Hydrothermal Isometric Tension Research Articles

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.

Empirical evidence that bone collagen molecules denature as a result of bone fracture

Cortical bone tissue, primarily composed of collagen, hydroxyapatite, and water, is a strong and tough natural, structural biomaterial. The integrity of the collagenous phase (native triple helix vs. damaged/denatured coil) has previously been correlated via various means, including hydrothermal isometric tension testing and FTIR and Raman spectroscopy, with the capability of cortical bone to undergo stable fracture. Collagen is a relatively stable protein, requiring around 70 J/g to thermally denature its native triple helix structure, through the melting of hydrogen bonds. It is widely thought that bone collagen molecules denature (unravel) during fracture, acting as a molecular-scale mechanical toughening mechanism, but this has not been empirically demonstrated to date. A new technology, fluorescently-labeled collagen hybridizing peptides (F–CHP), enables imaging that specifically detects denatured collagen. This provides an opportunity to empirically test whether bone collagen molecules do denature during bone fracture. Here, F–CHP was used to stain fracture surfaces produced by transverse Mode-I fracture of chevron-notched bovine and human cortical bone beams. The fracture surfaces demonstrated increased staining, above the level of rigorous paired controls, and the staining directly correlated with the work-to-fracture (WFx) of bovine bone beams. This increased denaturation signal was also constrained to a rough textured region visible on the fracture surface, which is known to correspond with stable tearing. Similar staining was also detected on the fracture surfaces of human cortical bone. Increased staining was not detected on the fracture surfaces of specimens that were dehydrated prior to fracture, suggesting a role for water in the denaturation process. This study provides the first empirical evidence of bone collagen denaturation resulting from cortical bone fracture and extends our understanding of this mechanism towards the mechanical performance of cortical bone.

A linear systems model of the hydrothermal isometric tension test for assessing collagenous tissue quality

Collagen is the most abundant structural protein in the animal kingdom. Its thermal and thermomechanical properties are often measured using differential scanning calorimetry (DSC) and hydrothermal isometric tension (HIT) tests, respectively. In living tissues, not all collagenous structures (molecules, fibrils, etc.) have the same “quality,” and the heterogeneity among these structures in specific tissues increases with remodeling, aging, and/or disease states. In this paper, first, a peak-fitting analysis is carried out to separate and distinguish the sequential denaturation events in a DSC endotherm, which presumably stem from heterogeneity in the collagen fibrils. The fitting analysis uses one of two functions: a Gaussian function or a function proposed by Miles. The individual endotherms were then convolved with a physics-based parametric function, J(T), proposed by the authors, to model the development of the isometric tension in two stages: 1) tension development due to a sudden increase in conformational entropy as each collagen packet denatures, and 2) additional isometric tension development due to increasing temperature, consistent with rubber thermo-elasticity. The proposed function parameters were then found by fitting to actual HIT curves using a global optimization technique. This model provides a decoupling of the effects of denaturation kinetics and collagen network connectivity and therefore an improved interpretation of HIT test results during the temperature ramp from ambient temperature to 90 °C. The simple model outputs are two parameters, α and β, that have physical meaning and aid in assessing collagenous tissue quality in terms of connectivity and integrity.

ISSLS PRIZE IN BASIC SCIENCE 2020: Beyond microstructure-circumferential specialization within the lumbar intervertebral disc annulus extends to collagen nanostructure, with counterintuitive relationships to macroscale material properties.

To determine whether the annulus of lumbar intervertebral discs contains circumferential specialization in collagen nanostructure and assess whether this coincides with functional differences in macroscale material properties. Anterior and posterior disc wall samples were prepared from 38 mature ovine lumbar segments. Regional differences in molecular thermal stability and intermolecular network integrity of the annulus' tension-bearing collagen fibres were examined with hydrothermal isometric tension (HIT) analysis, with and without preceding NaBH4 treatment to stabilize labile crosslinks. Energetics of collagen denaturation were studied by differential scanning calorimetry (DSC). Tensile mechanics of annular lamellae were studied using oblique sagittal bone-disc-bone samples loaded to rupture. Annular failure characteristics of the ruptured test segments were compared via microscopy of serial sections. HIT showed that tension-bearing collagen fibres of the posterior annulus were composed of collagen molecules with significantly greater thermal stability and intermolecular network integrity than those of the anterior annulus. NaBH4 treatment confirmed that labile intermolecular crosslinks did not significantly contribute to network integrity in either region. Regional differences seen in DSC were smaller than those observed in HIT, indicating structural similarities in annular collagen outside of the main fibre bundles. Mechanical testing showed that the posterior annulus was significantly weaker than the anterior annulus. For both regions, ultimate tensile strengths of annular fibres were significantly greater than those previously reported. Ruptures in both regions were predominantly due to annular failure. Specializations in collagen nanostructure exist between different circumferential regions of the annulus and coincide with significant differences in material properties. These slides can be retrieved under Electronic Supplementary Material.

