Changes in the Fracture Resistance of Bone with the Progression of Type 2 Diabetes in the ZDSD Rat.

Individuals with type 2 diabetes (T2D) have a higher fracture risk compared to non-diabetics, even though their areal bone mineral density is normal to high. Identifying the mechanisms whereby diabetes lowers fracture resistance requires well-characterized rodent models of diabetic bone disease. Toward that end, we hypothesized that bone toughness, more so than bone strength, decreases with the duration of diabetes in ZDSD rats. Bones were harvested from male CD(SD) control rats and male ZDSD rats at 16 weeks (before the onset of hyperglycemia), at 22 weeks (5-6 weeks of hyperglycemia), and at 29 weeks (12-13 weeks of hyperglycemia). There were at least 12 rats per strain per age group. At 16 weeks, there was no difference in either body weight or glucose levels between the two rat groups. Within 2 weeks of switching all rats to a diet with 48 % of kcal from fat, only the ZDSD rats developed hyperglycemia (>250 mg/dL). They also began to lose body weight at 21 weeks. CD(SD) rats remained normoglycemic (<110 mg/dL) on the high-fat diet and became obese (>600 g). From micro-computed tomography (µCT) analysis of a lumbar vertebra and distal femur, trabecular bone volume did not vary with age among the non-diabetic rats but was lower at 29 weeks than at 16 weeks or at 22 weeks for the diabetic rats. Consistent with that finding, µCT-derived intra-cortical porosity (femur diaphysis) was higher for ZDSD following ~12 weeks of hyperglycemia than for age-matched CD(SD) rats. Despite an age-related increase in mineralization in both rat strains (µCT and Raman spectroscopy), material strength of cortical bone (from three-point bending tests) increased with age only in the non-diabetic CD(SD) rats. Moreover, two other material properties, toughness (radius) and fracture toughness (femur), significantly decreased with the duration of T2D in ZDSD rats. This was accompanied by the increase in the levels of the pentosidine (femur). However, pentosidine was not significantly higher in diabetic than in non-diabetic bone at any time point. The ZDSD rat, which has normal leptin signaling and becomes diabetic after skeletal maturity, provides a pre-clinical model of diabetic bone disease, but a decrease in body weight during prolonged diabetes and certain strain-related differences before the onset of hyperglycemia should be taken into consideration when interpreting diabetes-related differences.

The contribution of the extracellular matrix to the fracture resistance of bone.

The likelihood of suffering a bone fracture is not solely predicated on areal bone mineral density. As people age, there are numerous changes to the skeleton occurring at multiple length scales (from millimeters to submicron scales) that reduce the ability of bone to resist fracture. Herein is a review of the current knowledge about the role of the extracellular matrix (ECM) in this resistance, with emphasis on engineering principles that characterize fracture resistance beyond bone strength to include bone toughness and fracture toughness. These measurements of the capacity to dissipate energy and to resist crack propagation during failure precipitously decline with age. An age-related loss in collagen integrity is strongly associated with decreases in these mechanical properties. One potential cause for this deleterious change in the ECM is an increase in advanced glycation end products, which accumulate with aging through nonenzymatic collagen crosslinking. Potential regulators and diagnostic tools of the ECM with respect to fracture resistance are also discussed.

In vivo analysis of laser preconditioning in incisional wound healing of wild-type and HSP70 knockout mice with Raman spectroscopy.

BACKGROUND AND OBJECTIVE: Laser preconditioning augments incisional wound healing by reducing scar tissue and increasing maximum tensile load of the healed wound [Wilmink et al. (2009) J Invest Dermatol 129(1): 205-216]. Recent studies have optimized treatments or confirmed results using HSP70 as a biomarker. Under the hypothesis that HSP70 plays a role in reported results and to better understand the downstream effects of laser preconditioning, this study utilized a probe-based Raman spectroscopy (RS) system to achieve an in vivo, spatio-temporal biochemical profile of murine skin incisional wounds as a function of laser preconditioning and the presence of HSP70. STUDY DESIGN/MATERIALS AND METHODS: A total of 19 wild-type (WT) and HSP70 knockout (HSP70-/-) C57BL/6 mice underwent normal and laser preconditioned incisional wounds. Laser thermal preconditioning was conducted via previously established protocol (λ = 1.85 µm, H(0 ) = 7.64 mJ/cm(2) per pulse, spot diameter = 5 mm, Rep. rate = 50 Hz, τ(p) = 2 milliseconds, exposure time = 10 minutes) with an Aculight Renoir diode laser, with tissue temperature confirmed by real-time infrared camera measurements. Wound-healing progression was quantified by daily collection of a spatial distribution of Raman spectra. The results of RS findings were then qualified using standard histology and polarization microscopy. RESULTS: Raman spectra yielded significant differences (t-test; α = 0.05) in several known biochemical peaks between WT and HSP70 (-/-) mice on wounds and in adjacent tissue early in the wound-healing process. Analysis of peak ratios implied (i) an increase in protein configuration in and surrounding the wound in WT mice, and (ii) an increased cellular trend in WT mice that was prolonged due to laser treatment. Polarization microscopy confirmed that laser treated WT mice showed increased heterogeneity in collagen orientation. CONCLUSIONS: The data herein supports the theory that HSP70 is involved in normal skin protein configuration and the cellularity of early wound healing. Laser preconditioning extends cellular trends in the presence of HSP70. Despite study limitations, RS provided a non-invasive method for quantifying temporal trends in altered wound healing, narrowing candidates and design for future studies with clinically applicable instrumentation.

