Effect of short-term formaldehyde fixation on Raman spectral parameters of bone quality

Abstract

Medical knowledge of the skeleton including its structures has improved constantly over the past decades. Advanced imaging methods, mechanical testing and optical techniques have revealed insights into bone architecture and composition. Most of these advancements were possible due to the ex vivo investigation of biological tissues. Investigations of fresh tissue are generally preferred over preserved or fixed samples. However, chemical fixation is sometimes inevitable due to histological procedures or logistical reasons. The aim of this study was to investigate whether short-term chemical fixation with formaldehyde affects bone quality parameters obtained from Raman spectroscopy and if these effects last for intermediate sample storage of several hours. As formaldehyde induces cross-links to the organic components in bone tissue, we hypothesized that collagen-related parameters are particularly affected. Femurs of eight 17-week-old C57BL/6 mice were extracted and divided into two groups (N ¼ 8∕group). Samples of the first group were fixed by immersion in 4% formaldehyde (PFA-solution) for 12 h at 4°C (fixed group) while samples of the second group were left untreated (unfixed group). Raman spectroscopy was performed, and repeated after 4 h, to assess whether intermediate storage time influenced the obtained results. Based on resultant spectra, mineral-to-matrix ratio, carbonate-to-phosphate ratio, carbonate-to-amide I ratio, mineral crystallinity and collagen maturity were determined. Carbonate-to-phosphate ratio was the only parameter showing a significant difference between the first and the subsequent measurements. For both groups, ratios showed a decrease in carbonate substitution compared to the first measurement (percentage decrease: 3.1% in fixed, 4.7% in unfixed). Collagen maturity of samples, which were short-term fixed with formaldehyde, was significantly lower than of fresh, unfixed samples (percentage difference: 3.8%). Our study shows that Raman spectroscopy is able to detect changes in collagen structure initiated by formaldehyde and that changes in short-term fixed samples are minimally influencing bone material properties measured with Raman spectroscopy.

Effect of two (short-term) storage methods on load to failure testing of murine bone tissue

“…A close look at the literature shows that formalin causes changes in the organic matrix with irreversible alteration of material and mechanical properties 9,10 . Even immediate impairment (after 12 h) of biomechanical properties is described when using formaldehyde 19 or ethanol and therefore both substances are not recommended if material characteristics close to the in-vivo conditions are of interest 9 .…”

High-Resolution Chemical Mapping and Microbial Identification of Rhizosphere using Correlative Microscopy

“…46-49 Widely used fixatives such as glutaraldehyde and ethanol can cause significant changes to the Raman spectra of bacteria indicating chemical alterations, whereas formaldehyde and sodiumazide were better at preserving spectral features. 48 As rhizosphere consists of soil, root, bacteria and other organic and inorganic material, various components can be studied, and it is not trivial to adapt a single fixative method. We have used ethanol instead of acetone, to minimize the membrane damages by acetone.…”

Towards refining Raman spectroscopy-based assessment of bone composition

“…Here, human bone (exposed to formalin) and bovine bone (not exposed to formalin) display comparable Raman spectral characteristics in the context of the overlap between the organic and inorganic components. While long-term storage of bone in formaldehyde may induce various compositional alterations 28 , Raman bone quality parameters remain largely unaffected by short-term (~ 12 h) exposure to 4% paraformaldehyde although 3-4% decrease in collagen maturity (ratio between amide I sub-components at 1660 cm −1 and 1690 cm −1 ) has been reported 29 .…”