Simon Y. Tang

Age-related changes in the plasticity and toughness of human cortical bone at multiple length-scales

The structure of human cortical bone evolves over multiple length scales from its basic constituents of collagen and hydroxyapatite at the nanoscale to osteonal structures at near-millimeter dimensions, which all provide the basis for its mechanical properties. To resist fracture, bone’s toughness is derived intrinsically through plasticity (e.g., fibrillar sliding) at structural scales typically below a micrometer and extrinsically (i.e., during crack growth) through mechanisms (e.g., crack deflection/bridging) generated at larger structural scales. Biological factors such as aging lead to a markedly increased fracture risk, which is often associated with an age-related loss in bone mass (bone quantity). However, we find that age-related structural changes can significantly degrade the fracture resistance (bone quality) over multiple length scales. Using in situ small-angle X-ray scattering and wide-angle X-ray diffraction to characterize submicrometer structural changes and synchrotron X-ray computed tomography and in situ fracture-toughness measurements in the scanning electron microscope to characterize effects at micrometer scales, we show how these age-related structural changes at differing size scales degrade both the intrinsic and extrinsic toughness of bone. Specifically, we attribute the loss in toughness to increased nonenzymatic collagen cross-linking, which suppresses plasticity at nanoscale dimensions, and to an increased osteonal density, which limits the potency of crack-bridging mechanisms at micrometer scales. The link between these processes is that the increased stiffness of the cross-linked collagen requires energy to be absorbed by “plastic” deformation at higher structural levels, which occurs by the process of microcracking.

Characterization of the effects of x-ray irradiation on the hierarchical structure and mechanical properties of human cortical bone

Bone comprises a complex structure of primarily collagen, hydroxyapatite and water, where each hierarchical structural level contributes to its strength, ductility and toughness. These properties, however, are degraded by irradiation, arising from medical therapy or bone-allograft sterilization. We provide here a mechanistic framework for how irradiation affects the nature and properties of human cortical bone over a range of characteristic (nano to macro) length-scales, following x-ray exposures up to 630 kGy. Macroscopically, bone strength, ductility and fracture resistance are seen to be progressively degraded with increasing irradiation levels. At the micron-scale, fracture properties, evaluated using insitu scanning electron microscopy and synchrotron x-ray computed micro-tomography, provide mechanistic information on how cracks interact with the bone-matrix structure. At sub-micron scales, strength properties are evaluated with insitu tensile tests in the synchrotron using small-/wide-angle x-ray scattering/diffraction, where strains are simultaneously measured in the macroscopic tissue, collagen fibrils and mineral. Compared to healthy bone, results show that the fibrillar strain is decreased by ∼40% following 70 kGy exposures, consistent with significant stiffening and degradation of the collagen. We attribute the irradiation-induced deterioration in mechanical properties to mechanisms at multiple length-scales, including changes in crack paths at micron-scales, loss of plasticity from suppressed fibrillar sliding at sub-micron scales, and the loss and damage of collagen at the nano-scales, the latter being assessed using Raman and Fourier Transform Infrared spectroscopy and a fluorometric assay.

Bisphosphonate treatment modifies canine bone mineral and matrix properties and their heterogeneity

Bone loss and alterations in bone quality are major causes leading to bone fragility in postmenopausal women. Although bisphosphonates are well known to reduce bone turnover and prevent bone loss in postmenopausal osteoporosis, their effects on other bone properties are not fully characterized. Changes in bone mineral and matrix properties may contribute to the anti-fracture efficacy observed with bisphosphonate treatments. The aim of this work was to analyze the effect of a 1-year treatment with either alendronate or risedronate, at low and high doses, on spatially resolved bone material and compositional properties that could contribute to the fracture efficacy of these agents. Distal tibias from 30 normal beagles that had been treated daily for 1 year with oral doses of vehicle (Veh), alendronate (Aln) at 0.2 or 1 mg/kg, and risedronate (Ris) at 0.1 or 0.5 mg/kg were analyzed by Fourier Transform Infrared imaging (FTIRI) to assess the changes in both mineral and matrix properties in discrete bone areas. The widths at half maximum of the pixel histograms for each FTIRI parameter were used to assess the heterogeneity of the bone tissue. Aln and Ris increased the mineral content and the collagen maturity mainly in cancellous bone and at the endocortical surface. Significant differences were observed in the mineral content and in the hydroxyapatite crystallinity distribution in bone tissue, which can contribute to reduced ductility and micro-crack accumulation. No significant differences were observed between low and high dose nor between Aln and Ris treatments. These results show that pharmacologic suppression of bone turnover increases the mineral and matrix bone tissue maturity in normal cancellous and endocortical bone areas where bone turnover is higher. These positive effects for decreased fracture risk are also associated with a loss of bone heterogeneity that could be one factor contributing to increased bone tissue brittleness and micro-crack accumulation.

Non-enzymatic glycation alters microdamage formation in human cancellous bone.

