An overview of heat-induced molecular
changes in bone tissue
Master Forensic Science
Daphne Muijderman (12376388)
Supervisor: Tristan Krap MSc
Examiner: Prof. Dr. Roelof-Jan Oostra
Number of words: 7844
Table of contents
Abstract 3
Introduction 3
Burning process of bones 3
Composition of bone tissue 4
HI-changes of organic components in bone 5
Collagen and other proteins 5
Adipose tissue 6
Carbon and nitrogen 6
Carbonate 6
HI changes of inorganic components in bone 7
Calcium and phosphor 7
Hydrogen 7
Hydroxyapatite 7
Tricalcium phosphate 8
Calcium oxide 8
Discussion 9
Collagen and other proteins 9
Adipose tissue 9
Carbon, hydrogen and nitrogen 10
Carbonate 11
Other organic compounds 11
Calcium and phosphor 11
Hydroxyapatite 11
Tricalcium phosphate 12
Calcium oxide 12
Other inorganic compounds 13
Synthesis 13
Conclusion 14
References 14
Abstract
This literature thesis describes the alterations that occur in bone tissue when it is exposed to heat, such as in the case of a fire. The molecular composition of the bone matrix alters due to heat exposure, and these alterations could be indicators for estimating the heating temperature. This review describes the heat induced changes of collagen and other proteins, adipose tissue, carbon, hydrogen, nitrogen, carbonate, calcium, phosphor, hydroxyapatite, tricalcium hydroxyapatite and calcium oxide. The changes of these compounds are described in perspective to the different stages of burned bone: Dehydration, decomposition, inversion and fusion. Besides that, their relevance and value to the estimation of the temperature to which bones are exposed is explained.
Introduction
The recovery of bones is an important aspect in the fields of forensics and anthropology. In both research areas, investigators encounter the recovery of human remains that have been exposed to fire and have been altered due to the heat. In cases of accidents, suicides, and criminal offenses, human remains are an important source of information, since they can provide information concerning the events prior to death, and the cause of death (White & Folkens, 2005). The fact that the remains are burned is a challenge for the investigators of a case, since it hampers the recovery of bones, the identification process and the determination of the presence of trauma (Mamede Gonçalves, Marques & Batista de Carvalho, 2018; White & Folkens, 2005).
The content of the bone could provide the investigator with information. An example of this is the presence of larger amounts of blood in the burned bone, which could indicate that the bone was exposed to trauma while the person was still alive. This suggests that the person was injured before being exposed to the fire.
Different burning stages show different alterations to the bone. Several approaches are developed to obtain information from cremated remains, including light microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), x-ray diffraction (XRD), fourier transform infrared spectroscopy (FTIR) and raman spectroscopy (Ellingham et al., 2015). However, it is unclear which alterations are caused during which event of the burning process and at what specific temperature, which could add valuable information about the events around the time of death. A lot of research has been done on heat-induced (HI) changes of bone, but no complete overview has been published yet. Since a combination of all multiple alterations can provide more information than only one aspect that has changed due to burning, an overview of the possible changes in the perspective of increasing temperature would facilitate the prediction of burning temperature when certain features are analysed. Therefore, the aim of this literature thesis is to provide a figure which shows how the composition of bone is altered in relation to different burning stages. At first, the process of burning a bone will be explained by providing different burning stages. Secondly, the composition of the bone matrix will be discussed, to identify the components that can be altered due to heat. After that, an overview of the current literature about the heat induced (HI) changes of each organic compound will be given, followed by an overview of the inorganic compounds. Then, the literature will be discussed which will lead into a table which summarises the obtained results. Finally, a conclusion will be given with future research suggestions.
Burning process of bones
While investigating cremated human remains, the process of burning should be taken into account since it could explain the features that are observed (White & Folkens, 2005). During the burning process of a bone, several stages can be distinguished, and each stage can be linked to a temperature range causing alterations in bone tissue. The first stage is dehydration, in which hydroxy bonds are disrupted and water evaporates from the bone at temperatures of approximately 100°C and higher (Verkerk, 2013). Two phases within the event of dehydration can be distinguished. At first, the loss of free water content takes place, after which water that is chemically bound is released (Reidsma, Van
Hoesel, Van Os, Megens & Braadbaart, 2016). In the second stage, at temperatures of circa 500°C and higher, decomposition of the bone is taking place, in which pyrolysis is an important process. Pyrolysis is an endothermic reaction in which compounds are decomposed in the absence of oxygen, leading to the removal of organic compounds, including collagen fibres and other proteins present in bone tissue (Ellingham, Thompson, Islam & Taylor, 2015; Krap, Nota, Wilk, Van de Goot, Ruijter, Duijst & Oostra, 2017). Besides pyrolysis, there is combustion, in which compounds are decomposed in the presence of oxygen (Jackson & Jackson, 2008). During the third stage, around temperatures of 650°C and higher, inversion leads to the calcination of bone. This process of inversion is caused by a loss of carbonate, and in addition there is a release of magnesium that is present in the bone (Correia, 1997). Finally, the fourth stage is fusion, in which recrystallisation of inorganic matter occurs, while temperatures of approximately 1600°C and higher are present (Ellingham et al., 2015).
Composition of bone tissue
To create an overview of HI-changes in bone tissue, it is important to take all substances and structures that are present in bones into account. In adult bones, a distinction between two different structures can be made. The first structure is the cortical or compact bone, which is present on the external surfaces on the walls of bones and surrounds and protects the medullary cavity (Martini, 2006; White & Folkens, 2005). The second structure is the cancellous or trabecular bone, which is present on the epiphyses (Martini, 2006). Both structures consist of the same organic and inorganic elements and are molecularly build up in the same way. In both the cortical and cancellous bone, blood is present. In the cortical bone, blood is transported to osteons via vessels that are located in Haversian and Volkmann’s canals, while in cancellous bone there is an open network formed by trabeculae, in which nutrients are transported by diffusion in the absence of capillaries and venules (Martini, 2006). Additionally, red bone marrow is mainly present in the ends of long bones, where it is protected by the cancellous bone (Memmler & Rada, 1970). Red bone marrow functions as a producer of erythrocytes, lymphocytes and thrombocytes (Martini, 2006). The yellow bone marrow is mainly present in the central cavities of long bones and it consists of adipose tissue.
