Thermal Stability Of Collagen Research Articles

The impact of N-terminal modification of PAA with different chain lengths on the structure, thermal stability and pH sensitivity of succinylated collagen

The limitations of native collagen, such as thermal stability and solubility in physiological environments, can be improved by applying bioconjugation and synthetic chemistry techniques. However, the exquisite control of the modification site of collagen remains a challenge. In this work, pH-responsive poly(acrylic acid) (PAA) with different chain lengths was attached to the N-terminal α-amino groups of succinylated collagen using a site-specific modification strategy. Additionally, the structure, thermal stability, and pH sensitivity of succinylated collagen were explored. The modification rate of amino groups in the succinylated collagen-PAA bioconjugate (SPSC-PAA) was evaluated by the 2,4,6-trinitrobenzene sulfonic acid assay. The impact of N-terminal modification of PAA and its chain length on the thermal stability of collagen was explored by CD and DSC. These techniques revealed that the thermal stability of SPSC-Col is pH-responsive and closely related to the chain length of grafted PAA. The pH sensitivity of SPSC-PAA was further explored by rheology and turbidity. Subsquently, the critical pH and isoelectric point of SPSC-PAAs were also examined by turbidity and isoelectric point titration, respectively. This work provides a new insight into the N-terminal modification of collagen on its properties.Graphical abstract

Thermal stability of sea cucumber collagen and effects of gallic acid crosslinking

SummaryThe thermal processing characteristics of sea cucumber collagen (SCC) are closely related to its triple helix structure, self‐assembly and aggregation ability. The effect of heat treatment on SCC was studied by apparent viscosity, circular dichroism spectra and so on. As heating time increased from 10 to 160 min, apparent viscosity decreased from 400 to 300 mPa·s at 80 °C, indicating the structure of SCC changed. The decrease in SCC self‐assembly and aggregation ability showed the structural change. Positive peak at 220 nm was decreased from 24 to 0 mdeg at 60 °C for 10–160 min, which revealed that SCC triple helix structure was uncoiled and disappeared. Fluorescence intensity was increased and the absorption peak showed a red shift during thermal treatment. Gallic acid crosslinking could improve thermal stability and inhibit the degradation of collagen. It could provide theoretical guidance for sea cucumber processing.

Improvement of collagen self-assembly and thermal stability in the presence of trehalose

Trehalose, as a compatible osmolyte, can stabilize collagen and is beneficial to the self-assembly and mechanical properties of its products.

Physicochemical properties of acid‐soluble collagens from different tissues of large yellow croaker (Larimichthys crocea)

SummaryAcid‐soluble collagens (ASCs) were extracted and characterised from different tissues of large yellow croaker (Larimichthys crocea). The yields of ASCs in bones, skins, scales and muscles were 3.22 ± 0.47%, 42.30 ± 1.15%, 2.82 ± 0.31% and 0.89 ± 0.12%, respectively. SDS‐PAGE and ATR‐FTIR analysis showed that all ASCs were type I collagen with intact triple helical structure and consisted of α1,3‐chain, α2‐chain, β‐chain and γ‐chain. Quantitative analysis of SDS‐PAGE revealed that the ratio of β‐chains in ASC from scales was lower than other tissues. The imino acid contents of ASC from bones, skins, scales and muscles were 169, 167, 162 and 173 residues/1000 residues, respectively. The maximum transition temperature (Tm) of ASC from scales was 30.15 °C, lower than the other three ASCs, indicated that the thermal stability of collagen was affected by both the content of imino acid and β‐chains. The results also implied that thermal stability of ASC might exist the tissue specificity among different fish species.

Rare Missense Functional Variants at COL4A1 and COL4A2 in Sporadic Intracerebral Hemorrhage.

