Mineral and cross-linking in collagen fibrils: The mechanical behavior of bone tissue at the nano-scale.

The mineralized collagen fibril is the main building block of hard tissues and it directly affects the macroscopic mechanics of biological tissues such as bone. The mechanical behavior of the fibril itself is determined by its structure: the content of collagen molecules, minerals, and cross-links, and the mechanical interactions and properties of these components. Advanced-Glycation-Endproducts (AGEs) cross-linking between tropocollagen molecules within the collagen fibril is one important factor that is believed to have a major influence on the tissue. For instance, it has been shown that brittleness in bone correlates with increased AGEs densities. However, the underlying nano-scale mechanisms within the mineralized collagen fibril remain unknown. Here, we study the effect of mineral and AGEs cross-linking on fibril deformation and fracture behavior by performing destructive tensile tests using coarse-grained molecular dynamics simulations. Our results demonstrate that after exceeding a critical content of mineral, it induces stiffening of the collagen fibril at high strain levels. We show that mineral morphology and location affect collagen fibril mechanics: The mineral content at which this stiffening occurs depends on the mineral's location and morphology. Further, both, increasing AGEs density and mineral content lead to stiffening and increased peak stresses. At low mineral contents, the mechanical response of the fibril is dominated by the AGEs, while at high mineral contents, the mineral itself determines fibril mechanics.

Type 2 diabetes impairs annulus fibrosus fiber deformation and rotation under disc compression in the University of California Davis type 2 diabetes mellitus (UCD-T2DM) rat model.

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.

Advanced-Glycation Endproducts: How cross-linking properties affect the collagen fibril behavior.

Advanced-Glycation-Endproducts (AGEs) are known to be a major cause of impaired tissue material properties. In collagen fibrils, which constitute a major building component of human tissue, these AGEs appear as fibrillar cross-links. It has been shown that when AGEs accumulate in collagen fibrils, a process often caused by diabetes and aging, the mechanical properties of the collagen fibril are altered. However, current knowledge about the mechanical properties of different types of AGEs, and their quantity in collagen fibrils is limited owing to the scarcity of available experimental data. Consequently, the precise relationship between the nano-scale cross-link properties, which differ from type to type, their density in collagen fibrils, and the mechanical properties of the collagen fibrils at larger scales remains poorly understood. In our study, we use coarse-grained molecular dynamics simulations and perform destructive tensile tests on collagen fibrils to evaluate the effect of different cross-link densities and their mechanical properties on collagen fibril deformation and fracture behavior. We observe that the collagen fibril stiffens at high strain levels when either the AGEs density or the loading energy capacity of AGEs are increased. Based on our results, we demonstrate that this stiffening is caused by a mechanism that favors energy absorption via stretching rather than inter-molecular sliding. Hence, in these cross-linked collagen fibrils, the absorbed energy is stored rather than dissipated through friction, resulting in brittle fracture upon fibrillar failure. Further, by varying multiple AGEs nano-scale parameters, we show that the AGEs loading energy capacity is, aside from their density in the fibril, the unique factor determining the effect of different types of AGEs on the mechanical behavior of collagen fibrils. Our results show that knowing AGEs properties is crucial for a better understanding of the nano-scale origin of impaired tissue behavior. We further suggest that future experimental investigations should focus on the quantification of the loading energy capacity of AGEs as a key property for their influence on collagen fibrils.

Effect of in vitro ribosylation on the dynamic fracture behavior of mature bovine cortical bone.

In this study, the fracture behavior of ribosylated bovine cortical bone is investigated under loading conditions simulating a fall event. Single edge notched specimens, separated into a control group (n = 11) and a ribosylated group (n = 8), were extracted from the mid-diaphysis of a single bovine femur harvested from a mature cow. A seven-day ribosylation process results in the accumulation of Advanced-Glycation End Products (AGEs) cross-links and AGE adducts. Specimens were subjected to symmetric three point bending (opening mode) and an impact velocity of 1.6 m/s using a drop tower. Near-crack displacement fields up to fracture initiation are determined from high-speed images post-processed using digital image correlation. A constrained over-deterministic least squares regression and orthotropic material linear elastic fracture mechanics theory are used to extract the in-plane critical stress intensity factors at fracture initiation (i.e., fracture initiation toughness values). Statistically significant differences were not observed when comparing the in-plane fracture initiation toughness values (p≥0.96) or energy release rate (p=0.90) between the control and seven-day ribosylated groups. The intrinsic variability of bone may require high sample numbers in order to achieve an adequately powered experiment when assessing dynamic fracture behavior. While there are no detectable differences due to the ribosylation treatment investigated, this is likely due to the limited sample sizes utilized.