A distinction between organic and inorganic compounds can be made for the substances of which a bone consists of. Approximately 30% of the bone consists of organic components, while around 70% consists of inorganic components (Martini, 2006). The most important organic substance are collagen fibres. These fibres form a framework in which inorganic minerals can crystallise (Martini, 2006). Besides collagen, other proteins such as albumin and ɑ2-HS-glycoprotein are present in bone tissue as well (Clarke, 2008). Albumin plays a role in decreasing crystallization by which hydroxyapatite, an important mineral in bone tissue, is created, while ɑ2-HS-glycoprotein regulates the proliferation of bone cells (Clarke, 2008). Other organic compounds that can be found in bones are several hormones, such as calcitriol, growth hormone, thyroxine, calcitonin, parathyroid hormone, and sex hormones during growth; and several vitamins among which A, B12, C and K (Martini, 2006). The transportation of for example these hormones and vitamins to osteons occurs via blood vessels. Blood consists mainly of water, in which different organic compounds, such as plasma proteins, erythrocytes, lymphocytes and thrombocytes, can be found in the solution. Examples of plasma proteins are albumins, globulins and fibrinogen (Martini, 2006). Finally, the last organic substance is adipose tissue, which is located in the yellow bone marrow (Martini, 2006). Below, an overview of the organic components of the bone matrix are listed, the percentages are given as the contribution to the total amount of both organic and inorganic substances:
- Collagen fibres: 23.05% (Dequeker & Merlevede, 1971)
- Noncollagenous proteins (among which albumin and ɑ2-HS-glycoprotein): 1 – 5% (Clarke, 2008)
- Carbonate (CO32−): 9.8% (Martini, 2006)
The most redundant mineral that can be found in bone is apatite, which is a salt consisting of calcium and phosphor. In reaction with an ion such as hydrogen, hydroxyapatite (Ca10(PO4)6(OH)2) crystals will
form (Martini, 2006). Inorganic compounds, such as calcium carbonate (CaCO3), convert into calcium
oxide (CaO) and calcium hydroxyapatite, and the latter can be transformed into β-tricalcium phosphate (Correia, 1997). Besides these calcium phosphates, others among which brushite (CaHPO4·2H2O), octacalcium phosphate (Ca8H2(PO4)6·5H2O), calcium pyrophosphate dihydrate
(Ca2P2O7), and amorphous calcium phosphates, can be found in bone tissue. Additionally, magnesium
phosphates are present in bones, such a struvite (MgNH4PO4·6H2O) and amorphous calcium
magnesium pyrophosphates. These calcium and magnesium phosphates are only present in small amounts compared to calcium hydroxyapatite (LeGeros & LeGeros, 1984). Other minerals that can be found in the extracellular matrix of bones are magnesium, fluoride, calcium, phosphor, potassium, carbonate, chloride and pyrophosphate (LeGeros & LeGeros, 1984; Martini, 2006). Minerals that are present as trace elements are lead, strontium, barium, zinc and iron (LeGeros & LeGeros, 1984). In addition to that, blood is also a source of minerals that can be detected in bones. Chloride, calcium, natrium, potassium, magnesium and bicarbonate are dissolved in blood plasma. In erythrocytes, the red blood cells, the haemoglobin protein is the oxygen and carbon dioxide carrier, of which the binding is enabled by an iron ion (Martini, 2006). An overview of the amounts of inorganic compounds in relation to the total amount of bone substances in the bone matrix is listed below:
- Calcium hydroxyapatite (Ca10(PO4)6(OH)2): ~70% (Martini, 2006)
- Calcium oxide (CaO): 0%
- Tricalcium phosphate (Ca3(PO4)2): 0%
- Calcium (Ca2+)
- Phosphate (PO34−)
- Sodium (Na+): 0.7% (Martini, 2006)
- Potassium (K+): 0.2% (Martini, 2006)
- Magnesium (K+): 0.5% (Martini, 2006)
- Fluoride (F−)
- Chloride (Cl−)
- Trace elements: Strontium (Sr2+), lead (Pb2+), barium (Ba2+), iron (Fe2+) and zinc (Zn2+). This
summation of trace elements is not complete. HI-changes of organic components in bone
Collagen and other proteins
Collagen is the most common protein in bone tissue, and it is built out of amino acids (Martini, 2006). Besides collagen, albumin, ɑ2-HS-glycoprotein and other proteins can be found in bones as well. By using direct temperature-resolved mass spectrometry (DTMS), Reidsma et al. (2016) investigated the presence of collagen and other proteins in bovine bones with increasing temperature in circumstances of low amounts of oxygen, using an exposure time of 60 minutes. Between 200°C and 300°C, a decrease in collagen was observed, after which an increase of condensed aromatic compounds, phenols and alkylated benzenes was observed at temperatures of 300°C to 340°C. The appearance of these compounds could be a result of the breakdown of collagen. Furthermore, no proteins were detected at 370°C and higher, while other organic substances, among which condensed aromatic compounds, were still present at these temperatures. After 500°C, a decline in these compounds was detected and around 900°C, only a small amount of condensed aromatic compounds was still observable in bone tissue (Reidsma et al., 2016). Marques, Mamede, Vassalo, Makhoul, Cunha, Gonçalves, Parker & Batista de Carvalho (2018) exposed human femurs and humeri for 2 hours to temperatures of 400°C to 1000°C and they applied FTIR with attenuated total reflectance (ATR) and inelastic neutron scattering (INS) to investigate the content changes within the bones due to combustion. Results obtained by FTIR-ATR showed that the organic matrix was no longer observed at temperatures of 700°C and higher (Marques et al., 2018). In addition to that, experiments with INS showed that hardly any proteins were detectable at 700°C, and that the destruction of proteins was completed at 900°C (Marques et al., 2018). Castillo, Ubelaker, Acosta, & De la Fuente (2013)
published an article about a histological research using light microscopy on human bones that were exposed for 20 minutes to temperatures ranging from 100°C to 1100°C. Their results show that there was a separation of collagen fibres visible at 100°C, where after the fibres were more compact and rigid at temperatures of 200°C. Around 300°C, the collagen fibres were separating again, followed up by compression with other fibres at 400°C (Castillo et al., 2013). In another paper, by Castillo, Ubelaker, Acosta, De la Rosa & Garcia (2013) with the same experimental setup, it was showed that ''linear macromolecular crystalline polymers of collagen and extracellular matrix'' (Castillo et al., 2013, p. 580) were still observed at temperatures of 600°C.