Pregnancy-Induced Remodeling of Heart Valves

Although many cardiovascular tissues have been shown to remodel when exposed to chronic changes in hemodynamic stresses, little is known about the capacity of heart valves to adapt in a non-pathological state such as pregnancy. Pregnancy is a volume-overload state that triggers enlargement of the heart and valve orifices, thereby elevating valve leaflet stresses. The aim of this work was to (i) investigate pregnancy-induced alterations in valve leaflet biomechanics, (ii) perform structural studies defining the material basis for these changes, and (iii) link the observed structural and mechanical information using a structural constitutive model. Valve leaflets were harvested from non-pregnant heifers and pregnant cows. Gross leaflet structure was characterized by leaflet dimensions, and small-angle light scattering was used to assess changes in internal collagen fiber architecture. Tissue composition was determined using standard biochemical assays. Histological studies assessed architectural changes in cellular and matrix components. Leaflet mechanical properties were assessed using equibiaxial mechanical testing. Collagen thermal stability and crosslinking state was assessed using denaturation and hydrothermal isometric tension tests. We observed rapid and extensive heart valve remodeling in pregnancy, with alterations to leaflet geometry, fiber architecture, composition, biomechanics, and cellularity. All the valves expanded in pregnancy, via an increase in the production of collagen, with associated changes in tissue composition and structure of the collagen network (i.e. loss of crimp, crosslink maturation, and reduction in thermal stability). Our structural constitutive model suggests that there is an initial period of permanent set-like deformation where no remodeling occurs, followed by a remodeling phase that results in near-complete restoration of homeostatic tissue-level stresses and mechanical properties. Understanding the valvular adaptations to pregnancy may be fundamental both to developing interventions and treatments for valve disease and heart failure, as well as recognizing the implications of pregnancies on maternal long-term vascular risk.

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.

The thermal shrinkage force in perimysium from different beef muscles is not affected by post-mortem ageing

Differences in the thermal shrinkage and collagen solubility between bovine Semitendinosus (ST) and Pectoralis profundus (PP) muscles and their interactions with ageing were evaluated by studying collagen solubility, hydrothermal isometric tension and thermal denaturation properties of intramuscular connective tissue after 5–20days post-mortem storage at 4°C.Collagen solubility was higher in ST than in PP muscle at 5–13days, but the differences between the two muscles decreased at longer ageing times. A small decrease in the peak denaturation temperature of perimysium occurred with increasing ageing times in both muscles. Maximum force in isometrically-heated perimysium was broadly equivalent in both muscles. Although the amount and solubility of collagen varies between muscles and ageing decreases the stability of some of the collagen, thermal shrinkage forces in heated perimysium are not significantly diminished by ageing.These findings support the idea of one collagen fraction easily degraded by ageing and heat, and another more resistant fraction that determines the physical properties of the tissue after ageing and cooking.

Age-related changes of wall composition and collagen cross-linking in the rat carotid artery – In relation with arterial mechanics

