Abstract
Enzymatic cross-linking of the bone collagen is important to resist to crack growth and to increased flexural strength. In the present study, we proposed a new method for assessment of enzymatic cross-link based on Fourier transform infrared (FTIR) microspectroscopy that takes into account secondary structure of type I collagen. Briefly, femurs were collected from sham or ovariectomized mice and subjected either to high-performance liquid chromatography—mass spectrometry or embedded in polymethylmethacrylate, cut and analyzed by FTIR microspectroscopy. FTIR acquisition was recorded before and after ultraviolet (UV) exposure or acid treatment. In addition, femurs from a second animal study were used to compare gene expression of Plod2 and Lox enzymes and enzymatic cross-links determined by FTIR microspectroscopy. We evidenced here that intensities and areas of subbands located at ~1660, ~1680, and ~1690 cm−1 were positively and significantly associated with the concentration of pyridinoline (PYD), deoxypyridinoline, or immature dihydroxylysinonorleucine/hydroxylysinonorleucine cross-links. Seventy-two hours exposure to UV light significantly reduced by ~86% and ~89% the intensity and area of the ~1660 cm−1 subband. Similarly, 24 h of acid treatment significantly reduced by 78% and 76% the intensity and area of the ~1690 cm−1 subband. Plod2 and Lox expression were also positively associated to the signal of the ~1660 and ~1690 cm−1 subbands. In conclusion, our study provided a new method for decomposing the amide I envelope of bone section that positively correlates with PYD and immature collagen cross-links. This method allows for investigation of tissue distribution of enzymatic cross-links in bone section.
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Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Acknowledgements
We are thankful to Prof Peter Gardner (University of Manchester) for supplying the Mie scattering correction routine for Matlab. We are also grateful to the institutional animal lab SCAHU, SFR ICAT 4208, Univ Angers, for their help with animal care.
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This project was funded by an institutional grant from the University of Angers.
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AM: Investigation, Formal analysis and GM: Conceptualization, Investigation, Formal analysis, Writing—Review & Editing, Supervision, Funding acquisition, Data curation.
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Aleksandra Mieczkowska and Guillaume Mabilleau have no competing interests to declare that are relevant to the content of this article.
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All procedures were carried out in accordance with the European Union Directive 2010/63/EU for animal experiments and were approved by the regional ethical committee for animal use (authorization CEEA-PdL06-01740.01).
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Mieczkowska, A., Mabilleau, G. Validation of Fourier Transform Infrared Microspectroscopy for the Evaluation of Enzymatic Cross-Linking of Bone Collagen. Calcif Tissue Int 113, 344–353 (2023). https://doi.org/10.1007/s00223-023-01105-z
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DOI: https://doi.org/10.1007/s00223-023-01105-z