Adipose tissue
Adipose tissue is particularly present in the yellow bone marrow, and it consists of cells that have the storage of lipids as their main function (Martini, 2006). In an experiment with DTMS, Reidsma et al. (2016) investigated the thermal degradation of lipids in bovine bone tissue. They observed that the degradation of lipids was finalised at 340°C when the bones were exposed to heat for 60 minutes. Slightly different results were observed by Braadbaart, Wright, Van der Horst & Boon (2007), who found that there was no dissociation of lipids in the temperature range of 160°C to 340°C. In addition to that, in between 340°C to 370°C, the lipids were no longer observable due to evaporation (Braadbaart et al., 2007). For these experiments, the method of DTMS under electron ionisation (EI) conditions was applied with a duration of exposure of 60 minutes, and instead of bones, sunflower seeds were used (Braadbaart et al., 2007). An experiment on the heating of human femurs and humeri by Marques et al. (2018) with INS showed that lipids disappeared at temperatures of around 500°C when a duration of exposure of 2 hours was used, which is higher than what both Reidsma et
al. (2016) and Braadbaart et al. (2007) stated. Carbon and nitrogen
By using carbon, hydrogen and nitrogen (CHN) analysis in an environment that is low in oxygen, Reidsma et al. (2016) investigated the carbon, hydrogen and nitrogen content in bovine bone exposed to increasing temperatures for 60 minutes. In the temperature range of 200°C to 450°C, an increase of 15% of carbon content was observed. After increasing the temperature over 450°C, the carbon content remained stable (Reidsma et al., 2016). When increasing the temperature from 200°C to 450°C, a decrease of about 4% in nitrogen content was observed. At higher temperatures than 450°C, no changes were measured in the amount of nitrogen (Reidsma et al., 2016).
Carbonate
Carbonate (CO32−) is an organic compound that is present in the bone matrix, and more specifically, in
the hydroxyapatite crystals (Figueiredo, Fernando, Martins, Freitas, Judas & Figueiredo, 2010). Several researchers investigated the effect of heat to the carbonate content and concluded that a decrease is observable which mainly occurs during the third stage of burning bone at temperatures of 650°C and higher (Correia, 1997). Haberko, Bućko, Brzezińska-Miecznik, Haberko, Mozgawa, Panz, Pyda & Zarębski (2006) and Marques et al. (2018) state that there is a decline in carbonate content at temperatures of 700°C and higher. At 1000°C, only small amounts of carbonate were still present in the bone tissue (Haberko et al., 2006; Marques et al., 2018). Haberko et al. (2006) came to these conclusions by investigating the precipitation of CaCO3 in bovine bones while Marques et al. (2018)
used FTIR-ATR on human femurs and humeri. Figueiredo et al. (2010) applied FTIR on human bones and their results showed that there is a decrease in carbonate content at temperatures of at least 600°C when heated for 18 hours in vacuum conditions. Additionally, no carbonate was detected at temperatures of 900°C (Figueiredo et al., 2010). Research conducted by Reidsma et al. (2016), showed by using FTIR that there already is a small decrease in the amount of carbonate in bovine bones when exposing them to temperatures of 250°C to 340°C for 60 minutes. At temperatures of 600°C and higher, in particularly at 800°C to 900°C, a larger decrease is noticeable (Reidsma et al., 2016). Other research on human bones using FTIR-ATR showed that there is a structural loss of carbonate from temperatures of 500°C and higher, causing a release of CO2 (Snoeck, Lee-Thorp &
Schulting, 2014). This experiment was done for a temperature range of 500°C to 900°C with a duration of exposure of 30 minutes to 24 hours (Snoeck et al., 2014).
HI-changes of inorganic components in bone
Calcium and phosphor
Calcium and phosphor are two important minerals in bone tissue. Reidsma et al. (2016) used x-ray fluorescence (XRF) to measure their content in bovine bones. No changes in both calcium and phosphor content were observed when the temperature was increased up to 900°C (Reidsma et al., 2016).