In association with age-related changes in arterial wall mechanics, the composition of connective tissues, the fraction and size of vascular smooth muscle cells (SMCs), and the degree of collagen cross-linking were studied with common carotid arteries harvested from 8, 16, 32, and 64 week-old Wistar rats. For histomorphometric studies, each arterial segment was fixed under in vivo operating force condition, and then sequentially sliced into thin specimens, followed by selective staining for the observation of collagen, elastin, and SMCs. Then, the fraction of each component, and the number and size of SMCs were determined with an image analyzer. The content of collagen and elastin, their ratio, and the number and the area fraction of SMCs showed no significant correlations with age, while the density and the size of SMCs were significantly smaller and larger, respectively, in 64 week-old animals than in the others. The results of collagen and elastin cannot explain the biomechanical data obtained in our previous study using the same animal model, which showed that the elastic modulus and wall stiffness were significantly larger in 64 week-old animals compared to younger ones. To investigate the reason for the discrepancy between the histological and the biomechanical results, a hydrothermal isometric tension method was applied to the analysis of the cross-linking of collagen, and we found that the amount of cross-links was significantly greater in 64 week-old arteries than in the others. This result corresponded well with the biomechanical results, and therefore the higher wall stiffness and elastic modulus in older arteries might be ascribed to their larger amount of collagen cross-links.

Elastic–plastic fracture toughness and rising JR-curve behavior of cortical bone is partially protected from irradiation–sterilization-induced degradation by ribose protectant

ObjectiveThis study tested the hypothesis that pre-treating cortical bone with ribose would protect the rising fracture resistance curve behavior and crack initiation fracture toughness of both bovine and human cortical bone from the degrading effects of γ-irradiation sterilization. Materials and methodsA ribose pre-treatment (1.8 M for bovine, and 1.2 M for human, in PBS at 60 °C for 24 h) was applied to single-edge notched bending fracture specimens prior to sterilization with a 33 kGy dose of γ-irradiation. Fracture resistance curves were generated with a single specimen method using an optical crack length measurement technique. The effect of the treatment on overall fracture resistance behavior, crack initiation fracture toughness, and tearing modulus was compared with non-irradiated and conventionally irradiation sterilized controls. Hydrothermal isometric tension testing was used to examine collagen network connectivity and thermal stability to explore relationships between collagen network quality and fracture resistance. ResultsThe ribose pre-treatment successfully protected the crack growth initiation fracture toughness of bovine and human bone by 32% and 63%, respectively. The rising JR-curve behavior was also partially protected. Furthermore, collagen connectivity and thermal stability followed similar patterns to those displayed by fracture toughness. ConclusionsThis paper demonstrates that the fracture toughness of irradiation-sterilized bone tissue can be partially protected with a ribose pre-treatment. This new approach shows potential for the production and clinical application of sterilized allografts with improved mechanical performance and durability.

Collagen fibrils in functionally distinct tendons have differing structural responses to tendon rupture and fatigue loading

In this study we investigate relationships between the nanoscale structure of collagen fibrils and the macroscale functional response of collagenous tissues. To do so, we study two functionally distinct classes of tendons, positional tendons and energy storing tendons, using a bovine forelimb model. Molecular-level assessment using differential scanning calorimetry (DSC), functional crosslink assessment using hydrothermal isometric tension (HIT) analysis, and ultrastructural assessment using scanning electron microscopy (SEM) were used to study undamaged, ruptured, and cyclically loaded samples from the two tendon types. HIT indicated differences in both crosslink type and crosslink density, with flexor tendons having more thermally stable crosslinks than the extensor tendons (higher TFmax of >90 vs. 75.1±2.7°C), and greater total crosslink density than the extensor tendons (higher t1/2 of 11.5±1.9 vs. 3.5±1.0h after NaBH4 treatment). Despite having a lower crosslink density than flexor tendons, extensor tendons were significantly stronger (37.6±8.1 vs. 23.1±7.7MPa) and tougher (14.3±3.6 vs. 6.8±3.4MJ/m3). SEM showed that collagen fibrils in the tougher, stronger extensor tendons were able to undergo remarkable levels of plastic deformation in the form of discrete plasticity, while those in the flexor tendons were not able to plastically deform. When cyclically loaded, collagen fibrils in extensor tendons accumulated fatigue damage rapidly in the form of kink bands, while those in flexor tendons did not accumulate significant fatigue damage. The results demonstrate that collagen fibrils in functionally distinct tendons respond differently to mechanical loading, and suggests that fibrillar collagens may be subject to a strength vs. fatigue resistance tradeoff. Statement of SignificanceCollagen fibrils—nanoscale biological cables—are the fundamental load-bearing elements of all structural human tissues. While all collagen fibrils share common features, such as being composed of a precise quarter-staggered polymeric arrangement of triple-helical collagen molecules, their structure can vary significantly between tissue types, and even between different anatomical structures of the same tissue type. To understand normal function, homeostasis, and disease of collagenous tissues requires detailed knowledge of collagen fibril structure-function. Using anatomically proximate but structurally distinct tendons, we show that collagen fibrils in functionally distinct tendons have differing susceptibilities to damage under both tensile overload and cyclic fatigue loading. Our results suggest that the structure of collagen fibrils may lead to a strength versus fatigue resistance tradeoff, where high strength is gained at the expense of fatigue resistance, and vice versa.

