Structural role of osteocalcin and its modification in bone fracture.

Osteocalcin (OC), an abundant non-collagenous protein in bone extracellular matrix, plays a vital role in both its biological and mechanical function. OC undergoes post-translational modification, such as glycation; however, it remains unknown whether glycation of OC affects bone's resistance to fracture. Here, for the first time, we demonstrate the formation of pentosidine, an advanced glycation end-product (AGE) cross-link on mouse OC analyzed by ultra-performance liquid chromatography. Next, we establish that the presence of OC in mouse bone matrix is associated with lower interlamellar separation (distance) and thicker bridges spanning the lamellae, both of which are critical for maintaining bone's structural integrity. Furthermore, to determine the impact of modification of OC by glycation on bone toughness, we glycated bone samples in vitro from wild-type (WT) and osteocalcin deficient (Oc-/-) mice, and compared the differences in total fluorescent AGEs and fracture toughness between the Oc -/- glycated and control mouse bones and the WT glycated and control mouse bones. We determined that glycation resulted in significantly higher AGEs in WT compared to Oc-/- mouse bones (delta-WT > delta-OC, p = 0.025). This observed change corresponded to a significant decrease in fracture toughness between WT and Oc-/- mice (delta-WT vs delta-OC, p = 0.018). Thus, we propose a molecular deformation and fracture mechanics model that corroborates our experimental findings and provides evidence to support a 37%-90% loss in energy dissipation of OC due to formation of pentosidine cross-link by glycation. We anticipate that our study will aid in elucidating the effects of a major non-collagenous bone matrix protein, osteocalcin, and its modifications on bone fragility and help identify potential therapeutic targets for maintaining skeletal health.

Unmasking Fracture Risk in Type 2 Diabetes: The Association of Longitudinal Glycemic Hemoglobin Level and Medications.

CONTEXT: Fracture risk is underestimated in people with type 2 diabetes (T2D). OBJECTIVE: To investigate the longitudinal relationship of glycated hemoglobin (HbA1c) and common medications on fracture risk in people with T2D. METHODS: This retrospective population-based cohort study was conducted using de-identified claims and electronic health record data obtained from the OptumLabs Data Warehouse for the period January 1, 2007, to September 30, 2015. For each individual, the study was conducted within a 2-year HbA1c observation period and a 2-year fracture follow-up period. A cohort of 157 439 individuals with T2D [age ≥ 55 years with mean HbA1c value ≥ 6%] were selected from 4 018 250 US Medicare Advantage/Commercial enrollees with a T2D diagnosis. All fractures and fragility fractures were measured. RESULTS: With covariates adjusted, poor glycemic control in T2D individuals was associated with an 29% increase of all fracture risk, compared with T2D individuals who had adequate glycemic control (HR: 1.29; 95% CI, 1.22-1.36). Treatment with metformin (HR: 0.88; 95% CI, 0.85-0.92) and DPP4 inhibitors (HR: 0.93; 95% CI, 0.88-0.98) was associated with a reduced all fracture risk, while insulin (HR: 1.26; 95% CI, 1.21-1.32), thiazolidinediones (HR: 1.23; 95% CI, 1.18-1.29), and meglitinides (HR: 1.12; 95% CI, 1.00-1.26) were associated with an increased all fracture risk (All P value < 0.05). Bisphosphonates were associated similarly with increased fracture risk in the T2D and nondiabetic groups. CONCLUSION: Longitudinal 2-year HbA1c is independently associated with elevated all fracture risk in T2D individuals during a 2-year follow-up period. Metformin and DPP4 inhibitors can be used for management of T2D fracture risk.

Structural role of osteocalcin and osteopontin in energy dissipation in bone.

