Quantification of immature and mature collagen crosslinks by liquid chromatography–electrospray ionization mass spectrometry in connective tissues

Gineyts, Évelyne; Borel, Olivier; Chapurlat, R.; Garnero, Patrick

doi:10.1016/j.jchromb.2010.03.039

Cited by 47 publications

(46 citation statements)

References 26 publications

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“…2). These values were similar to reported values in human and bovine femurs of various ages, which ranged from 0.1-0.4 mol/mol for HP-to-collagen and 0.03-0.08 mol/mol for LP-to-collagen [29-31], and were not significantly different from previously reported values for the same bovine femur tissue of the same age used in this study ( p > 0.05; [21]). Taken together, these results demonstrate that the LC-MS/MS method in our study was sensitive to HP and LP crosslinks.…”

Section: Resultssupporting

confidence: 91%

“…Samples were prepared following the method of Gineyts et al [21]. Briefly, freshly excised chick tendon tissues, including an adult porcine Achilles tendon control group, were minced, washed in dH 2 O, and lyophilized for 1 week to obtain dry mass.…”

Section: Methodsmentioning

confidence: 99%

“…Marrow and soft tissue were removed, and samples were immersed in liquid nitrogen for 10 min and pulverized. Bone fragments were then demineralized in 0.5 M EDTA and 50 mM Tris (pH 7.4) for 21 days at 4° C with daily solution changes [21]. Demineralized bone samples were subsequently processed in an identical manner as tendon samples for LC-MS/MS analysis.…”

Section: Methodsmentioning

confidence: 99%

“…A triple quadrupole linear ion trap mass spectrometer (3200 QTRAP, AB SCIEX, Foster City, CA) coupled to a binary pump HPLC (1200 Series, Agilent, Santa Clara, CA) was used for the identification and quantification of HP, LP, pyridoxine and hydroxyproline following the method of Gineyts et al [21] with minor modifications. Prior to sample analysis, MS-specific parameters were optimized for each target analyte to identify the multiple reaction monitoring transition (precursor/product fragment ion pair) with the highest intensity under positive mode.…”

Section: Methodsmentioning

confidence: 99%

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Lysyl oxidase-mediated collagen crosslinks may be assessed as markers of functional properties of tendon tissue formation

et al. 2014

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Mechanical property elaboration of engineered tissues is often assumed on the basis of gene and protein characterizations, rather than mechanical testing. However, we recently demonstrated mechanical properties are not consistently correlated with matrix content and organization during embryonic tissue development. Based on this, mechanical properties should be assessed independently during natural or engineered tissue formation. Unfortunately, mechanical testing is destructive, and thus alternative means of assessing these properties are desirable. In this study, we examined lysyl oxidase (LOX)-mediated crosslinks as markers for mechanical properties during embryonic tendon formation and the potential to detect them non-destructively. We used tandem mass spectrometry (LC-MS/MS) to quantify changes in hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP) crosslink density in embryonic chick tendon as a function of developmental stage. In addition, we assessed a multiphoton imaging approach that exploits the natural fluorescence of HP and LP. With both techniques, we quantified crosslink density in normal and LOX-inhibited tendons, and correlated measurements with mechanical properties. HP and LP crosslink density varied as a function of developmental stage, with HP-to-dry mass ratio correlating highly to elastic modulus, even when enzymatic crosslink formation was inhibited. Multiphoton optical imaging corroborated LC-MS/MS data, identifying significant reductions in crosslink density from LOX inhibition. Taken together, crosslink density may be useful as a marker of tissue mechanical properties that could be assessed with imaging non-destructively and perhaps non-invasively. These outcomes could have significant scientific and clinical implications, enabling continuous and long-term monitoring of mechanical properties of collagen-crosslinked tissues or engineered constructs.

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Section: Resultssupporting

confidence: 91%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Lysyl oxidase-mediated collagen crosslinks may be assessed as markers of functional properties of tendon tissue formation

et al. 2014

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“…The sample was centrifuged at 12,000 g for 15 min at 4 1/4C and the clear aqueous phase was transferred to an Agilent clear screw top micro sampling LC/MS vial (P/N 5184-3550. Agilent Tech, Santa Clara, CA) for the UPLC/MS/MS assay of collagen crosslinks [42,43]. The sample was maintained at 4 "C in the autosampler and a volume of 5 fiL was loaded onto an ACQUITY UPLC HHS C18 column (2.1 mm inner diameter x 100 mm with 1.8 ^m particles.…”

Section: Ultra Performance Liquid Chromatography-tandem Mass Spectrommentioning

confidence: 99%

Cervical Collagen Network Remodeling in Normal Pregnancy and Disrupted Parturition in Antxr2 Deficient Mice