Hydrogen
Reidsma et al. (2016) investigated the hydrogen content by using CHN analysis. In their experiment they heated bones to temperatures in the range of 200°C to 900°C in an environment that is low of oxygen. During this heat exposure, no differences were detected in the hydrogen content (Reidsma et
al., 2016). Hydroxyapatite
In bones, the most common form of apatite is hydroxyapatite (HAp) (Ca10(PO4)6(OH)2), which has a
crystalline structure. These crystals are usually formed over time in the framework that is formed by collagen fibres (Martini, 2006). This process is accelerated due to heat and it will mainly occur in the third stage of the burning process (Krap et al., 2017; Shipman, Foster & Schoeninger, 1984). There is consensus that there is an increase in crystal size with increasing temperature. Shipman et al. (1984) used XRD to investigate the size of hydroxyapatite crystals in sheep and goat bones and they observed that bones burned at temperatures higher than 645°C contain larger crystals, compared to bones burned at temperatures lower than 525°C (Shipman et al., 1984). Research conducted by Haberko et al. (2006) using XRD confirms that the hydroxyapatite crystals become larger when bovine bones are exposed to higher temperatures. Castillo et al. (2013) used light microscopy to investigate the crystalline structures in bones. They state that there is an increase in crystallite size from 600°C and higher, and that the individual hydroxyapatite crystals are no longer visible due to fusion at temperatures of at least 900°C (Castillo et al., 2013). According to Holden, Phakey, & Clement (1995), this process of recrystallization, leading towards a decrease in hydroxyapatite, occurs at much higher temperatures, namely at 1600°C and higher.
The increase in crystallite size is not stable and differs with increasing temperature. Shipman et al. (1984) stated that the increase in crystal size was relatively slow in the range of room temperature to 525°C and that the increase was much larger in the range of 770°C to 800°C. This is in line with research performed by Haberko et al. (2006), who show that there is a larger increase at higher temperatures (±800°C) compared to lower temperatures (±700°C). Figueiredo et al. (2010) used XRD to measure the crystallite size in human, bovine and porcine bones, at 600°C, 900°C and 1200°C for 18 hours. The results for the human bone were 63 nm, 76 nm and 105 nm respectively (Figueiredo et
al., 2010). These results are in accordance with the research conducted by Haberko et al. (2006),
since it shows a small increase in crystallite size in the temperature range of 600°C to 900°C (13 nm increase) and a large increase in the temperature range of 900°C to 1200°C (29 nm increase). However, the results of the bovine and porcine bones were different compared to the human bones. In these two species, there was a larger increase in size in the 900°C to 1200°C range (38 nm and 39 nm respectively) than in the 600°C to 900°C range (13 nm and 15 nm respectively) (Figueiredo et al., 2010). Holden et al. (1995) investigated hydroxyapatite crystal sizes using SEM and they looked at the diameters and shapes of the crystals in human bones at 200°C, 600°C, 800°C, 1000°C, 1200°C, 1400°C and 1600°C, when exposed to heat for 2 hours. At 600°C, they observed newly formed crystals compared to the crystals that were already present at 200°C. The sizes of the crystals were very similar, they all had a spherical shape and their average diameter at 600°C was 0.064 ± 0.04 µm. At temperatures of 800°C and 1200°C these diameters were increased: 0.070 ± 0.010 µm and 0.200 ±
0.050 µm respectively. Additionally, crystals with a hexagonal shape appeared and they were larger compared to the spherical ones since they had diameters of 0.30 ± 0.05 µm at 800°C and 1.2 ± 0.10 µm at 1200°C. At temperatures of 1000°C to 1400°C, local fusion of hexagonal crystals was observed. Besides that, new crystals were formed with rhombohedral shapes and an average diameter of 0.300 µm to 6.0 µm. Around 1600°C, no separate crystals were visible, due to fusion (Holden et al., 1995). An overview of the results of Holden et al. (1995) are shown in table 1 and they are in accordance with the results of Figueiredo et al. (2010) and Haberko et al. (2006), since an increase in crystal growth is observed at increasing temperatures.
Table 1: An overview of the results of Holden et al. (1995).
Temperature (°C) Spherical
crystals (µm)
Hexagonal crystals (µm)
Rhombohedral crystals (µm)
600 0.064 ± 0.04 Not present Not present
800 0.070 ± 0.010 0.30 ± 0.05 Not present
1000 N/A N/A, start fusion 0.300 - 6.0
1200 0.200 ± 0.050 1.2 ± 0.10 0.300 - 6.0
1400 N/A N/A 0.300 - 6.0
1600 Fusion Fusion Fusion
Tricalcium phosphate
Hydroxyapatite can transform into β-tricalcium phosphate and ɑ-tricalcium phosphate (Ca3(PO4)2),
especially at high temperatures (Correia, 1997). As already described earlier, the morphology of the crystals in bone tissue alters with increasing temperatures. This could be explained by the formation of β- and ɑ-tricalcium phosphate (Bonucci & Graziani, 1975; Civjan, Selting, De Simon, Battistone & Grower, 1972). According to research performed by Civjan et al. (1972) on the bones of rhesus macaques, a part of the hydroxyapatite is transformed into β-tricalcium phosphate at temperatures of 600°C to 800°C. Bonucci & Graziani (1975) stated that they came to the same conclusion by doing experiments with ox bone. Holden et al. (1995) stated that crystals with an altered morphology were observed at temperatures of 1200°C and higher, which could be explained by, for example, β- and ɑ-tricalcium phosphate. For their research, human femurs obtained from cadavers were used and exposed to high temperatures for 2, 12, 18 and 24 hours. They followed the same explanation as Civjan et al. (1972), but they obtained these results only at higher temperatures. Besides that, Shipman et al. (1984) did not find any evidence for the formation of β-tricalcium phosphate in sheep and goat bones when exposing the bones to temperatures of 800°C and higher for 4 hours. In addition, Rogers & Daniels (2002) did not detect β-tricalcium phosphate in human bones at temperatures ranging from 20°C to 1200°C when heating them for 2 hours. Both Shipman et al. (1984) and Rogers & Daniels (2002) used XRD for their research.
