During exposure to high temperatures and fire, the chemical composition and structural properties of human bone are altered.

Imaizumi et al. as well as Van Hoensel et al. (2019) measured the mass loss of the bones during the (controlled) heating process (Fig. 3) [68, 69]. This will be the terminus a quo to describe the HI changes, since three stages with chemical changes can be clearly observed: (i) the loss of water below 250 °C (I), (ii) a decline in organic content between 200 and 600 °C (II), and (iii) changes of the bone mineral above 700 °C (III).

A graphical representation of the weight loss of bone with increasing temperature, as described by Imaizumi et al. [68] and Van Hoensel et al. (2019). The three stages are indicated by boxes with a corresponding roman number

During the first stage, below 250 °C, the main alteration in the bone is the loss of water [69, 70]. According to Etok et al. [70] and Van Hoensel et al. (2019), first adsorbed water will evaporate from the bone, up to 100 °C, where after the structural bound water from proteins and mineral surfaces is lost [69,70,71]. Both measured this with thermogravimetric analysis (TGA) and Fourier transform infrared spectroscopy (FTIR). However, the appositeness of methods, such as TGA, can be debated, since the bone sample is ramped (i.e., a repetition of increasing the temperature and measuring the chemical composition). Although the effect of ramping on the HI changes is still unknown, it is expected this does affect the temperature a HI change occurs, given that the exposure expressed in ATUs is much higher for ramped bone than for bone that is heated a single time. As can be seen in Fig. 3, most water has been evaporated at 250 °C, but in the second stage, around 400 °C, the concentration of water increases slightly. According to Shafizadeh et al. [72], water loss at higher temperatures is due to the loss of structural water from the organic layer and as a result of thermal degradation [72]. An increase can be seen around 400 °C, which could be explained by the major loss of organic compounds between 300 and 400 °C, where water is formed as reaction product of combustion. It is suggested that only a slight increase can be observed around 400 °C, because the water that is formed, evaporates immediately at these temperatures. As a result of the degradation into even smaller organic compounds, Van Hoensel et al. (2019) measured the formation and immediate evaporation of water with FTIR up to 700 °C [69]. Similar observations were done by Reidsma et al. [73] under reducing conditions [73].

In the second stage, the bone loses most of its weight, which is mainly due to the loss of organic components (Fig. 3). Aside from FTIR, Van Hoensel et al. (2019) performed pyrolysis mass spectrometry (pyMS) on the bone and observed minor changes in the organic composition up to 200 °C [69]. Between 200 and 350 °C, a decrease of the most abundant protein, collagen, is observed. The degradation of collagen is accompanied by the introduction of new compounds, namely alkylated phenols, alkylated benzenes, condensed aromatic compounds, and N-containing heterocyclic compounds between 300 and 340 °C [63, 68]. Reidsma et al. [73] found similar temperatures under reducing conditions with direct temperature-resolved mass spectrometry (DTMS) [73]. From 350 °C onwards, smaller thermally degraded compounds were observed, such as benzene, until these aromatic compounds were completely oxidized and not observable anymore with FTIR and TGA at 600 °C (Reidsma et al. [73]: 900 °C with DTMS under reducing conditions) [73]. Van Hoensel et al. (2019) have made a distinction between combusted bone (i.e., heated in the presence of oxygen) and charred bone (i.e., heated under reducing conditions). While in the former the organic phase is mostly (93.5%) combusted at 350 °C, the charred bone has lost a similar percentage of organic phase at 600–700 °C [69]. Moreover, above 600 °C or 700 °C, cyanamide (CH2N2) was detected with FTIR and inelastic neutron scattering (INS), only for the charred bone [73, 75]. The presence of organic phase at higher temperature is in agreement with the observation of Reidsma et al. [73] to find organic compounds at 900 °C, since these experiments were performed under reducing conditions [73]. Similar to the aromatic compounds, the concentration of formed nitrogen containing (inorganic) compounds, such as prussic acid (HCN) and acetonitrile (ACN), is strongly reduced above 350 °C. A visibly observable, and measurable, effect of the thermal degradation of collagen is the HI-change in colour of the bone, from ivory white fresh bone to black when carbonized and ashy grey to calcined white after exposure to temperatures above 500 °C [68, 76,77,78].