INTRODUCTION The accumulation of advanced glycation end-products (AGEs) in bone has been suggested to adversely affect the fracture resistance of bone with aging, diabetes, and pharmacological treatments. The formation of AGEs increases crosslinking in the organic matrix of bone but it is unknown how elevated levels of AGEs affect the mechanisms of fracture resistance such as microdamage formation. METHODS Human tibial cancellous bone cores were subjected to non-enzymatic glycation (NEG) by in vitro ribosylation and were mechanically loaded to pre- (0.6%) and post- (1.1%) yield apparent level strains. Loaded specimens were stained with lead-uranyl acetate and subjected to microCT-based 3D quantification and characterization of microdamage as either diffuse damage and linear microcracks. Damaged volume per bone volume (DV/BV) and damaged surface per damaged volume (DS/DV) ratios were used to quantify the volume and morphology of the detected microdamage, respectively. RESULTS In vitro ribosylation increased the microdamage morphology parameter (DS/DV) under both pre- (p<0.05; +51%) and post-yield loading (p<0.001; +38%), indicating that the alteration of bone matrix by NEG caused the formation of crack-like microdamage morphologies. Under post-yield loading, the NEG-mediated increase in DS/DV was coupled with the reductions in microdamage formation (DV/BV; p<0.001) and toughness (p<0.001). DISCUSSION Using a novel microCT technique to characterize and quantify microdamage, this study shows that the accumulation of AGEs in the bone matrix significantly alters the quantity and morphology of microdamage production and results in reduced fracture resistance.

Matrix metalloproteinase–13 is required for osteocytic perilacunar remodeling and maintains bone fracture resistance

Like bone mass, bone quality is specified in development, actively maintained postnatally, and disrupted by disease. The roles of osteoblasts, osteoclasts, and osteocytes in the regulation of bone mass are increasingly well defined. However, the cellular and molecular mechanisms by which bone quality is regulated remain unclear. Proteins that remodel bone extracellular matrix, such as the collagen‐degrading matrix metalloproteinase (MMP)‐13, are likely candidates to regulate bone quality. Using MMP‐13–deficient mice, we examined the role of MMP‐13 in the remodeling and maintenance of bone matrix and subsequent fracture resistance. Throughout the diaphysis of MMP‐13–deficient tibiae, we observed elevated nonenzymatic cross‐linking and concentric regions of hypermineralization, collagen disorganization, and canalicular malformation. These defects localize to the same mid‐cortical bone regions where osteocyte lacunae and canaliculi exhibit MMP‐13 and tartrate‐resistant acid phosphatase (TRAP) expression, as well as the osteocyte marker sclerostin. Despite otherwise normal measures of osteoclast and osteoblast function, dynamic histomorphometry revealed that remodeling of osteocyte lacunae is impaired in MMP‐13−/− bone. Analysis of MMP‐13−/− mice and their wild‐type littermates in normal and lactating conditions showed that MMP‐13 is not only required for lactation‐induced osteocyte perilacunar remodeling, but also for the maintenance of bone quality. The loss of MMP‐13, and the resulting defects in perilacunar remodeling and matrix organization, compromise MMP‐13−/− bone fracture toughness and postyield behavior. Taken together, these findings demonstrate that osteocyte perilacunar remodeling of mid‐cortical bone matrix requires MMP‐13 and is essential for the maintenance of bone quality.

The tissue diagnostic instrument

Tissue mechanical properties reflect extracellular matrix composition and organization, and as such, their changes can be a signature of disease. Examples of such diseases include intervertebral disk degeneration, cancer, atherosclerosis, osteoarthritis, osteoporosis, and tooth decay. Here we introduce the tissue diagnostic instrument (TDI), a device designed to probe the mechanical properties of normal and diseased soft and hard tissues not only in the laboratory but also in patients. The TDI can distinguish between the nucleus and the annulus of spinal disks, between young and degenerated cartilage, and between normal and cancerous mammary glands. It can quantify the elastic modulus and hardness of the wet dentin left in a cavity after excavation. It can perform an indentation test of bone tissue, quantifying the indentation depth increase and other mechanical parameters. With local anesthesia and disposable, sterile, probe assemblies, there has been neither pain nor complications in tests on patients. We anticipate that this unique device will facilitate research on many tissue systems in living organisms, including plants, leading to new insights into disease mechanisms and methods for their early detection.

The relative contributions of non-enzymatic glycation and cortical porosity on the fracture toughness of aging bone.

The risk of fracture increases with age due to the decline of bone mass and bone quality. One of the age-related changes in bone quality occurs through the formation and accumulation of advanced glycation end-products (AGEs) due to non-enzymatic glycation (NEG). However as a number of other changes including increased porosity occur with age and affect bone fragility, the relative contribution of AGEs on the fracture resistance of aging bone is unknown. Using a high-resolution nonlinear finite element model that incorporate cohesive elements and micro-computed tomography-based 3d meshes, we investigated the contribution of AGEs and cortical porosity on the fracture toughness of human bone. The results show that NEG caused a 52% reduction in propagation fracture toughness (R-curve slope). The combined effects of porosity and AGEs resulted in an 88% reduction in propagation toughness. These findings are consistent with previous experimental results. The model captured the age-related changes in the R-curve toughening by incorporating bone quantity and bone quality changes, and these simulations demonstrate the ability of the cohesive models to account for the irreversible dynamic crack growth processes affected by the changes in post-yield material behavior. By decoupling the matrix-level effects due to NEG and intracortical porosity, we are able to directly determine the effects of NEG on fracture toughness. The outcome of this study suggests that it may be important to include the age-related changes in the material level properties by using finite element analysis towards the prediction of fracture risk.