Calcium oxide
Calcium oxide (CaO) is one of the products that arises mainly during the third phase of burning bones (Correia, 1997). This compound is visible as a white powder, and it will remain in solid state till very high temperatures (Verkerk, 2013). Calcium oxide arises due to the reaction that takes place when carbonate containing apatites are heated, which results in the formation of calcium oxide, carbon dioxide and water (Holden, Clement & Phakey, 1995; Rogers & Daniels, 2002). Rogers & Daniels (2002) demonstrate, using XRD, that calcium oxide can be observed in human bones at temperatures of 700°C or higher. This was confirmed by Haberko et al. (2006), who showed the appearance of
calcium oxide at temperatures of at least 700 °C. However, LeGeros & LeGeros (1984) state, by using the same method, that calcium oxide is only present in human bones at temperatures of at least 950°C. According to Figueiredo et al. (2010), calcium oxide could only be detected in human bones in small amounts (0.1 - 0.2%). Their results were obtained by exposing bones to temperatures of 1200°C for 18 hours, and by using XRD. In addition, it is stated by Rogers & Daniels (2002) that there is an increase in the relative amount of calcium oxide with increasing temperature.
Discussion
Collagen and other proteins
In several studies, different methods were used to analyse the destruction of collagen fibres and other proteins that were present in bone tissue due to heat. Examples of these methods are DTMS, FTIR-ATR, INS and light microscopy. Reidsma et al. (2016) used DTMS and observed that there were no proteins present in bovine tissue after an exposure to 370°C with a duration of 60 minutes. Other organic substances, such as condensed aromatic compounds, were still detected until 900°C (Reidsma
et al., 2016). By using FTIR-ATR, Marques et al. (2018) showed that there was no organic matter
present when bones were heated up to 700°C or higher. They did not go into detail about which organic compounds would disappear at which investigated temperatures. Experiments with INS showed that although there were barely any organic compounds left, some were still detected at 700°C and all of them were destroyed at 900°C. Within one article, Marques et al. (2018) came to slightly different conclusions by using different methods, which could be explained by the accuracy of these methods. Although INS is a reliable method to investigate inorganic compounds of the bone matrix, it is not suitable to measure the contents of organic compounds, as stated by Mamede et al. (2018). This method is used to detect the presence of hydroxyl groups, which are present in proteins and other organic compounds. However, hydroxyl groups are also present in the inorganic hydroxyapatite, which affects the reliability of the measurements on the protein content (Mamede et
al., 2018). Therefore, the results obtained by using FTIR-ATR are more valuable compared to the
results obtained by INS. What should be mentioned is another difference within the methods, is that Reidsma et al. (2016) investigated the HI-changes due to pyrolysis and Marques et al. (2018) due to combustion. In general, the results of Marques et al. (2018) are in line with the results of the research done by Reidsma et al. (2016), who also state that exposing bones to 900°C or higher there was no organic matter left. Experiments conducted with light microscopy show different results compared to molecular analyses. Castillo et al. (2013) observed collagen polymers at temperatures of 600°C, while DTMS-experiments showed that proteins are all degraded around 370°C. Histological methodologies are less objective and the structures that were observed could have been organic products resulting from the heating of collagen, instead of the collagen fibres itself. Therefore, the results obtained from the research done by Reidsma et al. (2016) and Marques et al. (2018) are more reliable than the histological investigation done by Castillo et al. (2013). That is why it could be concluded that collagen fibres and other proteins disappear from the bone, by forming other organic products and that collagen will completely disappear when the bone is heated to 370°C while the other organic products will remain present till around 800°C to 900°C (figure 1).
Adipose tissue
Reidsma et al. (2016) observed that lipids in bovine bones were degraded at 340°C, while Braadbaart
et al. (2007) stated that this occurred in sunflower seeds in the temperature range of 340°C to 370°C.
A comparable method (DTMS and DTMS-EI) was used in these experiment, the time of exposure was the same and both looked at the process of pyrolysis. The fact that sunflower seeds were used instead of bones could explain the difference in temperature of the lipid disappearance. Bovine bones are expected to be more similar to human bones than sunflower seeds, which makes the results of Reidsma et al. (2016) more reliable. Besides these two studies an experiment conducted by Marques
et al. (2018) showed that lipids disappeared at much higher temperatures than stated by Reidsma et al. (2016) and Braadbaart et al. (2007). As Reidsma et al. (2016) observed a total degradation of lipids
be the starting temperature on which the experiment was based. The lowest temperature that was used by Marques et al. (2018) was namely 400°C, while lipids should no longer be present in bone tissue at these temperatures according to Reidsma et al. (2016). Besides that, a different method was used to investigate the lipid content in human bones. As described earlier, the method used by Marques et al. (2018), INS, is not very suitable for the determination of the content of organic components (Mamede et al., 2018). To conclude, the results of Reidsma et al. (2016) are the most reliable, since they are based on bones instead of sunflower seeds and are obtained with DTMS, which is a more suitable method compared to INS in investigating organic compounds, it is the most likely that the disappearance of lipids in bone is finished around temperatures of 340°C (figure 1).
O rg an ic co m p o n en ts General changes In o rg an ic co m p o n e n ts Carbon Nitrogen Protein Adipose tissue Carbonate Organic products HAp CaO 0 2 0 0 4 0 0 6 0 0 8 0 0 10 0 0 12 0 0 14 0 0 16 0 0 Temperature (°C) Dehydration Decomposition Inversion [1h] [1h] [1h] [1h–2h] [20min–2h] [2h–24h] [2h-24h] [2h] Fusion (9, 11) (5, 120 23) (23) (13, 20, 23) (13, 18, 24) (13, 15) (23) (23) (23)
Figure 1: Overview of the general trends of HI-changes of components in the bone matrix. On the top of the figure, the general HI-changes of bone are shown. Below that, the HI-changes of different (in-)organic components are shown. The alterations are visualised as an abstract line and represent a general trend. In the right row, the duration of exposure range in which the research is conducted is shown between square brackets. References, which are shown as a number corresponding with the reference list, on which the results are based are placed between round brackets.