New recommendations for measuring collagen solubility

The heat-solubility of intramuscular collagen is usually conducted in 1/4 Ringer's solution at pH7.4, despite this ionic strength and pH being inappropriate for post-rigor meat. The current work studied the percentage of soluble collagen and hydrothermal isometric tension characteristics of perimysial strips on bovine semitendinosus muscles in either 1/4 Ringer's solution, distilled water, PBS, or a solution of the same salt concentration as 1/4 Ringer's but at pH5.6. Values of % soluble collagen were lower at pH7.4 than 5.6. Increasing ionic strength reduced % soluble collagen. The maximum perimysial isometric tension was independent of the bathing medium, but the percent relaxation was higher at pH7.4 than at pH5.6, and increased with ionic strength of the media. It is recommended that future measurements of collagen solubility and tests on connective tissue components of post-rigor meat should be carried out in a solution of concentrations NaCl and KCl equivalent to those in 1/4 Ringer's, but at pH5.6, a pH relevant to post-rigor meat.

γ-Irradiation sterilized bone strengthened and toughened by ribose pre-treatment

ObjectiveThis study tested the hypothesis that a ribose-based pre-treatment would protect the strength, ductility and toughness of γ-irradiation sterilized cortical bone. MethodsExperiment 1: The effects of ribose pre-treatment (1.8M in PBS at 60°C for 24h) prior to 33kGy of irradiation on strength, ductility and toughness (beams in three-point bending) and fracture toughness (J-integral at instability in single edge notched (bending)) were tested against matched non-irradiated and irradiated controls from bovine tibiae. Experiment 2: Three-point bending tests were conducted using beams from human femora (males, 59–67 years). Bone collagen thermal stability and network connectivity were examined using hydrothermal isometric tension testing. ResultsRibose pre-treatment protected the strength, ductility and toughness of irradiation sterilized bovine and human specimens to differing degrees. Their ultimate strength was not detectably different from non-irradiated control levels; toughness in bovine and human specimens was protected by 57 and 76%, respectively. Untreated human bone was less affected by irradiation and ribose pre-treatment was more effective in human bone than bovine bone. ConclusionsThis paper presents the first proof-of-principle that irradiation-sterilized bone with improved mechanical properties can be produced through the application of a ribose pre-irradiation treatment, which provides a more stable and connected collagen network than found in conventionally irradiated controls.

Bone embrittlement and collagen modifications due to high-dose gamma-irradiation sterilization