ObjectiveTo test the genetic contribution of rare missense variants in COL4A1 and COL4A2 in which common variants are genetically associated with sporadic intracerebral hemorrhage (ICH), we performed rare variant analysis in multiple sequencing data for the risk for sporadic ICH.MethodsWe performed sequencing across 559 Kbp at 13q34 including COL4A1 and COL4A2 among 2,133 individuals (1,055 ICH cases; 1,078 controls) in United States–based and 1,381 individuals (192 ICH cases; 1,189 controls) from Scotland-based cohorts, followed by sequence annotation, functional impact prediction, genetic association testing, and in silico thermodynamic modeling.ResultsWe identified 107 rare nonsynonymous variants in sporadic ICH, of which 2 missense variants, rs138269346 (COL4A1I110T) and rs201716258 (COL4A2H203L), were predicted to be highly functional and occurred in multiple ICH cases but not in controls from the United States–based cohort. The minor allele of rs201716258 was also present in Scottish patients with ICH, and rs138269346 was observed in 2 ICH-free controls with a history of hypertension and myocardial infarction. Rs138269346 was nominally associated with nonlobar ICH risk (p = 0.05), but not with lobar ICH (p = 0.08), while associations between rs201716258 and ICH subtypes were nonsignificant (p > 0.12). Both variants were considered pathogenic based on minor allele frequency (<0.00035 in European populations), predicted functional impact (deleterious or probably damaging), and in silico modeling studies (substantially altered physical length and thermal stability of collagen).ConclusionsWe identified rare missense variants in COL4A1/A2 in association with sporadic ICH. Our annotation and simulation studies suggest that these variants are highly functional and may represent targets for translational follow-up.

Electro-deposition synthesis of tube-like collagen–chitosan hydrogels and their biological performance

Electro-deposition is a smart, safe and efficient method for biomaterial manufacturing. Collagen, a functional protein with excellent biocompatibility and biosafety, is a promising candidate for tissue engineering and biomedical applications. However, there are few reports on electro-deposition of biomaterials using collagen without electrically or magnetically active nanoparticles. In this study, electro-deposition was employed to swiftly fabricate tube-like collagen–chitosan hydrogels in a mild environment. Fourier transform infrared spectroscopy was employed to analyze the ingredients of the tube-like hydrogels. The result showed that the hydrogels contained both collagen and chitosan. The distribution and content of collagen in the hydrogels was further measured by hematoxylin–eosin staining and hydroxyproline titration. Collagen was distributed homogeneously and its content was related to the initial collagen:chitosan ratio. The tension resistance of the composite gels and the thermal stability of collagen in the composites were obviously enhanced by the chitosan doping. Meanwhile, the tube-like hydrogels retained a good ability to promote cell proliferation of collagen. This method offers a convenient approach to the design and fabrication of collagen-based materials, which could effectively retain the bioactivity and biosafety of collagen and furnish a new way to enhance the stability of collagen and the tensile strength of collagen-based materials.

Development of collagen/nanohydroxyapatite scaffolds containing plant extract intended for bone regeneration

In this study scaffolds of nanohydroxyapatite (nHA) and anionic collagen (C) combined with plant extracts intended for bone tissue repair were developed. Grape seed (P), pomegranate peel (R) and jabuticaba peel (J) extracts were used as collagen crosslinker agents in order to improve the materials properties. All crude extracts were effective against Staphylococcus aureus, but only for CR scaffold inhibition zone was noticed. The extracts acted as crosslinking agents, increasing enzymatic resistance and thermal stability of collagen. The extracts showed cytotoxicity at the concentrations tested, while nHA increased cell viability. The scaffolds presented porosity and pore size appropriate for bone growth. CR, CnHAP, CnHAR and CnHAJ increased the cell viability after 24 h. The combination of collagen, nHA and plant extracts offers a promising strategy to design novel biomaterials for bone tissue regeneration.

SEM, FTIR and DSC Investigation of Collagen Hydrolysate Treated Degraded Leather

Abstract It is essential to establish an appropriate conservation method to reduce the deterioration of leather-made artifact and extend its lifetime. The restoration and consolidation of historic leather have always been a challenge for conservators. This paper discussed the new conservation treatment by applying skin collagen hydrolysate as the consolidation material while glutaraldehyde and tannin as cross-linking agents onto the degraded leather. The fresh leather samples were artificially aged to simulate the deterioration of historical leather. Then the simulated samples were treated with the skin collagen hydrolysate and cross linked by glutaraldehyde and tannin to improve the collagen fiber structures and thermal stability. The analytical approaches combining Ultra-depth three-dimensional Microscope, Scanning Electron Microscope (SEM), Fourier Transform Infrared Spectroscopy (FTIR) and Differential Scanning Calorimetry (DSC) were undertaken to identify the significant differences of the collagen fiber structures and thermal stability between untreated and treated leather samples. The results indicated that the thermal stability and the fibers cohesion of the treated sample were improved. It overall confirmed the use of the skin collagen hydrolysate isan effective consolidation strategy for the degraded leather.

