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Raman spectroscopy (RS) is sensitive to the accumulation of advanced glycation end-products (AGEs), and it measures matrix-sensitive properties that correlate with the fracture toughness of human cortical bone. However, it is unclear whether sugar-mediated accumulation of AGEs affects the fracture toughness of human cortical bone in a manner that is consistent with the negative correlations between amide I sub-peak ratios and fracture toughness. Upon machining 64 single-edge notched beam (SENB) specimens from cadaveric femurs (8 male and 7 female donors between 46 years and 61 years of age), pairs of SENB specimens were incubated in 15 mL of phosphate buffered saline with or without 0.1 M ribose for 4 weeks at 37 °C. After acquiring 10 Raman spectra per bone specimen (n = 32 per incubation group), paired SENB specimens were loaded in three-point bending at a quasi-static or a high loading rate approximating 10 -4 s -1 or 10 -2 s -1 , respectively (n = 16 per incubation group per loading rate). While 2 amide I sub-peak ratios, I 1670 /I 1640 and I 1670 /I 1610 , decreased by 3-5% with a 100% increase in AGE content, as confirmed by fluorescence measurements, the ribose incubation to accumulate AGEs in bone did not affect linear elastic (K Ic ) nor non-linear elastic (K Jc ) measurements of bone's ability to resist crack growth. Moreover, AGE accumulation did not affect the change in these properties when the loading rate changed. Increasing the loading rate increased K Ic but decreased K Jc . Ribose incubation did not affect mineral-related RS properties such as mineral-to-matrix ratios, Type B carbonate substitutions, and crystallinity. It did however increase the thermal stability of demineralized bone (differential scanning calorimetry), without affecting the network connectivity of the organic matrix (i.e., maximum slope during a hydrothermal isometric tension test of demineralized bone). In conclusion, RS is sensitive to AGE accumulation via the amide I band (plus the hydroxyproline-to-proline ratio), but the increase in AGE content due to ribose incubation was not sufficient to affect the fracture toughness of human cortical bone., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Three-to-four percent of children with neurofibromatosis type 1 (NF1) present with unilateral tibia bowing, fracture, and recalcitrant healing. Alkaline phosphatase (ALP) enzyme therapy prevented poor bone mineralization and poor mechanical properties in mouse models of NF1 skeletal dysplasia; but transition to clinical trials is hampered by the lack of a technique that (i) identifies NF1 patients at risk of tibia bowing and fracture making them eligible for trial enrollment and (ii) monitors treatment effects on matrix characteristics related to bone strength. Therefore, we assessed the ability of matrix-sensitive techniques to provide characteristics that differentiate between cortical bone from mice characterized by postnatal loss of Nf1 in Osx-cre Tet-Off ;Nf1 flox/flox osteoprogenitors (cKO) and from wild-type (WT) mice. Following euthanasia at two time points of bone disease progression, femur and tibia were harvested from both genotypes (n ≥ 8/age/sex/genotype). A reduction in the mid-diaphysis ultimate force during three-point bending at 20 weeks confirmed deleterious changes in bone induced by Nf1 deficiency, regardless of sex. Pooling females and males, low bound water (BW), and low cortical volumetric bone mineral density (Ct.vBMD) were the most accurate outcomes in distinguishing cKO from WT femurs with accuracy improving with age. Ct.vBMD and the average unloading slope (Avg-US) from cyclic reference point indentation tests were the most sensitive in differentiating WT from cKO tibias. Mineral-to-matrix ratio and carbonate substitution from Raman spectroscopy were not good classifiers. However, when combined with Ct.vBMD and BW (femur), they helped predict bending strength. Nf1 deficiency in osteoprogenitors negatively affected bone microstructure and matrix quality with deficits in properties becoming more pronounced with duration of Nf1 deficiency. Clinically measurable without ionizing radiation, BW and Avg-US are sensitive to deleterious changes in bone matrix in a preclinical model of NF1 bone dysplasia and require further clinical investigation as potential indicators of an onset of bone weakness in children with NF1. © 2022 American Society for Bone and Mineral Research (ASBMR)., (© 2022 American Society for Bone and Mineral Research (ASBMR).)