According to Reidsma et al. [73], no proteins were detected anymore under reducing conditions at temperatures of more than 370 °C, while Correia [79] reported the presence of collagen at temperatures up to 800 °C after microscopic observations, Castillo et al. [29] up to 600 °C, and Marques et al. [80] reported a complete combustion of proteins between 700 and 900 °C with FTIR-attenuated total reflectance (ATR) and INS [29, 79, 80]. The inconsistency between the authors here could be explained by the definition of ‘a complete combustion of proteins’. On the one hand, this could include the combustion of their organic degradation products. On the other hand, this could solely be the combustion until no protein is left anymore. The difference between the results of Reidsma et al. [73] and the other authors could be explained by the availability of oxygen. However, keeping in mind the other HI changes, the temperature is high. The results of Reidsma et al. [73] are considered more reliable than the results of Correia [79] and Castillo et al. [29], since analytical methods are preferred over subjective interpretation of histological features [81, 82]. So, although proteins, such as collagen, have shown to endure high temperatures, it is expected that these will be broken down before the third stage at 700 °C, and will certainly not be present in their original form, neither under oxidizing conditions [47].

Consensus exists regarding the thermal degradation of adipose tissue: Van Hoensel et al. (2019) measured the HI change of lipids in the bone: the thermal degradation of lipids is suggested to be completed below 300 °C after pyMS analysis, since no lipid markers were found above this temperature [69]. Braadbaart [74] reported a similar temperature between 340 and 370 °C for the evaporation of lipids (in sunflower seeds) [83]. Reidsma et al. [73] reported the completion of the thermal degradation of lipids at a temperature of 340 °C under reducing conditions after DTMS analysis [73]. So, the lipid content of the human bone is completely degraded in the second stage.

Several hormones and vitamins can be found in the bone [37,38,39, 41, 42]. To the best of our knowledge and based on the literature search, no research has been performed on the thermal degradation of these compounds in bone. Considering the chemical structure of vitamins and hormones, it is supposed that the thermal degradation will be similar to the organic compounds that originated from protein degradation, meaning a complete degradation around 600 °C in the presence of oxygen and 900 °C under reducing conditions [69, 73]. Note, vitamin B12, that can also be found in the bone (mostly in the red blood cells and red bone marrow), is a coordination complex of cobalt (Co+) and certain ligands. The cobalt ion will not be thermally degraded and will remain in the bone [37].

As discussed, a major part of the organic phase is thermally degraded between 300 and 400 °C. Although some HI physical conversions, or denaturations, within the blood are described in literature, such as the HI haemolysis at 52 °C and DNA denaturation between approximately 60 and 100 °C, to the best of our knowledge, no literature can be found on HI chemical conversions of specifically blood and/or red bone marrow components [84, 85]. Therefore, it is assumed that the proteins in blood and red bone marrow as well as the other organic components (e.g., cellular constituents) and water in the blood follow the same reaction pathways as respectively the organic components and water within the bone matrix. However, the electrolytes sodium (Na+), chloride (Cl−), magnesium (Mg2+), potassium (K+), and carbonate (CO32−), that can be found in the plasma, will not be degraded [37]. Moreover, the main component of blood, haemoglobin, is an iron-containing metalloprotein consisting of heme groups that contain four pyrrole molecules and an iron ion (Fe2+ or Fe3+ for met-haemoglobin) [30]. These cations will still be present in the bone after thermal degradation of the organic phase. So, from the blood and red bone marrow, only some ions will still be present during the calcination stage (Correia [79]: 700–1100 °C).