Bone allografts are often used in orthopedic reconstruction of skeletal defects resulting from trauma, bone cancer or revision of joint arthroplasty. γ-Irradiation sterilization is a widely-used biological safety measure; however it is known to embrittle bone. Irradiation has been shown to affect the post-yield properties, which are attributed to the collagen component of bone. In order to find a solution to the loss of toughness in irradiated bone allografts, it is important to fully understand the effects of irradiation on bone collagen. The objective of this study was to evaluate changes in the structure and integrity of bone collagen as a result of γ-irradiation, with the hypothesis that irradiation fragments collagen molecules leading to a loss of collagen network connectivity and therefore loss of toughness.Using cortical bone from bovine tibiae, sample beams irradiated at 33kGy on dry ice were compared to native bone beams (paired controls). All beams were subjected to three-point bend testing to failure followed by characterization of the decalcified bone collagen, using differential scanning calorimetry (DSC), hydrothermal isometric tension testing (HIT), high performance liquid chromatography (HPLC) and gel electrophoresis (SDS-PAGE). The carbonyl content of demineralized bone collagen was also measured chemically to assess oxidative damage. Barium sulfate staining after single edge notch bending (SEN(B)) fracture testing was also performed on bovine tibia bone beams with a machined and sharpened notch to evaluate the fracture toughness and ability of irradiated bone to form micro-damage during fracture.Irradiation resulted in a 62% loss of work-to-fracture (p≤0.001). There was significantly less micro-damage formed during fracture propagation in the irradiated bone. HPLC showed no significant effect on pentosidine, pyridinoline, or hydroxypyridinoline levels suggesting that the loss of toughness is not due to changes in these stable crosslinks. For DSC, there was a 20% decrease in thermal stability (p<0.001) with a 100% increase (p<0.001) in enthalpy of denaturation (melting). HIT testing also showed a decrease in thermal stability (20% lower denaturation temperature, p<0.001) and greatly reduced measures of collagen network connectivity (p<0.001). Interestingly, the increase in enthalpy of denaturation suggests that irradiated collagen requires more energy to denature (melt), perhaps a result of alterations in the hydrogen bonding sites (increased carbonyl content detected in the insoluble collagen) on the irradiated bone collagen.Altogether, this new data strongly indicates that a large loss of overall collagen connectivity due to collagen fragmentation resulting from γ-irradiation sterilization leads to inferior cortical bone toughness. In addition, notable changes in the thermal denaturation of the bone collagen along with chemical indicators of oxidative modification of the bone collagen indicate that the embrittlement may be a function not only of collagen fragmentation but also of changes in bonding.

Riboflavin-sensitized photo-crosslinking of collagen using a dental curing light.

Photo-crosslinking of biomolecules such as collagen and fibrinogen is an emerging area of research interest. The use of a small dental curing light with a non-toxic photosensitizer represents a novel, practical approach to periodontal wound treatment. This study evaluated the effects of riboflavin-sensitized photo-oxidation using a dental curing light on two collagenous biomaterials, as a preliminary step towards developing a medical technology for wound closure/healing. A collagenous biomaterial (DBP) and type I collagen gels were treated by this photo-oxidative technique and characterized by hydrothermal isometric tension (HIT) analysis, amino acid analysis, SDS-PAGE, and rheology. HIT analysis suggested that dental curing light exposure for 300 s with riboflavin produced heavily crosslinked DBP. Dental curing light exposure for 300 s with riboflavin also showed a reduction in lysine concentration of DBP. SDS-PAGE showed that dental curing light exposure for 30 or 300 s with riboflavin resulted in crosslinked collagen gels. Dental curing light exposure for 30 s with riboflavin yielded a collagen gel with the strongest rheological characteristics. This novel approach to wound treatment has potential for wide adoption and clinical use, particularly because dental curing lights, riboflavin, and collagen biomaterials are all used clinically, but not yet combined together as one technology for broad application.

Cross‐link stabilization does not affect the response of collagen molecules, fibrils, or tendons to tensile overload

We investigated whether immature allysine-derived cross-links provide mechanically labile linkages by exploring the effects of immature cross-link stabilization at three levels of collagen hierarchy: damaged fibril morphology, whole tendon mechanics, and molecular stability. Tendons from the tails of young adult steers were either treated with sodium borohydride (NaBH₄) to stabilize labile cross-links, exposed only to the buffer used during stabilization treatment, or maintained as untreated controls. One-half of each tendon was then subjected to five cycles of subrupture overload. Morphologic changes to collagen fibrils resulting from overload were investigated using scanning electron microscopy, and changes in the hydrothermal stability of collagen molecules were assessed using hydrothermal isometric tension testing. NaBH4 cross-link stabilization did not affect the response of tendon collagen to tensile overload at any of the three levels of hierarchy studied. Cross-link stabilization did not prevent the characteristic overload-induced mode of fibril damage that we term discrete plasticity. Similarly, stabilization did not alter the mechanical response of whole tendons to overload, and did not prevent an overload-induced thermal destabilization of collagen molecules. Our results indicate that hydrothermally labile cross-links may not be as mechanically labile as was previously thought.

