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 = 8 / group). Samples of the first group were fixed by immersion in 4% formaldehyde (PFA-solution) for 12h at 4°C (fixed group) while samples of the second group were left untreated (unfixed group). Raman spectroscopy was performed, and repeated after 4h, 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.
Highlights
Raman spectroscopy has been established as a powerful tool for bone characterization by means of identifying molecule-specific vibrational modes of the sample’s chemical components.[1,2] In contrast to biomechanical testing, Raman spectroscopy requires only little sample preparation and allows nondestructive examination of bone material properties
Our aim is to investigate whether short-term chemical fixation with formaldehyde has an influence on bone quality parameters obtained from Raman spectroscopy and to show if Raman spectroscopy is sufficiently sensitive to detect these alterations
We investigated whether short-term chemical fixation with formaldehyde has an influence on bone quality parameters obtained from Raman spectroscopy
Summary
Raman spectroscopy has been established as a powerful tool for bone characterization by means of identifying molecule-specific vibrational modes of the sample’s chemical components.[1,2] In contrast to biomechanical testing, Raman spectroscopy requires only little sample preparation and allows nondestructive examination of bone material properties. Chemical fixation is a commonly performed procedure to hinder degenerative processes and to preserve the ultrastructure of biological tissue. The choice of the fixation protocol is not a trivial one and is essential for the outcome of an experiment, as the fixation process might alter certain tissue properties.[10] The most commonly used fixative is formaldehyde. This agent preserves biological tissue by cross-linking proteins present in the sample. A great number of different fixation protocols applying formaldehyde-solutions are found. Weeks of fixation are common for intermediate storage between measurements, and months and years of fixation are performed for embalmment and long-term preservation.[13]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have