Yoshida

Reeves²,

Vink

et al. 2014

Journal of Biomechanical Engineering

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The remodeling of the cervix from a rigid barrier into a compliant structure, which dilates to allow for delivery, is a critical process for a successful pregnancy. Changes in the mechanical properties of cervical tissue during remodeling are hypothesized to be related to the types of collagen crosslinks within the tissue. To further understand normal and abnormal cervical remodeling, we quantify the material properties and collagen crosslink density of cervical tissue throughout pregnancy from normal wild-type and Anthrax Toxin Receptor 2 knockout (Antxr2-/-) mice. Antxr2-/- females are known to have a parturition defect, in part, due to an excessive accumulation of extracellular matrix proteins in the cervix, particularly collagen. In this study, we determined the mechanical properties in gestation-timed cervical samples by osmotic loading and measured the density of mature collagen crosslink, pyridinoline (PYD), by liquid chromatography tandem mass spectrometry (LC-MSMS). The equilibrium material response of the tissue to loading was investigated using a hyperelastic material model where the stresses in the material are balanced by the osmotic swelling tendencies of the glycosaminoglycans and the tensile restoring forces of a randomly-oriented crosslinked collagen fiber network. This study shows that the swelling response of the cervical tissue increased with decreasing PYD density in normal remodeling. In the Antxr2-/- mice, there was no significant increase in swelling volume or significant decrease in crosslink density with advancing gestation. By comparing the ECM-mechanical response relationships in normal and disrupted parturition mouse models this study shows that a reduction of collagen crosslink density is related to cervical softening and contributes to the cervical remodeling process.

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Bone Fracture Toughness and Strength Correlate With Collagen Cross-Link Maturity in a Dose-Controlled Lathyrism Mouse Model

McNerny

Gong

Morris

et al. 2014

Journal of Bone and Mineral Research

121

117

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Collagen cross-linking is altered in many diseases of bone, and enzymatic collagen cross-links are important to bone quality as evidenced by losses of strength following lysyl oxidase inhibition (lathyrism). We hypothesized that cross-links also contribute directly to bone fracture toughness. A mouse model of lathyrism using subcutaneous injection of up to 500mg/kg β-aminopropionitrile (BAPN) was developed and characterized (60 animals across 4 dosage groups). Three weeks of 150 or 350 mg/kg BAPN treatment in young growing mice significantly reduced cortical bone fracture toughness, strength, and pyridinoline cross-link content. Ratios reflecting relative cross-link maturity were positive regressors of fracture toughness (HP/[DHLNL+HLNL] r2=0.208, p<0.05; [HP+LP]/[DHNL+HLNL] r2=0.196, p<0.1), whereas quantities of mature pyridinoline cross-links were significant positive regressors of tissue strength (lysyl pyridinoline r2=0.159, p=0.014; hydroxylysyl pyridinoline r2=0.112, p<0.05). Immature and pyrrole cross-links, which were not significantly reduced by BAPN, did not correlate with mechanical properties. The effect of BAPN treatment on mechanical properties was dose specific, with the greatest impact found at the intermediate (350mg/kg) dose. Calcein labeling was used to define locations of new bone formation, allowing for the identification of regions of normally cross-linked (preexisting) and BAPN treated (newly formed, cross-link-deficient) bone. Raman spectroscopy revealed spatial differences due to relative tissue age and effects of cross-link inhibition. Newly deposited tissues had lower mineral/matrix, carbonate/phosphate and Amide I cross-link (matrix maturity) ratios compared to preexisting tissues. BAPN treatment did not affect mineral measures, but significantly increased the cross-link (matrix maturity) ratio compared to newly formed control tissue. Our study reveals that spatially localized effects of short term BAPN cross-link inhibition can alter the whole bone collagen cross-link profile to a measureable degree, and this cross-link profile correlates with bone fracture toughness and strength. Thus, cross-link profile perturbations associated with bone disease may provide insight into bone mechanical quality and fracture risk.

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Quantification of immature and mature collagen crosslinks by liquid chromatography–electrospray ionization mass spectrometry in connective tissues

Cited by 47 publications

References 26 publications

Lysyl oxidase-mediated collagen crosslinks may be assessed as markers of functional properties of tendon tissue formation

Lysyl oxidase-mediated collagen crosslinks may be assessed as markers of functional properties of tendon tissue formation

Cervical Collagen Network Remodeling in Normal Pregnancy and Disrupted Parturition in Antxr2 Deficient Mice

Bone Fracture Toughness and Strength Correlate With Collagen Cross-Link Maturity in a Dose-Controlled Lathyrism Mouse Model

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