During the whole second phase, a strong presence of carbon monoxide (CO) and carbon dioxide (CO2) was observed, which is between 250 and 500 °C mostly due to the (incomplete) combustion of the, by protein degradation, formed compounds, the proteins themselves, and the lipids (Eq. 1) (between 300 and 500 °C, according to Etok et al. [70]) [39, 69, 70].

$$\mathrm a\;___+\mathrm b\;_2\xrightarrow\mathrm c\;}_2+\mathrm d\;\mathrm+\mathrm e\;_2\mathrm O$$

(1)

Equation 1. A chemical representation of a general incomplete combustion of an organic compound into carbon dioxide, carbon monoxide, and water. The coefficients and subscripts are denoted as letters.

As can be seen from the upper equation, and Fig. 3, water is formed during combustion, which is also observed with FTIR (until 700 °C) [69]. However, this will evaporate immediately at these temperatures (see also: 4.1 Water).

According to i.a. Mamede et al.[39], a second fraction (50% according to Etok et al. [70]) of carbon dioxide was released at temperatures of more than 500 °C. Structural carbonate (CO32−) loss from the bone matrix (i.e., in BAp) is suggested as source [39, 55, 70, 86]. Reidsma et al. [73] measured a minor carbonate loss under reducing conditions at much lower temperatures, namely between 250 and 340 °C, with FTIR. However, most carbonate loss was measured above 600 °C [73]. Van Hoensel et al. (2019) confirmed the carbonate loss with FTIR and TGA under oxidizing conditions and also observed a release of water up to 700 °C and therefore proposed the following equations:

$$\mathrm_3^+_2\mathrm O\xrightarrow}_2+2\;\mathrm^-$$

(2)

$$\mathrm_3^+2\;\mathrm\mathrm O_4^\xrightarrow}_2+2\;\mathrm^-+_2\mathrm O_7^$$

(3)

Equations 2 and 3. A chemical representation of two possible reaction equations for the conversion of carbonate into carbon dioxide and the rehydroxylation of bioapatite [69].

Equation 2. was considered the most likely, since no evidence was found for the presence of pyrophosphate (P2O74−) with FTIR [69]. Moreover, Mamede et al. [14] measured an increasing hydroxyl content in bioapatite between 700 and 900 °C with FTIR-ATR [14]. This is further supported by the observation of carbon trioxide (CO3) by Van Hoensel et al. [69] and Madupalli et al. [87], of which a first peak is observed at 600 °C with FTIR. It is suggested that the carbonate ions reorganized into carbon trioxide to create space for the formed hydroxyl ions [69, 87]. Under reducing conditions, Reidsma et al. [73] did not found any evidence for Eq. 3 [73].

Amongst others, Marques et al. [80] and Haberko et al. [88]reported again a carbonate loss in the third stage, between 700 and 1100 °C, with the major loss below 1000 °C [79, 80, 88]. This was measured with different methods, such as carbonate precipitation from the bone and FTIR-ATR. At the same time, Piga et al. (2011) observed the appearance of calcium oxide or lime (CaO) at temperatures of 775 °C with powder X-ray diffraction (p-XRD), increasing up to 1000 °C [89]. With general XRD, Van Hoensel et al. (2019) observed the formation of lime above 800 °C, Rogers and Daniels. [90] and Haberko et al. [88] above 700 °C, and others above 900 °C [64, 69, 88, 90, 91]. The discrepancy between temperatures can be explained by the differences in the age of the donors [90]. Best et al. [92] suggest that lime is formed during the formation of β-TCP when the Ca/P-ratio is higher than 1.67 [92]. Piga et al. (2011, 2018) therefore proposed the following chemical reaction:

$$2\;}_5}_4)_3\mathrm\xrightarrow3\;}_3}_4)_2+\mathrm+_2\mathrm O$$

(4)

Equation 4. A chemical representation of the conversion of HAp into β-TCP, calcium oxide, and water [89, 93].

In the case that water does not evaporate completely in Eq. 4, which is dependent on the speed of cooling after the burning process, also rehydrate