toughness and material properties. Elements of bone's density and size contribute to bone quantity (or bone mass), whereas elements of bone's material composition, material properties, internal structure, and organization describe bone quality. Bone quantity and quality can be degraded by factors such as aging, disease, treatments, and irradiation, compromising its ability to resist fracture and sustain loading. Accessing the morphology and architecture of bone at the microscale to quantify microstructural features and assess the degree of mineralization and path of crack propagation in bone provides crucial information on how these factors are influencing bone quantity and quality. Synchrotron radiation micro-computed tomography (SRμCT) was first used to assess bone structure at the end of the 1990's. One of the main advantages of the technique is that it enables accurate three-dimensional (3D), non-destructive quantification of structure while traditional histomorphometry on histological sections is inherantly destructive to the sample and two-dimensional (2D). Additionally, SRμCT uses monochromatic, high-flux X-ray beams to provide high-resolution and high-contrast imaging of bone samples. This allows the quantification of small microstructural features (e.g. osteocyte lacunae, canals, trabeculae, microcracks) and direct gray value compositional mapping (e.g. mineral quantification, cement lines) with greater speed and fidelity than lab-based micro-computed tomography. In this article, we review how SRμCT has been applied to bone research to elucidate the mechanisms by which bone aging, disease, and other factors affect bone fragility and resistance to fracture.

Through a process called perilacunar remodeling, bone-embedded osteocytes dynamically resorb and replace the surrounding perilacunar bone matrix to maintain mineral homeostasis. The vital canalicular networks required for osteocyte nourishment and communication, as well as the exquisitely organized bone extracellular matrix, also depend upon perilacunar remodeling. Nonetheless, many questions remain about the regulation of perilacunar remodeling and its role in skeletal disease. Here, we find that suppression of osteocyte-driven perilacunar remodeling, a fundamental cellular mechanism, plays a critical role in the glucocorticoid-induced osteonecrosis. In glucocorticoid-treated mice, we find that glucocorticoids coordinately suppress expression of several proteases required for perilacunar remodeling while causing degeneration of the osteocyte lacunocanalicular network, collagen disorganization, and matrix hypermineralization; all of which are apparent in human osteonecrotic lesions. Thus, osteocyte-mediated perilacunar remodeling maintains bone homeostasis, is dysregulated in skeletal disease, and may represent an attractive therapeutic target for the treatment of osteonecrosis.

This research aims to further the understanding of the structure and mechanical properties of the dermal armor of the boxfish (Lactoria cornuta). Structural differences between collagen regions underlying the hexagonal scutes were observed with confocal microscopy and microcomputed tomography (μ-CT). μ-CT revealed a tapering of the mineral plate from the center of the scute to the interface between scutes, suggesting the structure allows for more flexibility at the interface. High-resolution μ-CT revealed, for the first time, a 3D image of the dermal armor's complex collagen structure. Helical interfibrillar gaps in the collagen base were found that are similar to the Bouligand-type structure of the lobster, Homarus americanus, thereby suggesting that the collagen in the boxfish is also of a Bouligand-type structure. In situ scanning electron microscopy tests were performed in shear and tension between two connected scutes and suggest that the interfacial collagen is structurally designed to preferentially absorb energy during deformation to protect the internal collagen. Similarly, in situ small-angle x-ray scattering was performed in shear and tension and further corroborated the complex collagen structure. Lastly, these experimental results are coupled with finite element simulations that characterize the interfacial collagen and corroborate the non-linear deformation response seen during in situ testing. Overall, these findings further the understanding of the structure and mechanics of the dermal armor of the boxfish which may help provide a basis to synthesize bioinspired composites for impact-resistant materials, specifically with bioinspired Bouligand-type structures to create novel fiber-reinforced composites.