Following severe injury, biomineralization is disrupted and limited therapeutic options exist to correct these pathologic changes. This study utilized a clinically relevant murine model of polytrauma including a severe injury with concomitant musculoskeletal injuries to identify when bisphosphonate administration can prevent the paradoxical decrease of biomineralization in bone and increased biomineralization in soft tissues, yet not interfere with musculoskeletal repair., Introduction: Systemic and intrinsic mechanisms in bone and soft tissues help promote biomineralization to the skeleton, while preventing it in soft tissues. However, severe injury can disrupt this homeostatic biomineralization tropism, leading to adverse patient outcomes due to a paradoxical decrease of biomineralization in bone and increased biomineralization in soft tissues. There remains a need for therapeutics that restore the natural tropism of biomineralization in severely injured patients. Bisphosphonates can elicit potent effects on biomineralization, though with variable impact on musculoskeletal repair. Thus, a critical clinical question remains as to the optimal time to initiate bisphosphonate therapy in patients following a polytrauma, in which bone and muscle are injured in combination with a severe injury, such as a burn., Methods: To test the hypothesis that the dichotomous effects of bisphosphonates are dependent upon the time of administration relative to the ongoing biomineralization in reparative bone and soft tissues, this study utilized murine models of isolated injury or polytrauma with a severe injury, in conjunction with sensitive, longitudinal measure of musculoskeletal repair., Results: This study demonstrated that if administered at the time of injury, bisphosphonates prevented severe injury-induced bone loss and soft tissue calcification, but did not interfere with bone repair or remodeling. However, if administered between 7 and 21 days post-injury, bisphosphonates temporally and spatially localized to sites of active biomineralization, leading to impaired fracture callus remodeling and permanence of soft tissue calcification., Conclusion: There is a specific pharmacologic window following polytrauma that bisphosphonates can prevent the consequences of dysregulated biomineralization, yet not impair musculoskeletal regeneration., (© 2021. International Osteoporosis Foundation and National Osteoporosis Foundation.)

Developing clinical tools that assess bone matrix quality could improve the assessment of a person's fracture risk. To determine whether Raman spectroscopy (RS) has such potential, we acquired Raman spectra from human cortical bone using microscope- and fiber optic probe-based Raman systems and tested whether correlations between RS and fracture toughness properties were statistically significant. Calculated directly from intensities at wavenumbers identified by second derivative analysis, Amide I sub-peak ratio I 1670 /I 1640 , not I 1670 /I 1690 , was negatively correlated with K init (N = 58; R 2  = 32.4%) and J-integral (R 2  = 47.4%) when assessed by Raman micro-spectroscopy. Area ratios (A 1670 /A 1690 ) determined from sub-band fitting did not correlate with fracture toughness. There were fewer correlations between RS and fracture toughness when spectra were acquired by probe RS. Nonetheless, the I 1670 /I 1640 sub-peak ratio again negatively correlated with K init (N = 56; R 2  = 25.6%) and J-integral (R 2  = 39.0%). In best-fit general linear models, I 1670 /I 1640, age, and volumetric bone mineral density explained 50.2% (microscope) and 49.4% (probe) of the variance in K init . I 1670 /I 1640 and v 1 PO 4 /Amide I (microscope) or just I 1670 /I 1640 (probe) were negative predictors of J-integral (adjusted-R 2  = 54.9% or 37.9%, respectively). While Raman-derived matrix properties appear useful to the assessment of fracture resistance of bone, the acquisition strategy to resolve the Amide I band needs to be identified.