Changes in cortical bone response to high-fat diet from adolescence to adulthood in mice

SummaryDiabetic obesity is associated with increased fracture risk in adults and adolescents. We find in both adolescent and adult mice dramatically inferior mechanical properties and structural quality of cortical bone, in agreement with the human fracture data, although some aspects of the response to obesity appear to differ by age.IntroductionThe association of obesity with bone is complex and varies with age. Diabetic obese adolescents and adult humans have increased fracture risk. Prior studies have shown reduced mechanical properties as a result of high-fat diet (HFD) but do not fully address size-independent mechanical properties or structural quality, which are important to understand material behavior.MethodsCortical bone from femurs and tibiae from two age groups of C57BL/6 mice fed either HFD or low-fat diet (LFD) were evaluated for structural and bone turnover changes (SEM and histomorphometry) and tested for bending strength, bending stiffness, and fracture toughness. Leptin, IGF-I, and non-enzymatic glycation measurements were also collected.ResultsIn both young and adult mice fed on HFD, femoral strength, stiffness, and toughness are all dramatically lower than controls. Inferior lamellar and osteocyte alignment also point to reduced structural quality in both age groups. Bone size was largely unaffected by HFD, although there was a shift from increasing bone size in obese adolescents to decreasing in adults. IGF-I levels were lower in young obese mice only.ConclusionsWhile the response to obesity of murine cortical bone mass, bone formation, and hormonal changes appear to differ by age, the bone mechanical properties for young and adult groups are similar. In agreement with human fracture trends, adult mice may be similarly susceptible to bone fracture to the young group, although cortical bone in the two age groups responds to diabetic obesity differently.

How tough is brittle bone? Investigating osteogenesis imperfecta in mouse bone.

The multiscale hierarchical structure of bone is naturally optimized to resist fractures. In osteogenesis imperfecta, or brittle bone disease, genetic mutations affect the quality and/or quantity of collagen, dramatically increasing bone fracture risk. Here we reveal how the collagen defect results in bone fragility in a mouse model of osteogenesis imperfecta (oim), which has homotrimeric α1(I) collagen. At the molecular level, we attribute the loss in toughness to a decrease in the stabilizing enzymatic cross‐links and an increase in nonenzymatic cross‐links, which may break prematurely, inhibiting plasticity. At the tissue level, high vascular canal density reduces the stable crack growth, and extensive woven bone limits the crack‐deflection toughening during crack growth. This demonstrates how modifications at the bone molecular level have ramifications at larger length scales affecting the overall mechanical integrity of the bone; thus, treatment strategies have to address multiscale properties in order to regain bone toughness. In this regard, findings from the heterozygous oim bone, where defective as well as normal collagen are present, suggest that increasing the quantity of healthy collagen in these bones helps to recover toughness at the multiple length scales.

Differences in non-enzymatic glycation and collagen cross-links between human cortical and cancellous bone.

SummaryIt is important to establish the relationship between pentosidine and advanced glycation endproducts (AGEs) in bone. We found the relationship between pentosidine and AGEs and their magnitude of accumulation were dependent on bone’s surface-to-volume ratio. Results illustrate the importance of measuring pentosidine and AGEs separately in cancellous and cortical bone.IntroductionAccumulation of collagen cross-links (AGEs) produced by non-enzymatic glycation deteriorates bone’s mechanical properties and fracture resistance. Although a single AGE, pentosidine, is commonly used as a representative marker, it is unclear whether it quantitatively reflects total fluorescent AGEs in bone. The goal of this study was to establish the relationship between pentosidine and total AGEs in cancellous and cortical bone.MethodsPentosidine and total AGEs were quantified in 170 human bone samples. Total fluorescent AGEs were measured in 28 additional cancellous and cortical bone specimens of the same apparent volume that were incubated in control or in vitro glycation solutions. Correlations between pentosidine and total AGEs and differences between cortical and cancellous groups were determined.ResultsPentosidine was correlated with total AGEs in cancellous bone (r = 0.53, p < 0.0001) and weakly correlated in cortical bone (r = 0.23, p < 0.05). There was more pentosidine (p < 0.01) and total AGEs (p < 0.001) in cancellous than in cortical bone. The in vitro glycation substudy showed that cancellous bone accumulated more AGEs than cortical bone (p < 0.05).ConclusionThe relationship between pentosidine and total AGEs and their magnitude of accumulation differed in cancellous and cortical bone of the same apparent volume, and were dependent on the surface-to-volume ratios of each sample. It is important to consider the bone types as two separate entities, and it is crucial to quantify total AGEs in addition to pentosidine to allow for more comprehensive analysis of the effects of non-enzymatic glycation in bone.