Measuring differences in compositional properties of bone tissue by confocal Raman spectroscopy.

The full range of fracture risk determinants arise from each hierarchical level comprising the organization of bone. Raman spectroscopy is one tool capable of characterizing the collagen and mineral phases at a near submicron-length scale, but the ability of Raman spectra to distinguish compositional differences of bone is not well defined. Therefore, we analyzed multiple Raman peak intensities and peak ratios to characterize their ability to distinguish between the typically less mineralized osteonal tissue and the more mineralized interstitial tissue in intracortical human bone. To further assess origins of variance, we collected Raman spectra from embedded specimens and for two orientations of cut. Per specimen, Raman peak intensities or ratios were averaged among multiple sites within five osteons and five neighboring interstitial tissue. The peak ratios of ν(1) phosphate (PO(4)) to proline or amide III detected the highest increases of 15.4 or 12.5%, respectively, in composition from osteonal to interstitial tissue. The coefficient of variance was less than 5% for each as opposed to a value of ~8% for the traditional ν(1)PO(4)/amide I, a peak ratio that varied the most between transverse and longitudinal cuts for each tissue type. Although embedding affected Raman peaks, it did not obscure differences in most peak ratios related to mineralization between the two tissue types. In studies with limited sample size but sufficient number of Raman spectra per specimen for spatial averaging, ν(1)PO(4)/amide III or ν(1)PO(4)/proline is the Raman property that is most likely to detect a compositional difference between experimental groups.

Applying Full Spectrum Analysis to a Raman Spectroscopic Assessment of Fracture Toughness of Human Cortical Bone.

A decline in the inherent quality of bone tissue is a † Equal contributors contributor to the age-related increase in fracture risk. Although this is well-known, the important biochemical factors of bone quality have yet to be identified using Raman spectroscopy (RS), a nondestructive, inelastic light-scattering technique. To identify potential RS predictors of fracture risk, we applied principal component analysis (PCA) to 558 Raman spectra (370-1720 cm-1) of human cortical bone acquired from 62 female and male donors (nine spectra each) spanning adulthood (age range = 21-101 years). Spectra were analyzed prior to R-curve, nonlinear fracture mechanics that delineate crack initiation (Kinit) from crack growth toughness (Kgrow). The traditional ν1phosphate peak per amide I peak (mineral-to-matrix ratio) weakly correlated with Kinit (r = 0.341, p = 0.0067) and overall crack growth toughness (J-int: r = 0.331, p = 0.0086). Sub-peak ratios of the amide I band that are related to the secondary structure of type 1 collagen did not correlate with the fracture toughness properties. In the full spectrum analysis, one principal component (PC5) correlated with all of the mechanical properties (Kinit: r = - 0.467, Kgrow: r = - 0.375, and J-int: r = - 0.428; p < 0.0067). More importantly, when known predictors of fracture toughness, namely age and/or volumetric bone mineral density (vBMD), were included in general linear models as covariates, several PCs helped explain 45.0% (PC5) to 48.5% (PC7), 31.4% (PC6), and 25.8% (PC7) of the variance in Kinit, Kgrow, and J-int, respectively. Deriving spectral features from full spectrum analysis may improve the ability of RS, a clinically viable technology, to assess fracture risk.

Prevalent role of porosity and osteonal area over mineralization heterogeneity in the fracture toughness of human cortical bone.