Carbon, hydrogen and nitrogen
Since proteins are built out of amino acids, which consists of carbon, hydrogen and nitrogen, an explanation of the development of the presence of these atoms with increasing temperature could be provided. Condensed aromatic compounds are molecules that consists of a carbon and hydrogen ions structured in an hydrogonal shape. Phenols (C6H5OH) and benzenes (C6H6) also do not contain
any nitrogen atoms, which is in line with the decrease in nitrogen content with increasing temperature. The hydrogen ions are reused in mainly the phenols, which explains the results of Reidsma et al. (2016) that the hydrogen content remains stable. However, an increase in carbon content was detected, which cannot be explained by the conversion of collagen and other proteins into other carbon-containing compounds. Another source of these carbon-based molecules could be the vanished lipids from the adipose tissue. When lipids are broken down, carbon will be released, which could explain the increase in carbon content.
Carbonate
Consensus is reached about the decrease of the organic component carbonate when bones are exposed to heat. In researches by Haberko et al. (2006) and Marques et al. (2018) it is concluded that there is a decrease in the amount of carbonate from 700°C and higher temperatures. However, Figueiredo et al. (2010) stated that carbonate loss was initiated at 600°C and according to Snoeck et
al. (2014), this process was already started at 500°C. Besides this, Reidsma et al. (2016) observed a
small release of carbonate at temperatures between 250°C to 340°C, with a larger decline at temperatures of 600°C and higher. These differences in temperatures could be explained by the accuracy of the methods, since both Reidsma et al. (2016) and Figueiredo et al. (2010) did their experiments in an oxygen free environment. The loss of carbonate between 250°C and 340°C might be negligible compared to the amounts that are released at higher temperatures; therefore, it might be possible that in not all the experiments this decrease was detected or taken into account. Since this decline was only measured by Reidsma et al. (2016), it could be the case that this release of carbonate was either a coincidence or negligible. Therefore, a reasonable decline occurs around temperatures of 600°C to 700°C which stops around 900°C due to a total degradation of carbonate (Figueiredo et al., 2010) (figure 1).
Other organic compounds
Besides collagen fibres, other proteins such as albumin and ɑ2-HS-glycoprotein can also be found in bone tissue (Clarke, 2008). Usually collagen degradation due to HI-changes is investigated or protein degradation in general, and no research is conducted on differences between different proteins (Marques et al., 2018; Reidsma et al., 2016). Since proteins mainly consist of carbon, hydrogen and nitrogen, it could be assumed that they all have very similar properties and that the differences among degradation due to heating are negligible. Therefore, it could be difficult and maybe not even necessary to add more compounds in the future. In addition to that, no research has been done on the effects of heat on blood present in bone. Major components in blood are water, which will evaporate at relatively low temperatures during dehydration, and haemoglobin. Since haemoglobin is a protein, it is very likely that it will be disappeared around temperatures of 370°C (Reidsma et al., 2016). Another organic compound that can be found in bones are several hormones, among which calcitriol and thyroxine (Martini, 2006). However, hormone concentrations differ substantially among people, which makes it complicated to estimate the evaporation of these compounds. The same holds for vitamins, of which the amounts that are present depend on the amounts that are consumed. Therefore, hormone and vitamin evaporation are not reliable for the estimation of the temperature to which the bones are exposed.
Calcium and phosphor
Reidsma et al. (2016) detected no changes in the content of calcium and phosphor when bovine bones were exposed to heat. However, it is not clear how these contents were determined and whether only free calcium and phosphor or that also connections with these compounds were taken into account. Both calcium and phosphor can incorporate in different molecules that can be found in bone tissue, such as hydroxyapatite and tricalcium phosphate. It could be expected that there is a relocation of calcium and phosphor as an effect of heat exposure.
Hydroxyapatite
In general, there is consensus about the increase in size, and eventually the fusion, of hydroxyapatite crystals and that this increase is not linear (Castillo et al., 2013; Holden et al., 1995; Shipman et al., 1984). In the research that is done, other temperature ranges are used but all come to the same conclusion: There is a larger increase in crystal formation with increasing temperature. In addition to that, the fusion of the crystals and the formation of hydroxyapatite into other compounds such as tricalcium phosphate and calcium carbonates, leads to a decrease in hydroxyapatite (Civjan et al., 1972; Holden et al.,1995) (figure 1). As can be seen in table 1, the results of Holden et al. (1995) show
that there is an increase in crystal size and a change in the shape of these crystals. The crystals that are formed at first have a spherical shape, and when the temperature increases hexagonal and later rhombohedral shaped crystals appear (Holden et al., 1995). However, these observations are considered to be subjective since it depends on the investigator how the crystals are classified. This makes these results less reliable than the methods that measure the sizes of the crystals rather than classifying them based on the shape. Additionally, the relevance of the crystal shape could be considered, and it is important to mention that there are examples known of compounds influencing the amount of crystals. Since carbonate is located in the hydroxyapatite crystals, the content of carbonate that is built in the hydroxyapatite affects the amount of crystallisation, in a way that larger amounts of carbonate decrease the number of crystals, leading towards their degradation (Barrère, Van Blitterswijk & De Groot, 2006).