Effect of non-enzymatic glycation on collagen nanoscale mechanisms in diabetic and age-related bone fragility.

Age and diabetes have long been known to induce an oxidative reaction between glucose and collagen, leading to the accumulation of advanced glycation end-products (AGEs) cross-links in collagenous tissues. More recently, AGEs content has been related to loss of bone quality, independent of bone mass, and increased fracture risk with aging and diabetes. Loss of bone quality is mostly attributed to changes in material properties, structural organization, or cellular remodeling. Though all these factors play a role in bone fragility disease, some common recurring patterns can be found between diabetic and age-related bone fragility. The main pattern we will discuss in this viewpoint is the increase of fibrillar collagen stiffness and loss of collagen-induced plasticity with AGE accumulation. This study focused on recent related experimental studies and discusses the correlation between fluorescent AGEs content at the molecular and fibrillar scales, collagen deformation mechanisms at the nanoscale, and resistance to bone fracture at the macroscale.

Advanced-Glycation Endproducts: How cross-linking properties affect the collagen fibril behavior.

Advanced-Glycation-Endproducts (AGEs) are known to be a major cause of impaired tissue material properties. In collagen fibrils, which constitute a major building component of human tissue, these AGEs appear as fibrillar cross-links. It has been shown that when AGEs accumulate in collagen fibrils, a process often caused by diabetes and aging, the mechanical properties of the collagen fibril are altered. However, current knowledge about the mechanical properties of different types of AGEs, and their quantity in collagen fibrils is limited owing to the scarcity of available experimental data. Consequently, the precise relationship between the nano-scale cross-link properties, which differ from type to type, their density in collagen fibrils, and the mechanical properties of the collagen fibrils at larger scales remains poorly understood. In our study, we use coarse-grained molecular dynamics simulations and perform destructive tensile tests on collagen fibrils to evaluate the effect of different cross-link densities and their mechanical properties on collagen fibril deformation and fracture behavior. We observe that the collagen fibril stiffens at high strain levels when either the AGEs density or the loading energy capacity of AGEs are increased. Based on our results, we demonstrate that this stiffening is caused by a mechanism that favors energy absorption via stretching rather than inter-molecular sliding. Hence, in these cross-linked collagen fibrils, the absorbed energy is stored rather than dissipated through friction, resulting in brittle fracture upon fibrillar failure. Further, by varying multiple AGEs nano-scale parameters, we show that the AGEs loading energy capacity is, aside from their density in the fibril, the unique factor determining the effect of different types of AGEs on the mechanical behavior of collagen fibrils. Our results show that knowing AGEs properties is crucial for a better understanding of the nano-scale origin of impaired tissue behavior. We further suggest that future experimental investigations should focus on the quantification of the loading energy capacity of AGEs as a key property for their influence on collagen fibrils.

The influence of AGEs and enzymatic cross-links on the mechanical properties of collagen fibrils.