Structural-Mechanical Changes in the Pericardium During Pregnancy

The pericardium’s collagenous structure limits over-expansion of the heart, couples the left and right ventricles, and keeps the heart in place in the chest cavity. Experimental volume overload studies in dogs have demonstrated consequent pericardial remodeling. By comparison, the maternal heart is subjected to large, natural volume overload but with altered hormonal levels. No current literature exists on the resulting pericardial changes. This study investigates those effects in a bovine model that mimics human pregnancy. Pericardial samples were collected from 10 never-pregnant and 17 pregnant cattle from 13 weeks gestation or later. Biaxial mechanics, hydrothermal isometric tension (HIT) testing, crosslink analysis, histology and biochemical tests for water and collagen contents were conducted. There were no changes in the collagen and water contents, or in the crimp length between pericardia of pregnant and non-pregnant animals. In late pregnancy, pericardial thickness increased with only slight decreases in biomechanical stretch ratios, mainly in the base-to-apex direction. Pericardium from pregnant cattle had a 14.8% decrease in cell density, 1.3° lower collagen denaturation temperature, and a surprising 32.0% lower index of immature crosslinking. Indeed, hydrothermal studies of collagen from the pregnant animals’ pericardia showed greater mature and total crosslinking (40.9 and 22.4% increases in isothermal load decay half-times) respectively. Overall, then, there was evidence on the molecular level of remodeling and growth in the pericardium in early pregnancy (<13 weeks gestation). This is the first study to demonstrate that pericardium remodels and grows to adapt to changes in pregnancy, perhaps as a means to maintaining its functional properties.

Repeated subrupture overload causes progression of nanoscaled discrete plasticity damage in tendon collagen fibrils

A critical feature of tendons and ligaments is their ability to resist rupture when overloaded, resulting in strains or sprains instead of ruptures. To treat these injuries more effectively, it is necessary to understand how overload affects the primary load-bearing elements of these tissues: collagen fibrils. We have investigated how repeated subrupture overload alters the collagen of tendons at the nanoscale. Using scanning electron microscopy to examine fibril morphology and hydrothermal isometric tension testing to look at molecular stability, we demonstrated that tendon collagen undergoes a progressive cascade of discrete plasticity damage when repeatedly overloaded. With successive overload cycles, fibrils develop an increasing number of kinks along their length. These kinks-discrete zones of plastic deformation known to contain denatured collagen molecules-are accompanied by a progressive and eventual total loss of D-banding along the surface of fibrils, indicating a loss of native molecular packing and further molecular denaturation. Thermal analysis of molecular stability showed that the destabilization of collagen molecules within fibrils is strongly related to the amount of strain energy dissipated by the tendon after yielding during tensile overload. These novel findings raise new questions about load transmission within tendons and their fibrils and about the interplay between crosslinking, strain-energy dissipation ability, and molecular denaturation within these structures.

In vitro non-enzymatic ribation reduces post-yield strain accommodation in cortical bone

Non-enzymatic glycation (NEG) and advanced glycation endproducts (AGEs) may contribute to bone fragility in various diseases, ageing, and other conditions by modifying bone collagen and causing degraded mechanical properties. In this study, we sought to further understand how collagen modification in an in vitro non-enzymatic ribation model leads to loss of cortical bone toughness. Previous in vitro studies using non-enzymatic ribation reported loss of ductility in the cortical bone. Increased crosslinking is most commonly blamed for these changes; however, some studies report positive correlations between measures of total collagen crosslinking and work-to-fracture/toughness measurements whilst correlations between general NEG and measures of ductility are often negative.Fifteen bone beam triplets were cut from bovine metatarsi. Each provided one native non-incubated control, one incubated control and one ribated specimen. Incubation involved simulated body fluid±ribose for fourteen days at 37°C. Pentosidine and pyridinoline crosslinks were measured using HPLC. Three-point bending tests quantified mechanical properties. Fracture surfaces were examined using scanning electron microscopy. The effects of ribation on bone collagen molecular stability and intermolecular connectivity were investigated using differential scanning calorimetry and hydrothermal isometric tension testing.Ribation caused increased non-enzymatic collagen modification and pentosidine content (16mmol/mol collagen) and inferior post-yield mechanical behaviour, especially post-yield strain and flexural toughness. Fracture surfaces were smoother with less collagen fibril deformation or tearing than observed in controls. In the ribated group only, pentosidine content and thermomechanical measures of crosslinking were positively correlated with measures of strain accommodation and energy absorption before failure.Non-enzymatic ribation and the resulting modifications reduce cortical bone pseudo-plasticity through a reduced capacity for post-yield strain accommodation. However, the positive correlations we have found suggest that increased crosslinking may not provide a complete explanation for this embrittlement.