Changes in the distribution of bone mineralization occurring with aging, disease, or treatment have prompted concerns that alterations in mineralization heterogeneity may affect the fracture resistance of bone. Yet, so far, studies assessing bone from hip fracture cases and fracture-free women have not reached a consensus on how heterogeneity in tissue mineralization relates to skeletal fragility. Owing to the multifactorial nature of toughening mechanisms occurring in bone, we assessed the relative contribution of heterogeneity in mineralization to fracture resistance with respect to age, porosity, and area fraction of osteonal tissue. The latter parameters were extracted from quantitative backscattered electron imaging of human cortical bone sections following R-curve tests of single-edge notched beam specimens to determine fracture toughness properties. Microstructural heterogeneity was determined as the width of the mineral distribution (bulk) and as the sill of the variogram (local). In univariate analyses of measures from 62 human donors (21 to 101 years), local but not bulk heterogeneity as well as pore clustering negatively correlated with fracture toughness properties. With age as covariate, heterogeneity was a significant predictor of crack initiation, though local had a stronger negative contribution than bulk. When considering all potential covariates, age, cortical porosity and area fraction of osteons explained up to 50% of the variance in bone׳s crack initiation toughness. However, including heterogeneity in mineralization did not improve upon this prediction. The findings of the present work stress the necessity to account for porosity and microstructure when evaluating the potential of matrix-related features to affect skeletal fragility.

Age-related changes in the fracture resistance of male Fischer F344 rat bone.

In addition to the loss in bone volume that occurs with age, there is a decline in material properties. To test new therapies or diagnostic tools that target such properties as material strength and toughness, a pre-clinical model of aging would be useful in which changes in bone are similar to those that occur with aging in humans. Toward that end, we hypothesized that similar to human bone, the estimated toughness and material strength of cortical bone at the apparent-level decreases with age in the male Fischer F344 rat. In addition, we tested whether the known decline in trabecular architecture in rats translated to an age-related decrease in vertebra (VB) strength and whether non-X-ray techniques could quantify tissue changes at micron and sub-micron length scales. Bones were harvested from 6-, 12-, and 24-month (mo.) old rats (n=12 per age). Despite a loss in trabecular bone with age, VB compressive strength was similar among the age groups. Similarly, whole-bone strength (peak force) in bending was maintained (femur) or increased (radius) with aging. There was though an age-related decrease in post-yield toughness (radius) and bending strength (femur). The ability to resist crack initiation was actually higher for the 12-mo. and 24-mo. than for 6-mo. rats (notch femur), but the estimated work to propagate the crack was less for the aged bone. For the femur diaphysis region, porosity increased while bound water decreased with age. For the radius diaphysis, there was an age-related increase in non-enzymatic and mature enzymatic collagen crosslinks. Raman spectroscopy analysis of embedded cross-sections of the tibia mid-shaft detected an increase in carbonate subsitution with advanced aging for both inner and outer tissue.

Combined treatment with a transforming growth factor beta inhibitor (1D11) and bortezomib improves bone architecture in a mouse model of myeloma-induced bone disease.

Multiple myeloma (MM) patients frequently develop tumor-induced bone destruction, yet no therapy completely eliminates the tumor or fully reverses bone loss. Transforming growth factor-ß (TGF-ß) activity often contributes to tumor-induced bone disease, and pre-clinical studies have indicated that TGF-ß inhibition improves bone volume and reduces tumor growth in bone metastatic breast cancer. We hypothesized that inhibition of TGF-ß signaling also reduces tumor growth, increases bone volume, and improves vertebral body strength in MM-bearing mice. We treated myeloma tumor-bearing (immunocompetent KaLwRij and immunocompromised Rag2-/-) mice with a TGF-ß inhibitory (1D11) or control (13C4) antibody, with or without the anti-myeloma drug bortezomib, for 4weeks after inoculation of murine 5TGM1 MM cells. TGF-ß inhibition increased trabecular bone volume, improved trabecular architecture, increased tissue mineral density of the trabeculae as assessed by ex vivo micro-computed tomography, and was associated with significantly greater vertebral body strength in biomechanical compression tests. Serum monoclonal paraprotein titers and spleen weights showed that 1D11 monotherapy did not reduce overall MM tumor burden. Combination therapy with 1D11 and bortezomib increased vertebral body strength, reduced tumor burden, and reduced cortical lesions in the femoral metaphysis, although it did not significantly improve cortical bone strength in three-point bending tests of the mid-shaft femur. Overall, our data provides rationale for evaluating inhibition of TGF-ß signaling in combination with existing anti-myeloma agents as a potential therapeutic strategy to improve outcomes in patients with myeloma bone disease.

