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Tracking calcification in tissue-engineered bone using synchrotron micro-FTIR and SEM

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Abstract

One novel tissue engineering approach to mimic in vivo bone formation is the use of aggregate or micromass cultures. Various qualitative and quantitative techniques, such as histochemical staining, protein assay kits and RT-PCR, have been used previously on cellular aggregate studies to investigate how these intricate arrangements lead to mature bone tissue. However, these techniques struggle to reveal spatial and temporal distribution of proliferation and mineralization simultaneously. Synchrotron-based Fourier transform infrared microspectroscopy (micro-FTIR) offers a unique insight at the molecular scale by coupling high IR sensitivity to organic matter with the high spatial resolution allowed by diffraction limited SR microbeam. This study is set to investigate the effects of culture duration and aggregate size on the dynamics and spatial distribution of calcification in engineered bone aggregates by a combination of micro-FTIR and scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDX). A murine bone cell line has been used, and small/large bone aggregates have been induced using different chemically treated culture substrates. Our findings suggest that bone cell aggregate culturing can greatly increase levels of mineralization over short culture periods. The size of the aggregates influences mineralisation rates with larger aggregates mineralizing at a faster rate than their smaller counterparts. The micro-FTIR mapping has demonstrated that mineralization in the larger aggregates initiated from the periphery and spread to the centre, whilst the smaller aggregates have more minerals in the centre at the early stage and deposited more in the periphery after further culturing, implying that aggregate size influences calcification distribution and development over time. SEM/EDX data correlates well with the micro-FTIR results for the total mineral content. Thus, synchrotron-based micro-FTIR can accurately track mineralization process/mechanism in the engineered bone.

FTIR mapping images of PO4 regions showing the big intensity and distribution difference between small and large aggregates cultured for 72 h.

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Acknowledgments

The authors are grateful for the financial support from NHS Orthopaedic Charity funding (001502) and Diamond light source for beamtime on B22 (Diamond Light Source fund SM8637).

Conflict of interest

There is no conflict of interest of the study.

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Authors and Affiliations

  1. Institute for Science and Technology in Medicine, School of Medicine, Keele University, Stoke-on-Trent, ST4 7QB, UK

    Anthony J. Deegan, Sandeep Konduru & Ying Yang

  2. Diamond Light Source, Harwell Science and Innovation Campus, Chilton-Didcot, Oxon, OX11 0DE, UK

    Gianfelice Cinque & Katia Wehbe

  3. Orthopaedics Department, University Hospital of Staffordshire, Stoke-on-Trent, ST4 6QG, UK

    Sandeep Konduru

Authors
  1. Anthony J. Deegan
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  2. Gianfelice Cinque
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  3. Katia Wehbe
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  4. Sandeep Konduru
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  5. Ying Yang
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Correspondence to Ying Yang.

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Deegan, A.J., Cinque, G., Wehbe, K. et al. Tracking calcification in tissue-engineered bone using synchrotron micro-FTIR and SEM. Anal Bioanal Chem 407, 1097–1105 (2015). https://doi.org/10.1007/s00216-014-8316-4

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  • DOI: https://doi.org/10.1007/s00216-014-8316-4

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