Alexander J. Makowski

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 1H 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 R2 = 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.

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.

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 ν1 phosphate (PO4) 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 ν1PO4/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, ν1PO4/amide III or ν1PO4/proline is the Raman property that is most likely to detect a compositional difference between experimental groups.

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.

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.

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.

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.

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.

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-β (TGF-β) activity often contributes to tumor-induced bone disease, and pre-clinical studies have indicated that TGF-β inhibition improves bone volume and reduces tumor growth in bone metastatic breast cancer. We hypothesized that inhibition of TGF-β 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-β inhibitory (1D11) or control (13C4) antibody, with or without the anti-myeloma drug bortezomib, for 4weeks after inoculation of murine 5TGM1 MM cells. TGF-β 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-β signaling in combination with existing anti-myeloma agents as a potential therapeutic strategy to improve outcomes in patients with myeloma bone disease.

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%.