Bone Molecular Modifications Induced by Diagenesis Followed-Up for 12 Months

Abstract

After death, diagenesis takes place. Numerous processes occur concomitantly, which makes it difficult to identify the diagenetic processes. The diagenetic processes refer to all processes (chemical or physical) that modify the skeletal remains. These processes are highly variable depending on the environmental factors (weather, temperature, age, sex, etc.), especially in the early stages. Numerous studies have evaluated bone diagenetic processes over long timescales (~millions of years), but fewer have been done over short timescales (between days and thousands of years). The objective of the study is to assess the early stages of diagenetic processes by Raman microspectroscopy over 12 months. The mineral and organic matrix modifications are monitored through physicochemical parameters. Ribs from six humans were buried in soil. The modifications of bone composition were followed by Raman spectroscopy each month. The decrease in the mineral/organic ratio and carbonate type-B content and the increase in crystallinity reveal that minerals undergo dissolution-recrystallization. The decrease in collagen cross-linking indicates that collagen hydrolysis induces the fragmentation of collagen fibres over 12 months.

Keywords: bone diagenesis; collagen; cross-links; mineral; raman spectroscopy; taphonomy.

Figure 1

Description of the protocol of preparation of the rib for Raman analysis. This protocol is repeated 12 times for 12 months for each rib. (a) Left panel: schema of a rib before the cutting procedure at M0. The dashed lines represent the limits of the cut. Right panel: rib after cutting procedure. The wax protects the outer proximal surface. (b) Zoom on the sample analysed by Raman microspectroscopy. The proximal transversal surface is protected by the wax. The distal transversal surface is not in contact with the wax and is analysed by Raman microspectroscopy. (c) Representation of a plastic bin with soil and one rib. One rib is represented to simplify the representation. It shows that a new 5 mm thick section of the same rib is cut each month and analysed by Raman. To do the cutting procedure, the rib is exhumed and reburied each month. (d) Piece of rib after the cutting procedure. The visible side corresponds to the distal transversal surface. (e) Optical image of the piece of rib under Raman microscope. The visible side corresponds to the distal transversal surface analysed by Raman microspectroscopy. The insets (f,g) show examples of each type of bone and the area analysed by Raman microspectroscopy. (f) Example of trabecular bone analysed. (g) Example of cortical bone showing periost, secondary osteon and interstitial bone analysed. The red dot represents the localization of the analysis. For each type of bone, 10 spectra were acquired at 10 different locations.

Figure 2

(a) Plastic bin with lid to protect from scavengers and rain; (b) plastic bin filled with clay soil typical of northern France carrying the rib. The rib is covered by 1 cm of soil; (c) ribs nos. 1 and 4 after 1 month of burial time.

Figure 3

(a) Averaged outdoor temperature (°C) per month during 12 months for each body. (b) Averaged RH (%) during the 12 months for each body. The month M0 is different depending on the body. For body 1, M0 = September 2013; body 2, M0 = November 2013; bodies 3 and 4, M0 = December 2013; bodies 5 and 6, M0 = April 2014.

Figure 4

Comparison of averaged Raman spectra at baseline (M0) and after 12 months (M12). Each Raman spectrum is averaged over the 6 bodies and both ribs. The spectrum M12 is shifted along the y-axis to facilitate the comparison of M0 and M12. The shift does not have a biological or instrumental significance.

Figure 5

Evolution of the 5 physicochemical variables (PPVs) as a function of burial time in months. One boxplot represents the variations in one parameter at one month. Each circle represents the mean value of a parameter of one body. The red cross represents the mean value of a parameter over the 6 bodies. The horizontal line represents the median value over the 6 bodies. (A) Mineral/organic ratio; (B) carbonatation type-B; (C) crystallinity; (D) hydroxyproline/proline ratio; (E) collagen cross-links.