Identifying Novel Clinical Surrogates to Assess Human Bone Fracture Toughness.

Fracture risk does not solely depend on strength but also on fracture toughness; ie, the ability of bone material to resist crack initiation and propagation. Because resistance to crack growth largely depends on bone properties at the tissue level, including collagen characteristics, current X-ray based assessment tools may not be suitable to identify age-related, disease-related, or treatment-related changes in fracture toughness. To identify useful clinical surrogates that could improve the assessment of fracture resistance, we investigated the potential of (1)H nuclear magnetic resonance spectroscopy (NMR) and reference point indentation (RPI) to explain age-related variance in fracture toughness. Harvested from cadaveric femurs (62 human donors), single-edge notched beam (SENB) specimens of cortical bone underwent fracture toughness testing (R-curve method). NMR-derived bound water showed the strongest correlation with fracture toughness properties (r = 0.63 for crack initiation, r = 0.35 for crack growth, and r = 0.45 for overall fracture toughness; p < 0.01). Multivariate analyses indicated that the age-related decrease in different fracture toughness properties were best explained by a combination of NMR properties including pore water and RPI-derived tissue stiffness with age as a significant covariate (adjusted R(2) = 53.3%, 23.9%, and 35.2% for crack initiation, crack growth, and overall toughness, respectively; p < 0.001). These findings reflect the existence of many contributors to fracture toughness and emphasize the utility of a multimodal assessment of fracture resistance. Exploring the mechanistic origin of fracture toughness, glycation-mediated nonenzymatic collagen crosslinks and intracortical porosity are possible determinants of bone fracture toughness and could explain the sensitivity of NMR to changes in fracture toughness. Assuming fracture toughness is clinically important to the ability of bone to resist fracture, our results suggest that improvements in fracture risk assessment could potentially be achieved by accounting for water distribution (quantitative ultrashort echo time magnetic resonance imaging) and by a local measure of tissue resistance to indentation, RPI.

Predicting mouse vertebra strength with micro-computed tomography-derived finite element analysis.

As in clinical studies, finite element analysis (FEA) developed from computed tomography (CT) images of bones are useful in pre-clinical rodent studies assessing treatment effects on vertebral body (VB) strength. Since strength predictions from microCT-derived FEAs (µFEA) have not been validated against experimental measurements of mouse VB strength, a parametric analysis exploring material and failure definitions was performed to determine whether elastic µFEAs with linear failure criteria could reasonably assess VB strength in two studies, treatment and genetic, with differences in bone volume fraction between the control and the experimental groups. VBs were scanned with a 12-µm voxel size, and voxels were directly converted to 8-node, hexahedral elements. The coefficient of determination or R (2) between predicted VB strength and experimental VB strength, as determined from compression tests, was 62.3% for the treatment study and 85.3% for the genetic study when using a homogenous tissue modulus (E t) of 18 GPa for all elements, a failure volume of 2%, and an equivalent failure strain of 0.007. The difference between prediction and measurement (that is, error) increased when lowering the failure volume to 0.1% or increasing it to 4%. Using inhomogeneous tissue density-specific moduli improved the R (2) between predicted and experimental strength when compared with uniform E t=18 GPa. Also, the optimum failure volume is higher for the inhomogeneous than for the homogeneous material definition. Regardless of model assumptions, µFEA can assess differences in murine VB strength between experimental groups when the expected difference in strength is at least 20%.

Polarization in Raman spectroscopy helps explain bone brittleness in genetic mouse models.

Raman spectroscopy (RS) has been extensively used to characterize bone composition. However, the link between bone biomechanics and RS measures is not well established. Here, we leveraged the sensitivity of RS polarization to organization, thereby assessing whether RS can explain differences in bone toughness in genetic mouse models for which traditional RS peak ratios are not informative. In the selected mutant mice - activating transcription factor 4 (ATF4) or matrix metalloproteinase 9 (MMP9) knock-outs - toughness is reduced but differences in bone strength do not exist between knock-out and corresponding wild-type controls. To incorporate differences in the RS of bone occurring at peak shoulders, a multivariate approach was used. Full spectrum principal components analysis of two paired, orthogonal bone orientations (relative to laser polarization) improved genotype classification and correlation to bone toughness when compared to traditional peak ratios. When applied to femurs from wild-type mice at 8 and 20 weeks of age, the principal components of orthogonal bone orientations improved age classification but not the explanation of the maturation-related increase in strength. Overall, increasing polarization information by collecting spectra from two bone orientations improves the ability of multivariate RS to explain variance in bone toughness, likely due to polarization sensitivity to organizational changes in both mineral and collagen.

