Fracture risk does not solely depend on strength but also on fracture toughness, i.e. 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-, disease-, 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, non-enzymatic collagen crosslinks and intra-cortical 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).
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 lower fracture resistance requires well-characterized rodent models of diabetic bone disease. Toward that end, we hypothesized that the 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-wks (before the onset of hyperglycemia), at 22-wks (5–6 wks of hyperglycemia), and at 29-wks (12–13 wks of hyperglycemia). There were at least 12 rats per strain per age group. At 16-wks, there was no difference in either body weight or glucose levels between the 2 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-wks. 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-wks than at 16-wks or at 22-wks for the diabetic rats. Consistent with that finding, µCT-derived intra-cortical porosity (femur diaphysis) was higher for ZDSD following ~12 wks 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 testing) 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 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 sub-micron 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 intra-cortical human bone. To further assess origins of variance, we collected Raman spectra from embedded specimens and for 2 orientations of cut. Per specimen, Raman peak intensities or ratios were averaged among multiple sites within 5 osteons and 5 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 (COV) was less than 5% for each as opposed to a COV 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 2 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.
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