DEGRADATION OF ORGANIC AND MINERAL PHASES IN BURIED HUMAN REMAINS: THE EARTH SCIENCES ANALYTICAL CHARACTERIZATION

The thesis focuses on the characterization of the alteration of the mineral and organic phases, investigated with different approaches, of human bone tissue from different burial contexts, with ages spanning from the Late Roman period to our time. This topic is very important in paleontological, archaeo-anthropological and forensic contexts in order to understand the taphonomic agents and then to provide biological data as possibly to discern human behavior in ancient funerary as well as in recent forensic contexts. It is well-known that peri and post mortem events may leave marks that have to be interpreted in the light of the state of the conservation or degradation of the skeletal remains. In fact, physical anthropologists are frequently required to date human bone remains, in order to recognize if osteological samples have an archaeological, historic or forensic interest. The determination of post mortem interval (PMI), the time elapsed between the death and the discovery of the corpse or skeletal remains, is extremely difficult to evaluate in absence of direct chronometric dating (e.g. C14), since bones might undergo several alterations, both structural and chemical, depending on the environment in which they deposited in. Because of bone tissue is an intimate association of mineral (carbonate-hydroxyapatite) and organic components (collagen) arranged in an ordinary structure, different levels of degradation are possible. Over time post mortem degradation is dominated by loss of structural collagen by collagenolytic enzymes, which caused a rapid swelling and hydrolysis of the protein fibers. Collagen dissolution is generally accompanied by the alteration of mineral crystals, which are vulnerable to diagenetic changes due to their small size. During diagenesis, the protein can be totally or partially removed and can replaced by inorganic precipitates, the most common beign hydroxyapatite, which in the process is subjected to recrystallization, ion exchange and substitution. As consequence, when depositional conditions are favorable for bone preservation, the mineral crystallinity increase, the porosity and chemical composition change. The quality and the assessement of organic and inorganic phase, can act positively or negatively both on bone mechanical properties in live, both on decomposition process after death, reducing or accelerating it. Several studies were performed to better understand the taphonomy of bone material during burial time. It appears that bone degradation depends on a wide range of environmental interactions, including biological, chemical and physical factors. These include: average temperature and humidity, microbilological composition and activity, soil chemistry (mineralogy and pH) and permeability, mechanical pressure and other numerous factors. Different type of bone degradation are observable at different scale of observation; particularly, in this study, bone preservation was investigated at macroscopic, biomolecular, microscopic, ultramicroscopic and chemical scale. The aim of this research is thus to further describe the impact of environmental conditions on bone preservation, and the effect of time, by applying and comparing the results from different analitical techniques. For this study 40 human skeletons of adult individuals from four different dated burial location in the Milan area were analyzed. The first one is a necropolis dated to the Late Roman age (3th-4th century AD), the second one is a 17th century AD mass grave, the third one is an ossuary contanining bones dated between 15th and 18th century AD, and the last one is a modern cemetery. The macroscopic analysis evaluated the general appearance of the remains and their state of preservation, through the observation of specific macroscopic parameters and morphological characteristics. The Luminol test, a fast and inexpensive method developed to detect blood traces, was performed to investigate the presence of haemoglobin preserved in bone. The histological analysis, conducted on calcified thin sections, considered the presence or absence of tunneling and bioerosion, in accordance to the Oxford Histological Index (OHI). Also, to evaluate the state of preservation of the organic component, primarily collagen, the samples were decalcified and stained with Hematoxylin and Eosin. Because of the lack of literature in this field, we created a new Decalcified Histological Index (DHI). Both calcified and decalcified bone thin sections were observed in transmitted and polarized light microscopy, in order to test the optical properties of structural components. Scanning electron microscope coupled with energy-dispersive X-ray spectroscopy (SEM-EDS) was used to evaluate exogenous chemical elements and minerals, adsorbed from burial environment, and histological changes, as well as recrystallization, tunnelling and fractures, due to fungal or bacteria action. X-ray micro-computed tomography of bone sections was performed at the SYRMEP beamline of the third-generation Synchrotron Light Laboratory (ELETTRA) located in Trieste (Italy), with the purpose to evaluate and quantify the preservation of bone structure, such as canals and lacunae, and the porosity changes due to diagenetic process. Fourier transform infrared spectrometry (FT-IR) and micro-spectrometry (mFTIR) were performed at Simon Fraser University (Burnaby) in Canada to investigate the preservation of both mineral and organic phases. Finally, 23 skeletons from the archaeological site of Travo (PC), dating from 7th-8th century AD, and their burial ground sediments were sampled and analyzed. Macroscopic, microscopic and chemical analyses were performed on bones to evaluate the tissue preservation state at different scales; the soil samples collected from the graves were characterized for color, particle size distribution, pH, organic carbon and calcium carbonate concentration. This study shows that macroscopic, biomolecular, microscopic, ultramicroscopic and chemical alterations follow independent paths that affect the bone preservation at different scales of observation. Therefore, the estimation of the diagenetic process cannot be limited to the macroscopic aspect of the bone tissue but must take into account biomolecular, microscopic and chemical alterations, since these may have affected the bone tissue differently at different scale. Bone degradation can be employed to estimate the post mortem interval, or to reconstruct the burial environment of human remains. As long as the evaluation of taphonomic alterations is performed at different scales with different ad hoc methodologies. In fact, age and environment can play an equal role on the degradation of organic and mineral phases, producing different effects on bone conservation at different levels.

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