Relationship between meat quality and intramuscular collagen characteristics of muscles from calf-fed, yearling-fed and mature crossbred beef cattle

Intramuscular Ehrlich Chromogen (EC) and pyridinoline (Pyr) concentrations in the gluteus medius (GM) and semitendinosus (ST) from crossbred Angus calf- (n = 14) and yearling-fed (n = 14) steer and mature cow (MC, n = 12) carcasses were related to collagen and intramuscular connective tissue (IMCT) thermal stability and peak Warner-Bratzler shear force (WBSF). In both muscles, Pyr density was greater in MC, while EC concentrations were comparable in calf- and yearling-fed steer muscles and lowest in MC muscles. Thermal denaturation temperature and enthalpy of IMCT were highest in both muscles when from MC, although only total collagen was correlated with WBSF in calf fed-yearling fed steer data. Results confirmed that EC concentration contributed to collagen thermal stability in steer muscles, but decreased it in MC muscles, while Pyr was consistently associated with collagen thermal stability.

Insights into the effect of artificial sweeteners on the structure, stability, and fibrillation of type I collagen

Artificial sweeteners (AS) are widely used as sugar substitutes because natural sweetener (sugar) leads to a number of health issues, including diabetes, obesity, and tooth decay. Since natural sugar (sucrose), diabetes and skin are highly interlinked, and also sucrose is known to inhibit the fibrillation of collagen, the major protein of the skin, a study on the impact of AS on collagen is important and essential. Herein, we have studied the influence of commonly used AS such as Sucralose (SUC), Aspartame (APM), and Saccharin (SAC) on the structure, stability, and fibrillation of collagen using various spectroscopic methods. The circular dichroism and turbidity results suggest that the AS does not disrupt the triple helix structure and also the fibrillar property of collagen, respectively. The fibrillar morphology was sustained, although there was a trivial difference in the entanglement of fibrils in the presence of SAC, compared to native collagen fibrils. The thermal stability of collagen is maintained in the presence of AS. Fluorescence and STD-NMR results indicate that the interaction between AS and collagen was weak, which supports the intact structure, stability, and fibrillation property of collagen. The current study thus suggests that the chosen AS does not influence collagen properties.

Thermal and vibrational biomarkers of porcine oral mucosa

The aim of this study was to develop an experimental protocol to determine specific and reproducible biomarkers of the oral mucosa using a combination of thermal and vibrational techniques. This works deals with the characterization of mandible and maxilla biopsies, 4 mm by diameter, from porcine mucosa in both the hydrated and lyophilized state. Thermogravimetric analysis was carried out to measure hydration level of these tissues and to define the onset of proteins degradation. By differential scanning calorimetry, thermal transitions of water and proteins were evidenced and used to quantify the hydric organization (unfreezable/freezable waters) and to evaluate collagen thermal stability (through denaturation parameters). To complete this protocol, Fourier transform infrared spectroscopy was also used to identify specific vibrational signatures of the main layers of oral mucosa. Total and freezable water amounts are significantly higher in maxilla, due to morphological differences at the macroscopic level, while unfreezable water amount is independent upon localization. Denaturation temperature (in peculiar the temperature corresponding to 50% of collagen denaturation) largely increases with dehydration. This temperature denaturation is also dependent upon localization whatever the hydration, possibly due to differences in cross-links or interactions with other proteins of oral mucosa. The acquisition of thermal and vibrational biomarkers of oral mucosa will contribute to a better knowledge of these soft tissues for further studies on aging.

Are aqueous solutions of choline-based ionic liquid biocompatible cross-linkers for collagen?