Greater understanding of the determinants of skeletal fragility is highly sought due to the great burden that bone affecting diseases and fractures have on economies, societies and health care systems. Being a complex, hierarchical composite of collagen type-I and non-stoichiometric substituted hydroxyapatite, bone derives toughness from its organic phase. In this study, we tested whether early observations that a strong correlation between bone collagen integrity measured by thermomechanical methods and work to fracture exist in a more general and heterogeneous sampling of the population. Neighboring uniform specimens from an established, highly characterized and previously published collection of human cortical bone samples (femur mid-shaft) were decalcified in EDTA. Fifty-four of the original 62 donors were included (26 male and 28 females; ages 21-101 years; aging, osteoporosis, diabetes and cancer). Following decalcification, bone collagen was tested using hydrothermal isometric tension (HIT) testing in order to measure the collagen's thermal stability (denaturation temperature, T d ) and network connectivity (maximum rate of isometric tension generation; Max.Slope). We used linear regression and general linear models (GLMs) with several explanatory variables to determine whether relationships between HIT parameters and generally accepted bone quality factors (e.g., cortical porosity, pentosidine content [pen], pyridinoline content [pyd]), age, and measures of fracture toughness (crack initiation fracture toughness, K init , and total energy release/dissipation rate evaluated at the point of unstable fast fracture, J-int) were significant. Bone collagen connectivity (Max.Slope) correlated well with the measures of fracture toughness (R 2  = 24-35%), and to a lesser degree with bound water fraction (BW; R 2  = 7.9%) and pore water fraction (PW; R 2  = 9.1%). Significant correlations with age, apparent volumetric bone mineral density (vBMD), and mature enzymatic [pyd] and non-enzymatic collagen crosslinks [pen] were not detected. GLMs found that Max.Slope and vBMD (or BW), with or without age as additional covariate, all significantly explained the variance in Kinit (adjusted-R 2  = 36.7-49.0%). Also, the best-fit model for J-int (adjusted-R 2  = 35.7%) included only age and Max.Slope as explanatory variables with Max.Slope contributing twice as much as age. Max.Slope and BW without age were also significant predictors of J-int (adjusted-R 2  = 35.5%). In conclusion, bone collagen integrity as measured by thermomechanical methods is a key factor in cortical bone fracture toughness. This study further demonstrates that greater attention should be paid to degradation of the overall organic phase, rather than a specific biomarker (e.g. [pen]), when seeking to understand elevated fracture rates in aging and disease., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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 (K init ) from crack growth toughness (K grow ). The traditional ν 1 phosphate peak per amide I peak (mineral-to-matrix ratio) weakly correlated with K init (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 (K init : r = - 0.467, K grow : 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 K init , K grow , 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.

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., Competing Interests: The authors do not have a conflict of interest with present work., (Published by Elsevier Ltd.)

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.

Accurately predicting fracture risk in the clinic is challenging because the determinants are multi-factorial. A common approach to fracture risk assessment is to combine X-ray-based imaging methods such as dual-energy X-ray absorptiometry (DXA) with an online Fracture Risk Assessment Tool (FRAX) that includes additional risk factors such as age, family history, and prior fracture incidents. This approach still does not adequately diagnose many individuals at risk, especially those with certain diseases like type 2 diabetes. As such, this study investigated bound water and pore water concentrations (Cbw and Cpw) from ultra-short echo time (UTE) magnetic resonance imaging (MRI) as new predictors of fracture risk. Ex vivo cadaveric arms were imaged with UTE MRI as well as with DXA and high-resolution micro-computed tomography (μCT), and imaging measures were compared to both whole-bone structural and material properties as determined by three-point bending tests of the distal-third radius. While DXA-derived areal bone mineral density (aBMD) and μCT-derived volumetric BMD correlated well with structural strength, they moderately correlated with the estimate material strength with gender being a significant covariate for aBMD. MRI-derived measures of Cbw and Cpw had a similar predictive ability of material strength as aBMD but did so independently of gender. In addition, Cbw was the only imaging parameter to significantly correlate with toughness, the energy dissipated during fracture. Notably, the strength of the correlations with the material properties of bone tended to be higher when a larger endosteal region was used to determine Cbw and Cpw. These results indicate that MRI measures of Cbw and Cpw have the ability to probe bone material properties independent of bone structure or subject gender. In particular, toughness is a property of fracture resistance that is not explained by X-ray based methods. Thus, these MRI-derived measures of Cbw and Cpw in cortical bone have the potential to be useful in clinical populations for evaluating fracture risk, especially involving diseases that affect material properties of the bone beyond its strength., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Published by Elsevier Inc.)

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., (© 2015 American Society for Bone and Mineral Research.)

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., (Published by Elsevier Inc.)