Collagen, one of the main building blocks for various tissues, derives its mechanical properties directly from its structure of cross-linked tropocollagen molecules. The cross-links are considered to be a key component of collagen fibrils as they can change the fibrillar behavior in various ways. For instance, enzymatic cross-links (ECLs), one particular type of cross-links, are known for stabilizing the structure of the fibril and improving material properties, while cross-linking AGEs (Advanced-Glycation Endproducts) have been shown to accumulate and impair the mechanical properties of collageneous tissues. However, the reasons for whether and how a given type of cross-link improves or impairs the material properties remain unknown, and the exact relationship between the cross-link properties and density, and the fibrillar behavior is still not well understood. Here, we use coarse-grained steered molecular models to evaluate the effect of AGEs and ECLs cross-links content on the deformation and failure properties of collagen fibrils. Our simulations show that the collagen fibrils stiffen at high strain levels when the AGEs content exceeds a critical value. In addition, the strength of the fibril increases with AGEs accumulation. By analyzing the forces within the different types of cross-links (AGEs and ECLs) as well as their failure, we demonstrate that a change of deformation mechanism is at the origin of these observations. A high AGEs content reinforces force transfer through AGEs cross-links rather than through friction between sliding tropocollagen molecules, which leads to failure by breaking of bonds within the tropocollagen molecules. We show that this failure mechanism, which is associated with lower energy dissipation, results in more abrupt failure of the collagen fibril. Our results provide a direct and causal link between increased AGEs content, inhibited intra-fibrillar sliding, increased stiffness, and abrupt fibril fracture. Therefore, they explain the mechanical origin of bone brittleness as commonly observed in elderly and diabetic populations. Our findings contribute to a better understanding of the mechanisms underlying impaired tissue behavior due to elevated AGEs content and could enable targeted measures regarding the reduction of specific collagen cross-linking levels.

The influence of AGEs and enzymatic cross-links on the mechanical properties of collagen fibrils.

Cross-links are considered to be a key component of collagen fibrils as they can change the fibrillar behavior in various ways. Advanced-Glycation Endproducts (AGEs), one particular type of cross-links, have been shown to accumulate and impair the mechanical properties of collageneous tissues, whereas enzymatic cross-links (ECLs) are known for stabilizing the structure of the fibril. However, the reasons for whether a given type of cross-link improves or impairs the material properties remain unknown. Here, we use coarse-grained steered molecular models to evaluate the effect of AGEs and ECLs cross-links content on the deformation and failure properties of collagen fibrils. Our simulations show that the collagen fibrils stiffen at high strain levels when the AGEs content exceeds a critical value. In addition, the strength of the fibril increases with AGEs accumulation. By analyzing the forces within the different types of cross-links (AGEs and ECLs) as well as their failure, we demonstrate that a change of deformation mechanism is at the origin of these observations. A high AGEs content reinforces force transfer through AGEs cross-links rather than through friction between sliding tropocollagen molecules. We show that this failure mechanism, which is associated with lower energy dissipation, results in more abrupt failure of the collagen fibril. Our results provide a direct and causal link between increased AGEs content, inhibited intra-fibrillar sliding, increased stiffness, and abrupt fibril fracture. Therefore, they explain the mechanical origin of bone brittleness as commonly observed in elderly and diabetic populations. Our findings contribute to a better understanding of the mechanisms underlying impaired tissue behaviour due to elevated AGEs content and could enable targeted measures regarding the reduction of specific collagen cross-linking levels.

Quantitative and qualitative bone imaging: A review of synchrotron radiation microtomography analysis in bone 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.

TGFß Regulation of Perilacunar/Canalicular Remodeling Is Sexually Dimorphic.

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 TGFß signaling, achieved using the 9.6-kb DMP1 promoter-driven Cre recombinase (TßRIIocy-/- mice), suppresses osteocyte perilacunar/canalicular remodeling (PLR) and compromises bone quality. Because systemic TGFß 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 TGFß. Moreover, because lactation induces PLR, we examined the effect of TGFß inhibition on the female skeleton during lactation. In contrast to males, female mice that possess an osteocyte-intrinsic defect in TGFß signaling were protected from TGFß-dependent defects in PLR and bone quality. The expression of requisite PLR enzymes, the lacunocanalicular network (LCN), and the flexural strength of female TßRIIocy-/- bone was intact. With lactation, however, bone loss and induction in PLR and osteocytic parathyroid hormone type I receptor (PTHR1) expression, were suppressed in TßRIIocy-/- bone, relative to the control littermates. Indeed, differential control of PTHR1 expression, by TGFß and other factors, may contribute to dimorphism in PLR regulation in male and female TßRIIocy-/- mice. These findings provide key insights into the sex-based differences in osteocyte PLR that underlie bone quality and highlight TGFß signaling as a crucial regulator of lactation-induced PLR. © 2020 American Society for Bone and Mineral Research.