Identification of biocompatible cross-linkers is extremely important in the field of biomaterials, as it aids in improving stability to protein but also elicits less immune response. Towards this, aqueous solution of choline-based ILs (CILs) has been keyed out as biocompatible cross-linkers with an aim to understand their impact and effect on stabilization of collagen. The changes in the hydration dynamics of collagen treated with aqueous solution of CILs has been probed using transverse NMR relaxation and impedance measurements. Upon treatment of aqueous solution of CILs with collagen, there is a reorientation in the surrounding water milieu suggesting compactness in the structure of collagen, which is indicative of crosslinking. Further, the conformational and thermal stability of collagen have been substantiated. It is ascertained that the helicity of collagen remains intact as witnessed from the Rpn values measured through circular dichroic measurements and shifts in frequencies of amide bands as measured through FT-IR measurements. A significant increase in the melting and denaturation temperature suggesting stability of collagen has been observed through DSC measurements. Aqueous solution of CILs treated collagen scaffolds have been further appraised for crosslinking efficiency, enzymatic stability, hemolytic and platelet attachment assays. The efficacy of aqueous solution of CILs treated scaffolds has been evaluated for biocompatibility with NIH-3T3 fibroblasts cells and further confirmed by the phase-contrast microscopic images of these cells. Interaction between the collagen and aqueous solution of choline-based ILs is typically anchored by non-covalent interactions like electrostatic, hydrogen, and van der Waals interactions. Therefore, the current study implies that the aqueous solution of CILs can be explored as biocompatible cross-linkers for collagen and thus can be employed for biomedical applications.

Collagen stabilization using ionic liquid functionalised cerium oxide nanoparticle

Nanoparticles owing to their size have a substantial influence on the biological behavior of collagen, thereby opening new channels to unfold the propensity of nanoparticles in terms of collagen stabilization. The present study aims to synthesize and characterize cerium oxide nanoparticles and to investigate their crosslinking efficiency on collagen. Cerium oxide nanoparticles, known biocatalysts, form an effective oxidation system due to their variable oxidation state. Ionic liquid functionalizes cerium oxide nanoparticles (IL-CONP) have been synthesis by the sonication method and characterizes using techniques such as Dynamic Light Scattering, and X-ray Diffraction. The hydrodynamic diameter, Zeta potential and polydispersity index of nanoparticles is 192.3 ± 2.14 nm, −13.76 ± 1.5 mV and 0.387, respectively. Changes in the secondary structure of collagen upon treatment with increasing concentration of IL-CONP indicate conformational modifications at the molecular level. Differential scanning calorimetry studies on rat tail tendon collagen fibers with IL-CONP indicate an increase in thermal stability of collagen from 61 to 87 °C. Thus, cerium oxide-based nanoparticle crosslinking invokes a considerable array of interest as a potential crosslinking agent for collagen.

Structure Restoration of Thermally Denatured Collagen by Ultrahigh Pressure Treatment

Thermal denaturation is the primary reason for the loss of excellent performance for collagen-based materials. It is particularly urgent to explore an effective structure restoration strategy for thermally denatured collagen. In this study, three different degrees of collagen thermal denaturation models were constructed; the effects of ultrahigh pressure treatments of varying intensities on the structure and properties of partially thermal denatured collagen were investigated. Those results showed that ultrahigh pressure partially restores the triple helix conformation and the α helix of low thermally denatured collagen molecules and moderately restores collagen thermal stability. However, this recovery is not a “perfect” structural restoration; it cannot effectively restore the fibrotic ability of collagen which leads to a decrease in the fiber diameter of the product and an increase in the storage modulus of the product gel. As the degree of thermal denaturation of collagen increases, the ultrahigh pressure recovery effect is gradually lost. For moderate thermally denatured collagen, ultrahigh pressure treatments only recover a small amount of its triple helical conformation. For high thermally denatured collagen, ultrahigh pressure treatments exacerbate the destruction of the three-dimensional structure of collagen molecules, thereby losing the order of molecular structure and the capability of fibrosis self-assembly.

Sterics and Stereoelectronics in Aza-Glycine: Impact of Aza-Glycine Preorganization in Triple Helical Collagen.

We previously demonstrated aza-glycine can serve as a reliable and general replacement of glycine in triple helical collagen. Aza-glycine considerably improves collagen's thermal stability and self-assembly properties without changing collagen's natural triple helix topology. We provided a firm structural basis for this stabilization with an atomic resolution crystal structure of collagen containing aza-glycine which revealed new cross-strand H-bonds within the triple helix interior. Here, using computational analysis, we show the enhanced properties of aza-glycine is a result of noncovalent forces (H-bonding) and backbone preorganization. The interplay of steric repulsion and hyperconjugative interactions in aza-glycine's backbone directly preorganizes the collagen peptide main-chain (φ, ψ) dihedrals for triple helical assembly. The synergy of multiple structural and electronic changes which originate at the residue level in the aza-glycine backbone and culminate at the macromolecular level of the triple helix lead to increased stability and faster refolding of collagen peptides containing aza-glycine.