The loss of activating transcription factor 4 (ATF4) reduces bone toughness and fracture toughness.

Even though age-related changes to bone tissue affecting fracture risk are well characterized, only a few matrix-related factors have been identified as important to maintaining fracture resistance. As a gene critical to osteoblast differentiation, activating transcription factor 4 (ATF4) is possibly one of these important factors. To test the hypothesis that the loss of ATF4 affects the fracture resistance of bone beyond bone mass and structure, we harvested bones from Atf4+/+ and Atf4-/- littermates at 8 and 20 weeks of age (n≥9 per group) for bone assessment across several length scales. From whole bone mechanical tests in bending, femurs from Atf4-/- mice were found to be brittle with reduced toughness and fracture toughness compared to femurs from Atf4+/+ mice. However, there were no differences in material strength and in tissue hardness, as determined by nanoindentation, between the genotypes, irrespective of age. Tissue mineral density of the cortex at the point of loading as determined by micro-computed tomography was also not significantly different. However, by analyzing local composition by Raman Spectroscopy (RS), bone tissue of Atf4-/- mice was found to have higher mineral to collagen ratio compared to wild-type tissue, primarily at 20 weeks of age. From RS analysis of intact femurs at 2 orthogonal orientations relative to the polarization axis of the laser, we also found that the organizational-sensitive peak ratio, ν1Phosphate per Amide I, changed to a greater extent upon bone rotation for Atf4-deficient tissue, implying bone matrix organization may contribute to the brittleness phenotype. Target genes of ATF4 activity are not only important to osteoblast differentiation but also in maintaining bone toughness and fracture toughness.

Asfotase-α improves bone growth, mineralization and strength in mouse models of neurofibromatosis type-1.

Individuals with neurofibromatosis type-1 (NF1) can manifest focal skeletal dysplasias that remain extremely difficult to treat. NF1 is caused by mutations in the NF1 gene, which encodes the RAS GTPase-activating protein neurofibromin. We report here that ablation of Nf1 in bone-forming cells leads to supraphysiologic accumulation of pyrophosphate (PPi), a strong inhibitor of hydroxyapatite formation, and that a chronic extracellular signal-regulated kinase (ERK)-dependent increase in expression of genes promoting PPi synthesis and extracellular transport, namely Enpp1 and Ank, causes this phenotype. Nf1 ablation also prevents bone morphogenic protein-2-induced osteoprogenitor differentiation and, consequently, expression of alkaline phosphatase and PPi breakdown, further contributing to PPi accumulation. The short stature and impaired bone mineralization and strength in mice lacking Nf1 in osteochondroprogenitors or osteoblasts can be corrected by asfotase-α enzyme therapy aimed at reducing PPi concentration. These results establish neurofibromin as an essential regulator of bone mineralization. They also suggest that altered PPi homeostasis contributes to the skeletal dysplasias associated with NF1 and that some of the NF1 skeletal conditions could be prevented pharmacologically.

Polarization control of Raman spectroscopy optimizes the assessment of bone tissue.

There is potential for Raman spectroscopy (RS) to complement tools for bone diagnosis due to its ability to assess compositional and organizational characteristics of both collagen and mineral. To aid this potential, the present study assessed specificity of RS peaks to the composition of bone, a birefringent material, for different degrees of instrument polarization. Specifically, relative changes in peaks were quantified as the incident light rotated relative to the orientation of osteonal and interstitial tissue, acquired from cadaveric femurs. In a highly polarized instrument (10(6)∶1 extinction ratio), the most prominent mineral peak (ν1 Phosphate at 961 cm(-1)) displayed phase similarity with the Proline peak at 856 cm(-1). This sensitivity to relative orientation between bone and light observed in the highly polarized regime persisted for certain sensitive peaks (e.g., Amide I at 1666 cm(-1)) in unaltered instrumentation (200∶1 extinction ratio). Though Proline intensity changed with bone rotation, the phase of Proline matched that of ν1 Phosphate. Moreover, when mapping ν1 Phosphate/Proline across osteonal-interstitial borders, the mineralization difference between the tissue types was evident whether using a 20x or 50x objectives. Thus, the polarization bias inherent in commercial RS systems does not preclude the assessment of bone composition when using phase-matched peaks.