Osteocyte dysfunction promotes osteoarthritis through MMP13-dependent suppression of subchondral bone homeostasis.

Osteoarthritis (OA), long considered a primary disorder of articular cartilage, is commonly associated with subchondral bone sclerosis. However, the cellular mechanisms responsible for changes to subchondral bone in OA, and the extent to which these changes are drivers of or a secondary reaction to cartilage degeneration, remain unclear. In knee joints from human patients with end-stage OA, we found evidence of profound defects in osteocyte function. Suppression of osteocyte perilacunar/canalicular remodeling (PLR) was most severe in the medial compartment of OA subchondral bone, with lower protease expression, diminished canalicular networks, and disorganized and hypermineralized extracellular matrix. As a step toward evaluating the causality of PLR suppression in OA, we ablated the PLR enzyme MMP13 in osteocytes while leaving chondrocytic MMP13 intact, using Cre recombinase driven by the 9.6-kb DMP1 promoter. Not only did osteocytic MMP13 deficiency suppress PLR in cortical and subchondral bone, but it also compromised cartilage. Even in the absence of injury, osteocytic MMP13 deficiency was sufficient to reduce cartilage proteoglycan content, change chondrocyte production of collagen II, aggrecan, and MMP13, and increase the incidence of cartilage lesions, consistent with early OA. Thus, in humans and mice, defects in PLR coincide with cartilage defects. Osteocyte-derived MMP13 emerges as a critical regulator of cartilage homeostasis, likely via its effects on PLR. Together, these findings implicate osteocytes in bone-cartilage crosstalk in the joint and suggest a causal role for suppressed perilacunar/canalicular remodeling in osteoarthritis.

Chronic kidney disease and aging differentially diminish bone material and microarchitecture in C57Bl/6 mice.

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.

Contributions of Material Properties and Structure to Increased Bone Fragility for a Given Bone Mass in the UCD-T2DM Rat Model of Type 2 Diabetes.

Adults with type 2 diabetes (T2D) have a higher fracture risk for a given bone quantity, but the mechanisms remain unclear. Using a rat model of polygenic obese T2D, we demonstrate that diabetes significantly reduces whole-bone strength for a given bone mass (µCT-derived BMC), and we quantify the roles of T2D-induced deficits in material properties versus bone structure; ie, geometry and microarchitecture. Lumbar vertebrae and ulnae were harvested from 6-month-old lean Sprague-Dawley rats, obese Sprague-Dawley rats, and diabetic obese UCD-T2DM rats (diabetic for 69 ± 7 days; blood glucose >200 mg/dL). Both obese rats and those with diabetes had reduced whole-bone strength for a given BMC. In obese rats, this was attributable to structural deficits, whereas in UCD-T2DM rats, this was attributable to structural deficits and to deficits in tissue material properties. For the vertebra, deficits in bone structure included thinner and more rod-like trabeculae; for the ulnae, these deficits included inefficient distribution of bone mass to resist bending. Deficits in ulnar material properties in UCD-T2DM rats were associated with increased non-enzymatic crosslinking and impaired collagen fibril deformation. Specifically, small-angle X-ray scattering revealed that diabetes reduced collagen fibril ultimate strain by 40%, and those changes coincided with significant reductions in the elastic, yield, and ultimate tensile properties of the bone tissue. Importantly, the biomechanical effects of these material property deficits were substantial. Prescribing diabetes-specific tissue yield strains in high-resolution finite element models reduced whole-bone strength by a similar amount (and in some cases a 3.4-fold greater amount) as the structural deficits. These findings provide insight into factors that increase bone fragility for a given bone mass in T2D; not only does diabetes associate with less biomechanically efficient bone structure, but diabetes also reduces tissue ductility by limiting collagen fibril deformation, and in doing so, reduces the maximum load capacity of the bone. © 2018 American Society for Bone and Mineral Research.

Fatigue as the missing link between bone fragility and fracture.

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.

Osteocyte-Intrinsic TGF-ß Signaling Regulates Bone Quality through Perilacunar/Canalicular Remodeling.