Non-collagenous proteins are a vital component of bone matrix. Amongst them, osteocalcin (OC) and osteopontin (OPN) hold special significance due to their intimate interaction with the mineral and collagenous matrix in bone. Both proteins have been associated with microdamage and fracture, but their structural role in energy dissipation is unclear. This study used bone tissue from genetic deficient mice lacking OC and/or OPN and subjected them to a series of creep-fatigue-creep tests. To this end, whole tibiae were loaded in four-point bending to 70% stiffness loss which captured the three characteristic phases of fatigue associated with initiation, propagation, and coalescence of microdamage. Fatigue loading preceded and followed creep tests to determine creep and dampening parameters. Microdamage in the form of linear microcracks and diffuse damage were analyzed by histology. It was shown that OC and OPN were 'activated' following stiffness loss associated with fatigue damage where they facilitated creep and dampening parameters (i.e. increased energy dissipation). More specifically, post-fatigue creep rate and dampening were significantly greater in wild-types (WTs) than genetic deficient mice (p < 0.05). These results were supported by microdamage analysis which showed significant increase in creep-associated diffuse damage formation in WTs compared to genetic deficient groups (p < 0.05). Based on these findings, we propose that during local yield events, OC and OPN rely on ionic interactions of their charged side chains and on hydrogen bonding to dissipate energy in bone.

Biomimetic matrices for rapidly forming mineralized bone tissue based on stem cell-mediated osteogenesis.

Bone regeneration, following fracture, relies on autologous and allogenic bone grafts. However, majority of fracture population consists of older individuals with poor quality bone associated with loss and/or modification of matrix proteins critical for bone formation and mineralization. Allografts suffer from same limitations and carry the risk of delayed healing, infection, immune rejection and eventual fracture. In this work, we apply a synergistic biomimetic strategy to develop matrices that rapidly form bone tissue - a critical aspect of fracture healing of weight bearing bones. Collagen matrices, enhanced with two selected key matrix proteins, osteocalcin (OC) and/or osteopontin (OPN), increased the rate and quantity of synthesized bone matrix by increasing mesenchymal stem/stromal cell (MSC) proliferation, accelerating osteogenic differentiation, enhancing angiogenesis and showing a sustained bone formation response from MSC obtained from a variety of human tissue sources (marrow, fat and umbilical cord). In vivo assessment of OC/OPN mineralized scaffolds in a critical sized-defect rabbit long-bone model did not reveal any foreign body reaction while bone tissue was being formed. We demonstrate a new biomimetic strategy to rapidly form mineralized bone tissue and secure a sustained bone formation response by MSC from multiple sources, thus facilitating faster patient recovery and treatment of non-union fractures in aging and diseased population. Acellular biomimetic matrices elicit bone regeneration response from MSC, obtained from multiple tissue sources, and can be used in variety of scaffolds and made widely available.

Biomolecular regulation, composition and nanoarchitecture of bone mineral.

Tough natural nanocomposites like bone, nacre and sea sponges contain within their hierarchy, a mineral (phosphate, silicate or carbonate) phase that interacts with an organic phase. In bone, the role of mineral ultrastructure (organization, morphology, composition) is crucial to the mechanical and biological properties of the tissue. Better understanding of mineral interaction with the organic matrix, in particular non-collagenous proteins, osteocalcin (OC) and osteopontin (OPN), can lead to better design of biomimetic materials. Using small angle x-ray scattering (SAXS) and wavelength dispersive spectroscopy (WDS) on single (OC-/- and OPN-/-) and double (OC-OPN-/-;-/-) genetic knockout mice bones, we demonstrate that both osteocalcin and osteopontin have specific roles in the biomolecular regulation of mineral in bone and together they are major determinants of the quality of bone mineral. Specifically, for the first time, we show that proteins osteocalcin and osteopontin regulate bone mineral crystal size and organization in a codependent manner, while they independently determine crystal shape. We found that OC is more dominant in the regulation of the physical properties of bone mineral, while OPN is more dominant in the regulation of the mineral composition.

A direct role of collagen glycation in bone fracture.