The effect of halloysite nanotubes dispersions on vegetable-tanned leather thermal stability

Leather artefacts in historical collections and old leather bookbindings in archives and libraries frequently show a variety of visible changes which alter their appearance and ability to be used, such as powdery surface, weakened structure, complete or partial loss of the grain layer. However, historical leather stabilization and consolidation yet represent a challenging problem for conservators due to the sensitivity of collagen to the action of most of the conservation and restoration formulations currently used. In this paper we report our recent research concerning the development of halloysite nanoparticles dispersions for enhancing the hydrothermal stability of weakened historical vegetable tanned leather. The effects of halloysite nanotubes (HNTs) dispersed in various aqueous and non-aqueous mediums on the thermal stability of collagen in historical leather were tested using thermal microscopy, thermogravimetry and micro differential scanning calorimetry. Unilateral nuclear magnetic resonance was used to evaluate the changes in water dynamics due to collagen matrix interaction with dispersion media. The treated samples were also characterized using infrared spectroscopy and scanning electron microscopy. The analytical assessment confirmed the leather thermal stability increase and a partial recovery of fibres cohesion, thus validating the use of HNT dispersions as a basic tool for the preservation of collagen-based materials. Further functionalisation and encapsulation using antimicrobials, fungicides and pH adjustment nanoparticles will deliver novel and more durable HNTs-based conservation treatments.

Improving catalase stability by its immobilization on grass carp (Ctenopharyngodon idella) scale collagen self-assembly films.

We extracted collagen (CL) from the scales of Ctenopharyngodon idella and fabricated a CL self-assembly film. Catalase (CAT) was immobilized on the films using cross-linking, adsorption, and embedding methods. The activity and operational stability of immobilized CAT were investigated, along with the influence of the concentration of glutaraldehyde (GTA) and of the initial concentration of embedded CAT. The results showed that the CL triple helix remained intact. Differential scanning calorimetry data showed that the thermal stability of CL was significantly improved by neutral salt-induced self-aggregation and GTA cross-linking. The immobilized enzyme had high activity and good operational stability. When the enzyme concentration reached 0.5 mg/mL, immobilized enzyme activity of grass carp scales reached a maximum of 2596 U/g, and after twenty-two uses, enzyme activity remained above 50%, and it could be reused >45 times (CAT = 0.5 mg/mL, GTA = 5%, Temperature = 30 °C, pH = 7). Moreover, the optimum temperature and pH of immobilized CAT were 35 °C and 7, respectively, while the same for free CAT was 30 °C and 7, respectively. This indicated that immobilization of CAT has a protective effect.

Feasibility Study of Tissue Transglutaminase for Self-Catalytic Cross-Linking of Self-Assembled Collagen Fibril Hydrogel and Its Promising Application in Wound Healing Promotion.

Collagen-based bio-hydrogels are undoubtedly a hot spot in the development of biological dressings for wound healing promotion. Herein, glutamine transaminase (TGase), a biological nontoxic cross-linker with high specific activity and reaction rate under mild conditions, was utilized for the self-catalytic cross-linking of the regenerated collagen (COL) fibril hydrogel fabricated through a molecular self-assembly method. The results showed that the natural triple helical conformation of COL remained completely integrated after self-catalytic cross-linking TGase, which was definitively the fundamental for maintaining its superior bioactivity. It was worth noting that TGase could promote the self-assembly process of COL building blocks into a higher order D-period cross-striated structure. Also, the reconstructed TGase cross-linked COL fibrils exhibited a higher degree of interfiber entanglements with more straight and longer fibrils. Meanwhile, the thermal stability of COL was significantly improved after introducing TGase. Besides, the cytocompatibility analysis suggested that the regenerated COL fibril hydrogel showed excellent cell growth activity and proliferation ability when the dosage of TGase is less than 40 U/g. Further, animal experiments indicated that the targeted COL fibril hydrogel could significantly promote skin wound healing, exhibiting better capacity of skin tissue for regeneration than the COL hydrogel untreated as expected. Therefore, the reconstructed TGase cross-linked COL fibril hydrogel could serve as a novel soft material for wound healing promotion.

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.