Poor bone quality contributes to bone fragility in diabetes, aging, and osteogenesis imperfecta. However, the mechanisms controlling bone quality are not well understood, contributing to the current lack of strategies to diagnose or treat bone quality deficits. Transforming growth factor beta (TGF-ß) signaling is a crucial mechanism known to regulate the material quality of bone, but its cellular target in this regulation is unknown. Studies showing that osteocytes directly remodel their perilacunar/canalicular matrix led us to hypothesize that TGF-ß controls bone quality through perilacunar/canalicular remodeling (PLR). Using inhibitors and mice with an osteocyte-intrinsic defect in TGF-ß signaling (TßRIIocy-/-), we show that TGF-ß regulates PLR in a cell-intrinsic manner to control bone quality. Altogether, this study emphasizes that osteocytes are key in executing the biological control of bone quality through PLR, thereby highlighting the fundamental role of osteocyte-mediated PLR in bone homeostasis and fragility.

Glucocorticoid suppression of osteocyte perilacunar remodeling is associated with subchondral bone degeneration in osteonecrosis.

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.

Parallel mechanisms suppress cochlear bone remodeling to protect hearing.

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.

Intrinsic mechanical behavior of femoral cortical bone in young, osteoporotic and bisphosphonate-treated individuals in low- and high energy fracture conditions.

Bisphosphonates are a common treatment to reduce osteoporotic fractures. This treatment induces osseous structural and compositional changes accompanied by positive effects on osteoblasts and osteocytes. Here, we test the hypothesis that restored osseous cell behavior, which resembles characteristics of younger, healthy cortical bone, leads to improved bone quality. Microarchitecture and mechanical properties of young, treatment-naïve osteoporosis, and bisphosphonate-treated cases were investigated in femoral cortices. Tissue strength was measured using three-point bending. Collagen fibril-level deformation was assessed in non-traumatic and traumatic fracture states using synchrotron small-angle x-ray scattering (SAXS) at low and high strain rates. The lower modulus, strength and fibril deformation measured at low strain rates reflects susceptibility for osteoporotic low-energy fragility fractures. Independent of age, disease and treatment status, SAXS revealed reduced fibril plasticity at high strain rates, characteristic of traumatic fracture. The significantly reduced mechanical integrity in osteoporosis may originate from porosity and alterations to the intra/extrafibrillar structure, while the fibril deformation under treatment indicates improved nano-scale characteristics. In conclusion, losses in strength and fibril deformation at low strain rates correlate with the occurrence of fragility fractures in osteoporosis, while improvements in structural and mechanical properties following bisphosphonate treatment may foster resistance to fracture during physiological strain rates.

Alendronate treatment alters bone tissues at multiple structural levels in healthy canine cortical bone.

Bisphosphonates are widely used to treat osteoporosis, but have been associated with atypical femoral fractures (AFFs) in the long term, which raises a critical health problem for the aging population. Several clinical studies have suggested that the occurrence of AFFs may be related to the bisphosphonate-induced changes of bone turnover, but large discrepancies in the results of these studies indicate that the salient mechanisms responsible for any loss in fracture resistance are still unclear. Here the role of bisphosphonates is examined in terms of the potential deterioration in fracture resistance resulting from both intrinsic (plasticity) and extrinsic (shielding) toughening mechanisms, which operate over a wide range of length-scales. Specifically, we compare the mechanical properties of two groups of humeri from healthy beagles, one control group comprising eight females (oral doses of saline vehicle, 1 mL/kg/day, 3 years) and one treated group comprising nine females (oral doses of alendronate used to treat osteoporosis, 0.2mg/kg/day, 3 years). Our data demonstrate treatment-specific reorganization of bone tissue identified at multiple length-scales mainly through advanced synchrotron x-ray experiments. We confirm that bisphosphonate treatments can increase non-enzymatic collagen cross-linking at molecular scales, which critically restricts plasticity associated with fibrillar sliding, and hence intrinsic toughening, at nanoscales. We also observe changes in the intracortical architecture of treated bone at microscales, with partial filling of the Haversian canals and reduction of osteon number. We hypothesize that the reduced plasticity associated with BP treatments may induce an increase in microcrack accumulation and growth under cyclic daily loadings, and potentially increase the susceptibility of cortical bone to atypical (fatigue-like) fractures.