Physiological remodeling of the mitral valve during pregnancy

There is growing evidence that heart valves are not passive structures but can remodel with left ventricular dysfunction. To determine if these tissues remodel under nonpathological conditions, we examined the mirtal valve anterior leaflet during the volume loading and cardiac expansion of pregnancy using a bovine model. We measured leaflet dimensions, chordal attachments, and biaxial mechanical properties of leaflets collected from never-pregnant heifers and pregnant cows (pregnancy duration estimated from fetal length). Hydrothermal isometric tension (HIT) tests were performed to assess the denaturation temperature (T(d)) associated with collagen molecular stability and the load decay half-time (t(1//2)) associated with intermolecular cross-linking. Histological changes were examined using Verhoeff-van Gieson and picrosirius red staining with polarized light. We observed striking changes to the structure and material properties of the mitral anterior leaflet during pregnancy. Leaflet area was increased 33%, with a surprising increase (nearly 25%) in chordae tendinae attachments. There was a biphasic change in leaflet extensibility: it rapidly decreased by 30% and then reversed to prepregnant values by late pregnancy. The 2°C decrease in T(d) in pregnancy was indicative of collagen remodeling, whereas the 70% increase in HIT t(1/2) indicated an increase in collagen cross-linking. Finally, histological results suggested transient increases in leaflet thickness and transient decreases in collagen crimp. This remodeling may compensate for the increased loading conditions associated with pregnancy by normalizing leaflet stress and maintaining coaptation. Understanding the mechanisms of mitral valve physiological remodeling in pregnancy could contribute to alternative treatments of pathological remodeling associated with left ventricular dysfunction.

Changes in Collagen With Aging Maintain Molecular Stability After Overload: Evidence From an In Vitro Tendon Model

Soft tissue injuries are poorly understood at the molecular level. Previous work using differential scanning calorimetry (DSC) has shown that tendon collagen becomes less thermally stable with rupture. However, most soft tissue injuries do not result in complete tissue rupture but in damaging fiber overextension. Covalent crosslinking, which increases with animal maturity and age, plays an important role in collagenous fiber mechanics. It is also a determinant of tissue strength and is hypothesized to inhibit the loss of thermal stability of collagen due to mechanical damage. Controlled overextension without rupture was investigated to determine if overextension was sufficient to reduce the thermal stability of collagen in the bovine tail tendon (BTT) model and to examine the effects of aging on the phenomenon. Baseline data from DSC and hydrothermal isometric tension (HIT) techniques were compared between two groups: steers aged 24-30 months (young group), and skeletally mature bulls and oxen aged greater than five years (old group). Covalent crosslinks were quantified by ion exchange chromatography. Overextension resulted in reduced collagen thermal stability in the BTT model. The Young specimens, showing detectably lower tissue thermomechanical competence, lost more thermal stability with overextension than did the old specimens. The effect on old specimens, while smaller, was detectable. Multiple overextension cycles increased the loss of stability in the young group. Compositional differences in covalent crosslinking corresponded with tissue thermomechanical competence and therefore inversely with the loss of thermal stability. HIT testing gave thermal denaturation temperatures similar to those measured with DSC. The thermal stability of collagen was reduced by overextension of the tendon--without tissue rupture--and this effect was amplified by increased cycles of overextension. Increased tissue thermomechanical competence with aging seemed to mitigate the loss of collagen stability due to mechanical overextension. Surprisingly, the higher tissue thermomechanical competence did not directly correlate with the concentration of endogenous enzymatically derived covalent crosslinking on a mole per mole of collagen basis. It did, however, correlate with the percentage of mature and thermally stable crosslinks. Compositional changes in fibrous collagens that occur with aging affect fibrous collagen mechanics and partially determine the nature of mechanical damage at the intermolecular level. As techniques develop and improve, this new information may lead to important future studies concerning improved detection, prediction, and modeling of mechanical damage at much finer levels of tissue hierarchy than currently possible.