Bone remodeling, a combination of bone resorption and formation, requires precise regulation of cellular and molecular signaling to maintain proper bone quality. Whereas osteoblasts deposit and osteoclasts resorb bone matrix, osteocytes both dynamically resorb and replace perilacunar bone matrix. Osteocytes secrete proteases like matrix metalloproteinase-13 (MMP13) to maintain the material quality of bone matrix through perilacunar remodeling (PLR). Deregulated bone remodeling impairs bone quality and can compromise hearing since the auditory transduction mechanism is within bone. Understanding the mechanisms regulating cochlear bone provides unique ways to assess bone quality independent of other aspects that contribute to bone mechanical behavior. Cochlear bone is singular in its regulation of remodeling by expressing high levels of osteoprotegerin. Since cochlear bone expresses a key PLR enzyme, MMP13, we examined whether cochlear bone relies on, or is protected from, osteocyte-mediated PLR to maintain hearing and bone quality using a mouse model lacking MMP13 (MMP13(-/-)). We investigated the canalicular network, collagen organization, lacunar volume via micro-computed tomography, and dynamic histomorphometry. Despite finding defects in these hallmarks of PLR in MMP13(-/-) long bones, cochlear bone revealed no differences in these markers, nor hearing loss as measured by auditory brainstem response (ABR) or distortion product oto-acoustic emissions (DPOAEs), between wild type and MMP13(-/-) mice. Dynamic histomorphometry revealed abundant PLR by tibial osteocytes, but near absence in cochlear bone. Cochlear suppression of PLR corresponds to repression of several key PLR genes in the cochlea relative to long bones. These data suggest that cochlear bone uniquely maintains bone quality and hearing independent of MMP13-mediated osteocytic PLR. Furthermore, the cochlea employs parallel mechanisms to inhibit remodeling by osteoclasts and osteoblasts, and by osteocytes, to protect hearing. Understanding the cellular and molecular mechanisms that confer site-specific control of bone remodeling has the potential to elucidate new pathways that are deregulated in skeletal disease.

Understanding the biomechanical behavior of the intervertebral disc is crucial for studying disease mechanisms and developing tissue engineering strategies for managing disc degeneration. We used synchrotron small-angle X-ray scattering to investigate how changes to collagen behavior contribute to alterations in the disc's ability to resist compression. Coccygeal motion segments from 6-month-old lean Sprague-Dawley rats ( n=7) and diabetic obese University of California Davis type 2 diabetes mellitus (UCD-T2DM) rats ( n=6, diabetic for 68±7 days) were compressed during simultaneous synchrotron scanning to measure collagen strain at the nanoscale (beamline 7.3.3 of the Advanced Light Source). After compression, the annulus fibrosus was assayed for nonenzymatic cross-links. In discs from lean rats, resistance to compression involved two main energy-dissipation mechanisms at the nanoscale: (1) rotation of the two groups of collagen fibrils forming the annulus fibrosus and (2) straightening (uncrimping) and stretching of the collagen fibrils. In discs from diabetic rats, both mechanisms were significantly impaired. Specifically, diabetes reduced fibril rotation by 31% and reduced collagen fibril strain by 30% (compared to lean discs). The stiffening of collagen fibrils in the discs from diabetic rats was consistent with a 31% higher concentration of nonenzymatic cross-links and with evidence of earlier onset plastic deformations such as fibril sliding and fibril-matrix delamination. These findings suggest that fibril reorientation, stretching, and straightening are key deformation mechanisms that facilitate whole-disc compression, and that type 2 diabetes impairs these efficient and low-energy elastic deformation mechanisms, thereby altering whole-disc behavior and inducing the earlier onset of plastic deformation.

Chronic kidney disease (CKD) is a common disease of aging and increases fracture risk over advanced age alone. Aging and CKD differently impair bone turnover and mineralization. We thus hypothesize that the loss of bone quality would be greatest with the combination of advanced age and CKD. We evaluated bone from young adult (6 mo.), middle-age (18 mo.), and old (24 mo.) male C57Bl/6 mice three months following either 5/6th nephrectomy, to induce CKD, or Sham procedures. CKD exacerbated losses of cortical and trabecular microarchitecture associated with aging. Aging and CKD each resulted in thinner, more porous cortices and fewer and thinner trabeculae. Bone material quality was also reduced with CKD, and these changes to bone material were distinct from those due to age. Aging reduced whole-bone flexural strength and modulus, micrometer-scale nanoindentation modulus, and nanometer-scale tissue and collagen strain (small-angle x-ray scattering [SAXS]. By contrast, CKD reduced work to fracture and variation in bone tissue modulus and composition (Raman spectroscopy), and increased percent collagen strain. The increased collagen strain burden was associated with loss of toughness in CKD. In addition, osteocyte lacunae became smaller, sparser, and more disordered with age for Sham mice, yet these age-related changes were not clearly observed in CKD. However, for CKD, larger lacunae positively correlated with increased serum phosphate levels, suggesting that osteocytes play a role in systemic mineral homeostasis. This work demonstrates that CKD reduces bone quality, including microarchitecture and bone material properties, and that loss of bone quality with age is compounded by CKD. These findings may help reconcile why bone mass does not consistently predict fracture in the CKD population, as well as why older individuals with CKD are at high risk of fragility.