An improved linear systems model of hydrothermal isometric tension testing to aid in assessing bone collagen quality: Effects of ribation and type-2 diabetes.

This study sought to further develop and validate a previously proposed physics-based model that maps denaturation kinetics from differential scanning calorimetry (DSC) to the isometric tension generated during hydrothermal isometric tension (HIT) testing of collagenous tissues. The primary objectives of this study were to verify and validate two physics-based model parameters: α, which indicates the amount of instantaneous isometric tension developed per unit of collagen denaturation, and ß, which captures the proportionality between temperature and the generated isometric tension post denaturation initiation. These parameters were used as measures of bone collagen quality, employing data from HIT and DSC testing of human bone collagen from two previous studies. Additionally, given the physical basis of the model, the study aimed to further validate Max.Slope, the rate of change in isometric tensile stress with change in temperature, as an independent measure of collagen network connectivity. Max.Slope has previously been positively correlated with measures of cortical bone fracture resistance. Towards this verification and validation, the hypotheses were a) that α would correlate strongly with HIT denaturation temperature, Td, and the enthalpy of melting (ΔH) from DSC, and b) that ß would correlate positively and strongly with Max.Slope. The model was employed in the analysis of HIT-DSC data from the testing of demineralized bone collagen isolated from cadaveric human femurs in two prior studies. In one study, data were collected from HIT-DSC testing of cortical bone collagen from 74 donors. Among them, 38 had a history of type 2 diabetes +/- chronic kidney disease, while the remaining 36 had no history of T2D again with or without CKD. Cortical bone specimens were extracted from the lateral mid-shaft. The second study involved 15 donor femora, with four cortical bone specimens extracted from each. Of these four, two specimens underwent a 4-week incubation in 0.1 M ribose at 37 °C to induce non-enzymatic ribation and advanced glycation endproducts, while the other two served as non-ribated controls. The examination involved investigating correlations between the model parameters α and ß and various measures, such as Max.Slope, Td, ΔH, age, and duration of type 2 diabetes. The results revealed positive correlations between the model parameter ß and Max.Slope (r = 0.55-0.58). The parameter α was found to be associated with Td, but also sensitive to the shape of the HIT curve around Td resulting in difficulties with variability and interpretation. As a result, while both hypotheses are confirmed, Max.Slope and ß are better indicators of bone collagen quality because they are measures of the connectivity or, more generally, the integrity of the bone collagen network.

Fracture Toughness: Bridging the Gap Between Hip Fracture and Fracture Risk Assessment.

PURPOSE OF REVIEW: This review surveys recent literature related to cortical bone fracture mechanics and its application towards understanding bone fragility and hip fractures. RECENT FINDINGS: Current clinical tools for hip fracture risk assessment have been shown to be insensitive in some cases of elevated fracture risk leading to the question of what other factors account for fracture risk. The emergence of cortical bone fracture mechanics has thrown light on other factors at the tissue level that are important to bone fracture resistance and therefore assessment of fracture risk. Recent cortical bone fracture toughness studies have shown contributions from the microstructure and composition towards cortical bone fracture resistance. A key component currently overlooked in the clinical evaluation of fracture risk is the importance of the organic phase and water to irreversible deformation mechanisms that enhance the fracture resistance of cortical bone. Despite recent findings, there is an incomplete understanding of which mechanisms lead to the diminished contribution of the organic phase and water to the fracture toughness in aging and bone-degrading diseases. Notably, studies of the fracture resistance of cortical bone from the hip (specifically the femoral neck) are few, and those that exist are mostly consistent with studies of bone tissue from the femoral diaphysis. Cortical bone fracture mechanics highlights that there are multiple determinants of bone quality and therefore fracture risk and its assessment. There is still much more to learn concerning the tissue-level mechanisms of bone fragility. An improved understanding of these mechanisms will allow for the development of better diagnostic tools and therapeutic measures for bone fragility and fracture.

Effect of ribose incubation on physical, chemical, and mechanical properties of human cortical bone.

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, I1670/I1640 and I1670/I1610, 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 (KIc) nor non-linear elastic (KJc) 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 KIc but decreased KJc. 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.

A critical evaluation of cortical bone fracture toughness testing methods.

Cortical bone fracture mechanics which quantifies the tissue's resistance to fracture is widely regarded as important to finding key determinants of bone fragility and fracture. Currently, the most widely used fracture mechanics approach is the J-integral resistance (J-R) curve as defined in ASTM E1820 standard. This standard employs an unloading compliance (UC) method to estimate crack extension, necessary for fracture toughness and resistance curve (R-curve) quantification. Further, this UC method requires a series of unload-reload cycles to be conducted during the fracture test. However, cortical bone violates some assumptions on which the UC method is based, which are: no energy loss during the unload-reload cycles and any change in unloading compliance is only due to crack extension. Consequently, the aim of this study was to examine the impact of the UC method on the accuracy of fracture toughness measurement for bovine cortical bone. Ten pairs of single edged notched bend specimens were prepared from the posterior diaphysis of bovine tibiae and underwent three-point bending fracture tests. The paired specimens were divided into two groups: a cyclic loaded group and a monotonic loaded group. Further, crack extension was determined by the UC method for the cyclic group and by an optical method for both the cyclic and monotonic groups. From these, three different approaches were used to generate J-R curves from which three fracture toughness parameters were computed and compared between the three approaches. This comparison allowed the impact of crack extension estimation by the UC method as well as the unload-reload cycles on the accuracy of the fracture toughness measures to be assessed. Results show that the UC method underestimates crack extension by an average error of 73%. In addition, the combined effects from crack extension estimation using the UC method and the unload-reload cycles lead to a significant overestimation of the specimen's fracture toughness measures. This highlights the need for more studies to establish a standardized approach to cortical bone fracture testing.

The impact of fall-related loading rate on the formation of micro-damage in human cortical bone fracture.

The quest for better predictive tools as well as new preventative and therapeutic measures for bone fragility and fracture has highlighted the need for greater mechanistic understanding of the bone fracture process. Cortical bone, the major load bearing part of the bone, employs different toughening mechanisms to either inhibit or slow down crack growth which leads to fracture. Among these toughening mechanisms, is the formation of a micro-damage process zone (MDPZ) around the region of the propagating crack. Investigations into the MDPZ to date have primarily been based on quasi-static or cyclic loading rate experiments which do not necessarily replicate physiological fracture rates. Consequently, the impact of fall-related loading rates on the formation of the micro-damage process zone was investigated comparing these to quasi-static loading rate equivalents. The size of MDPZ was found to be 42% smaller in the high-rate group compared to the quasi-static rate group. The smaller MDPZ size was associated with a brittle, unstable fracture behaviour and an overall smaller fracture resistance measure (Jmax). This result points to the possibility of a strain rate hardening mechanism at the heart of micro-damage formation, which is hampered under high loading rates, resulting in lower overall fracture resistance.

Bone collagen network integrity and transverse fracture toughness of human cortical bone.

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, Td) 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, Kinit, 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 (R2 = 24-35%), and to a lesser degree with bound water fraction (BW; R2 = 7.9%) and pore water fraction (PW; R2 = 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-R2 = 36.7-49.0%). Also, the best-fit model for J-int (adjusted-R2 = 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-R2 = 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.