Non-enzymatic glycation (NEG) is an age-related process accelerated by diseases like diabetes, and causes the accumulation of advanced glycation end-products (AGEs). NEG-mediated modification of bone's organic matrix, principally collagen type-I, has been implicated in impairing skeletal physiology and mechanics. Here, we present evidence, from in vitro and in vivo models, and establish a causal relationship between collagen glycation and alterations in bone fracture at multiple length scales. Through atomic force spectroscopy, we established that NEG impairs collagen's ability to dissipate energy. Mechanical testing of in vitro glycated human bone specimen revealed that AGE accumulation due to NEG dramatically reduces the capacity of organic and mineralized matrix to creep and caused bone to fracture under impact at low levels of strain (3000-5000 µstrain) typically associated with fall. Fracture mechanics tests of NEG modified human cortical bone of varying ages, and their age-matched controls revealed that NEG disrupted microcracking based toughening mechanisms and reduced bone propagation and initiation fracture toughness across all age groups. A comprehensive mechanistic model, based on experimental and modeling data, was developed to explain how NEG and AGEs are causal to, and predictive of bone fragility. Furthermore, fracture mechanics and indentation testing on diabetic mice bones revealed that diabetes mediated NEG severely disrupts bone matrix quality in vivo. Finally, we show that AGEs are predictive of bone quality in aging humans and have diagnostic applications in fracture risk.

Do Non-collagenous Proteins Affect Skeletal Mechanical Properties?

The remarkable mechanical behavior of bone is attributed to its complex nanocomposite structure that, in addition to mineral and collagen, comprises a variety of non-collagenous matrix proteins or NCPs. Traditionally, NCPs have been studied as signaling molecules in biological processes including bone formation, resorption, and turnover. Limited attention has been given to their role in determining the mechanical properties of bone. Recent studies have highlighted that NCPs can indeed be lost or modified with aging, diseases, and drug therapies. Homozygous and heterozygous mice models of key NCP provide a useful approach to determine the impact of NCPs on bone morphology as well as matrix quality, and to carry out detailed mechanical analysis for elucidating the pathway by which NCPs can affect the mechanical properties of bone. In this article, we present a systematic analysis of a large cohort of NCPs on bone's structural and material hierarchy, and identify three principal pathways by which they determine bone's mechanical properties. These pathways include alterations of bone morphological parameters crucial for bone's structural competency, bone quality changes in key matrix parameters (mineral and collagen), and a direct role as load-bearing structural proteins.

Multiscale imaging of bone microdamage.

Bone is a structural and hierarchical composite that exhibits remarkable ability to sustain complex mechanical loading and resist fracture. Bone quality encompasses various attributes of bone matrix from the quality of its material components (type-I collagen, mineral and non-collagenous matrix proteins) and cancellous microarchitecture, to the nature and extent of bone microdamage. Microdamage, produced during loading, manifests in multiple forms across the scales of hierarchy in bone and functions to dissipate energy and avert fracture. Microdamage formation is a key determinant of bone quality, and through a range of biological and physical mechanisms, accumulates with age and disease. Accumulated microdamage in bone decreases bone strength and increases bone's propensity to fracture. Thus, a thorough assessment of microdamage, across the hierarchical levels of bone, is crucial to better understand bone quality and bone fracture. This review article details multiple imaging modalities that have been used to study and characterize microdamage; from bulk staining techniques originally developed by Harold Frost to assess linear microcracks, to atomic force microscopy, a modality that revealed mechanistic insights into the formation diffuse damage at the ultrastructural level in bone. New automated techniques using imaging modalities, such as microcomputed tomography are also presented for a comprehensive overview.

Dilatational band formation in bone.

Toughening in hierarchically structured materials like bone arises from the arrangement of constituent material elements and their interactions. Unlike microcracking, which entails micrometer-level separation, there is no known evidence of fracture at the level of bone's nanostructure. Here, we show that the initiation of fracture occurs in bone at the nanometer scale by dilatational bands. Through fatigue and indentation tests and laser confocal, scanning electron, and atomic force microscopies on human and bovine bone specimens, we established that dilatational bands of the order of 100 nm form as ellipsoidal voids in between fused mineral aggregates and two adjacent proteins, osteocalcin (OC) and osteopontin (OPN). Laser microdissection and ELISA of bone microdamage support our claim that OC and OPN colocalize with dilatational bands. Fracture tests on bones from OC and/or OPN knockout mice (OC(-/-), OPN(-/-), OC-OPN(-/-;-/-)) confirm that these two proteins regulate dilatational band formation and bone matrix toughness. On the basis of these observations, we propose molecular deformation and fracture mechanics models, illustrating the role of OC and OPN in dilatational band formation, and predict that the nanometer scale of tissue organization, associated with dilatational bands, affects fracture at higher scales and determines fracture toughness of bone.