Quick and easy sample preparation without resin embedding for the bone quality assessment of fresh calcified bone using fourier transform infrared imaging.

Hiromi Kimura-Suda,Norimasa Iwasaki,Teppei Ito,Masahiro Ota,Kyosuke Kanazawa,Tomohiro Shimizu,Masahiko Takahata,Joseph M Wallace

doi:10.1371/journal.pone.0189650

Hiromi Kimura-Suda, Norimasa Iwasaki + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0189650

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Abstract

Fourier transform infrared (FTIR) imaging is a powerful tool for the assessment of bone quality; however, it requires the preparation of thin bone sections. Conventional poly(methyl methacrylate) (PMMA) embedding for the preparation of sections takes more than two weeks and causes denaturation of the bone. Development of a quick and easy sample preparation technique without denaturation is needed for accurate clinical evaluation of fresh calcified bone using FTIR imaging. Frozen sectioning allows the quick and easy preparation of thin sections without denaturation, but it requires a substrate with good chemical resistance and improved heat shock resistance. Polypropylene (PP) film afforded both good chemical resistance and greater heat shock resistance, and the 4-μm-thick PP film coated with glue was thin enough for the IR beam to pass through it, while the optical anisotropy of infrared bands overlapping with PO43- band was negligible. The bone quality of femoral thin sections prepared by the conventional PMMA embedding and sectioning procedure (RESIN-S) or the newly developed frozen sectioning procedure (FROZEN-S) was evaluated by FTIR imaging. The mineral-to-matrix ratio and crystallinity in the RESIN-S sections were higher than those in the FROZEN-S sections, whereas the carbonate-to-phosphate ratio in the RESIN-S sections was lower than that in the FROZEN-S sections. In RESIN-S, the increased mineral-to-matrix ratio could be caused by dehydration, and the increased crystallinity and decreased carbonate-to-phosphate ratio might be consequence of dissolution of bone mineral during PMMA embedding. Therefore, the combined use of PP film coated with glue and the frozen sectioning procedure without denaturation appears well suited to the assessment of the bone quality of fresh calcified bone using FTIR imaging.

Highlights

Osteoporosis is a bone disease characterized by increased skeletal fragility due to a reduction in bone strength
We characterized the PP film coated with glue (PP film + glue) and fresh bone mounted on the PP film using Fourier transform infrared (FTIR) imaging with a polarizer and aimed to demonstrate that the combined use of the thin PP film coated with glue as a substrate and the frozen sectioning procedure without denaturation of proteins by solvent or resin was suited to the assessment of the bone quality of fresh calcified bone using FTIR imaging in transmission mode, and that the newly developed sample preparation procedure afforded a powerful technique for clinical evaluation due to reductions in time and cost
The combined use of PP film + glue and the frozen sectioning procedure affords a powerful technique for FTIR imaging in a clinical setting due to reductions in time and cost. This is the first study on the assessment of the bone quality of fresh calcified bone using FTIR imaging in transmission mode

Summary

Introduction

Osteoporosis is a bone disease characterized by increased skeletal fragility due to a reduction in bone strength. The PMMA embedding procedure for bone takes more than two weeks, which hampers the assessment of bone quality using FTIR imaging as a clinical measurement. The assessment of bone quality using FTIR imaging requires the use of a frozen sectioning procedure that does not require fixation in order to achieve more accurate measurements. Such a frozen sectioning procedure without fixation maybe shorten the time and cost required in comparison to the PMMA embedding and sectioning procedure

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