Tricalcium phosphate
There is no consensus reached among the formation of β- and ɑ-tricalcium phosphate. Several studies suggest that hydroxyapatite is transformed into tricalcium phosphate when it is exposed to high temperatures (Bonucci & Graziani, 1975; Civjan et al., 1972; Holden et al., 1995); however, the temperatures at which these compounds are detected are not clear. Holden et al. (1995) found tricalcium phosphates at higher temperatures (1200°C and higher) compared to Civjan et al. (1972) and Bonucci & Graziani (1975) (600°C to 800°C). An explanation for this could be a difference in the methods that were used, since Bonucci & Graziani (1975) and Civjan et al. (1972) used thermogravimetry, while Holden et al. (1995) used SEM to investigate the crystallite formation. Shipman et al. (1984) and Roger & Daniels (2002) however, did not find any evidence for the conversion of hydroxyapatite into β- and ɑ-tricalcium phosphate. Since they both used XRD in their research (Shipman et al., 1984; Roger & Daniels, 2002), the difference in the methods that were applied could declare why their results did not support the statement that hydroxyapatite was transformed into β-tricalcium phosphate. Another possible explanation for the dissimilarity in the results is the difference in exposure time. Unfortunately, Civjan et al. (1972) and Bonucci & Graziani (1975) did not mention the duration of exposure that was used in their experiments. In case the exposure time was longer than the time used by Shipman et al. (1984) and Roger & Daniels (2002), 4 hours and 2 hours respectively, it could be stated that β- and ɑ-tricalcium phosphate does not form when it is not exposed to high temperatures for long enough. However, this could explain the different outcomes, it is important to mention on what the conclusions of the experiments conducted by Civjan et al. (1972), Bonucci & Graziani (1975) and Holden et al. (1995) are based. All that was observed were changes in the inorganic matter which could be explained by the formation of tricalcium phosphate. In none of their experiments it was proven that tricalcium phosphate was detected, it was only thought or assumed to be present. No actual measurements were done to confirm which compounds were present at these high temperatures. Other compounds among which calcium carbonates and magnesium ions, could also explain the observed alterations in the inorganic matter of bones (Civjan et al., 1972). Since tricalcium phosphate is not detected with certainty in any of the experiments and it is was only thought to be present in several experiments, it could be concluded that there is no proof for the formation of tricalcium phosphate when bones are exposed to high temperatures. More research should be done on the possible formation of this compound and identification of other possible burning products in crystals.
Calcium oxide
Several researches showed that calcium oxide is formed when bones are exposed to heat (Figueiredo
et al., 2010; Haberko et al., 2006; Holden et al., 1995; Rogers & Daniels, 2002). However, among the
temperature at which this compound can be found, no consensus is reached (700°C or higher (Haberko et al., 2006; Rogers & Daniels, 2002) or 950°C or higher (LeGeros & LeGeros, 1984)). Both the results of Rogers & Daniels (2002) and LeGeros & LeGeros (1984) were obtained by using XRD and human bones, these aspects of the method cannot explain the difference in their outcomes. Haberko
exposure may explain why LeGeros & LeGeros (1984) found calcium oxide formation only at higher temperatures, but unfortunately, the exposure time was not mentioned in neither their article nor the article by Haberko et al. (2006). In the research conducted by Rogers & Daniels (2002), bones were exposed to heat for 2 hours. In case LeGeros & LeGeros (1984) used a shorter time in their experiment, it is possible that there was not enough time for the calcium oxide to form. Another possible explanation for the difference in outcome could be a difference in the age of the donors, since the mineral composition in bone alters due to ageing. A larger amount of rod-shaped hydroxyapatite crystals is observed in older individuals and it changes chronologically (Holden et al., 1995). The age range of the donors in the experiment done by Rogers & Daniels (2002) was 42 to 79 years, but the age of the donors of LeGeros & LeGeros (1984) is unknown. Therefore, it could be concluded that the initiation of calcium oxide formation around 700°C to 950°C, or more general, around 800°C (figure 1).
Other inorganic compounds
As mentioned earlier, other compounds such as calcium phosphates and magnesium phosphates are also present in the bone matrix. However, in relation to hydroxyapatite are these compounds only found in small amounts (LeGeros & LeGeros, 1984). No research that has been conducted, mentioned any of these compounds, but they could be located in the hydroxyapatite crystals and due to their small amounts, they might not be detected. Further research could be done on whether other calcium phosphates and magnesium phosphates are relevant indicators for the estimation of the temperature during heat exposure and what their role could be in this estimation. Research on other minerals that can be found in bones, among which sodium and fluoride, has not been done either. These minerals are present in only small amounts (Martini, 2006) and might therefore not be visible during investigation. These minerals might only be built in the bone matrix instead of calcium as a coincidence (Martini, 2006), and are present in the body due to consumed food or medicines. However, a release of magnesium is detected during the third stage of burning bones, inversion (Correia, 1997). More research should be done on the temperatures at which this release occurs to make magnesium applicable in determining the burning temperature. The same argumentation of problems with detecting small amounts of mineral applies to iron which is present in the haemoglobin of erythrocytes. As discussed earlier, it is expected that haemoglobin will disappear around 370°C (Reidsma et al., 2016). Iron, however, will melt at temperatures of at least 1538°C and evaporate at temperatures of at least 2862°C (Verkerk, 2013). Therefore, it could be expected that iron remains in bone tissue after heat exposure. No information among iron detection in heated or burned bones can be found in the literature. However, this could be important information, especially in cases of trauma and heat exposure. If known which amounts of iron are commonly found in burned bones, a distinction could be made between injured and uninjured bones.
Synthesis
As discussed earlier, there are many components of the bone matrix of which is little known about how they alter during heat exposure. The compounds that are investigated are researched in different manners which could result in different outcomes. Aspects that could influence the outcome of an experiment are for example the type of bone that is used, whether the bone originates from a human or an animal and the age of the bones. However, since the figure that is created shows a general trend, these aspects might be negligible in interpreting the data. An aspect that could have a higher relevance in the different outcomes of the experiments, which is not extensively discussed yet, is the difference between combustion and pyrolysis. While some researchers burned the bones in an environment that is low in oxygen, causing pyrolysis (Figueiredo et al., 2010; Reidsma et al., 2016), others exposed the bones to air while burning the bones, leading to combustion (Holden et al., 1995; Holden et al., 1995; Marques, 2018). In the presence or absence of oxygen other degradation processes are occurring, which could affect the substances that are detected and the temperatures at which these substances are detected. Another important aspect which could affect the results of an investigation, is the method in which bones are exposed to heat. Possible ways to heat up bones
would be in an oven or by using a fire, and this difference could alter the reactions that are occurring during degradation. Besides that, some researchers heated up a bone to the highest temperature and did measurements in between at the selected temperatures (Holden et al., 1995; Holden et al., 1995). Other researchers exposed different bones to different temperatures and then determined the difference between before and after exposure (Figueiredo et al., 2010; Reidsma et al., 2016). Hypothetically, it would be expected that the content of adipose tissue is altered when measuring the bone at 800°C. However, this adipose tissue already is disappeared around 340°C. This is something that should be taken into account when interpreting these kinds of results.
Conclusion
To summarise, several conclusions can be made concerning HI-changes to the organic components of the bone matrix. At first, there will be a 15% increase in carbon content and a 4% decrease in nitrogen content when exposing bones to temperatures of between 200°C to 450°C. At temperatures higher than 450°C, both the carbon and nitrogen content remain stable. Besides that, the hydrogen content is not altered when exposing bones to temperatures ranging from 200°C to 900°C. Thereafter, lipids in the adipose tissue will be completely disappeared around 340°C, followed by the disappearance of collagen fibres and other proteins at 370°C. The evaporation of these proteins will be finalised when heating the bone up to temperatures of around 800°C to 900°C. A large decrease in carbonate content will be observed at temperatures of 600°C to 700°C and at 900°C all carbonate will be degraded.
Additionally, various alterations of inorganic components of the bone matrix due to heat exposure are indicated. The content of calcium and phosphor is not changed when exposed to high temperatures, which could be explained by a relocation of these compounds in the inorganic matrix. At temperatures of 600°C, there is an increase in hydroxyapatite crystal formation, and this increase is becoming larger with increasing temperatures. At temperatures of approximately 800°C, carbon oxide will become visible in the bone tissue and its content will increase with increasing temperature. At temperatures of around 1600°C, a decrease of hydroxyapatite is observed, which could be explained by the formation of hydroxyapatite into tricalcium phosphate and calcium carbonates. However, there is no proof of the formation of tricalcium phosphate due to heat exposure, since the literature is contradictory.
As mentioned earlier, the bone consists out of many more components than the ones shown in figure 1. Further research could point out which compounds could be valuable to include in the temperature estimation of bones after heat exposure. Additionally, there is uncertainty among the formation of tricalcium phosphate, which could be an important indicator for high burning temperatures. Besides that, it is unclear whether other compounds are formed in the bone crystals at high temperatures when there is a decrease in hydroxyapatite. Another compound that could be investigated more is magnesium. This compound is usually detected during inversion and more research could make this compound applicable in estimating the temperatures to which bones were exposed. Besides that, the amount of iron found in burned bone could be a good indicator for the presence of trauma before being exposed to the fire.
In conclusion, an overview of organic and inorganic components in bone tissue and their value in estimating the heating temperature is described. The compounds that are considered valuable at this moment are collagen and other proteins, adipose tissue, carbon, nitrogen, carbonate, hydroxyapatite and calcium oxide. Additionally, the HI-changes of these components are extensively described and shown in a table.
References
1. Alunni, V., Grevin, G., Buchet, L., & Quatrehomme, G. (2014). Forensic aspect of cremations on wooden pyre. Forensic science international, 241, 167-172.
2. Barrère, F., van Blitterswijk, C. A., & de Groot, K. (2006). Bone regeneration: molecular and cellular interactions with calcium phosphate ceramics. International journal of nanomedicine,
1(3), 317.
3. Bonucci, E., & Graziani, G. (1975). Comparative thermogravimetric, X-ray diffraction and electron microscope investigations of burnt bones from recent, ancient and prehistoric age.
Atti della Accademia Nazionale dei Lincei. Classe di Scienze Fisiche, Matematiche e Naturali. Rendiconti, 59(5), 517-532.
4. Braadbaart, F., Wright, P. J., Van der Horst, J., & Boon, J. J. (2007). A laboratory simulation of the carbonization of sunflower achenes and seeds. Journal of Analytical and Applied
Pyrolysis, 78(2), 316-327.
5. Castillo, R. F., Ubelaker, D. H., Acosta, J. A. L., & De la Fuente, G. A. C. (2013). Effects of temperature on bone tissue. Histological study of the changes in the bone matrix. Forensic
science international, 226(1-3), 33-37.
6. Castillo, R. F., Ubelaker, D. H., Acosta, J. A. L., De la Rosa, R. J. E., & Garcia, I. G. (2013). Effect of temperature on bone tissue: histological changes. Journal of forensic sciences, 58(3), 578-582.
7. Civjan, S., Selting, W. J., De Simon, L. B., Battistone, G. C., & Grower, M. F. (1972). Characterization of osseous tissues by thermogravimetric and physical techniques. Journal of
dental research, 51(2), 539-542.
8. Clarke, B. (2008). Normal bone anatomy and physiology. Clinical journal of the American
Society of Nephrology, 3(Supplement 3), S131-S139.
9. Correia, P. M. (1997). Fire modification of bone: a review of the literature. Forensic
taphonomy: the postmortem fate of human remains, 275-293.
10. Dequeker, J., & Merlevede, W. (1971). Collagen content and collagen extractability pattern of adult human trabecular bone according to age, sex and amount of bone mass. Biochimica et Biophysica Acta (BBA)-General Subjects, 244(2), 410-420.
11. Ellingham, S. T., Thompson, T. J., Islam, M., & Taylor, G. (2015). Estimating temperature exposure of burnt bone—a methodological review. Science & Justice, 55(3), 181-188.
12. Figueiredo, M., Fernando, A., Martins, G., Freitas, J., Judas, F., & Figueiredo, H. (2010). Effect of the calcination temperature on the composition and microstructure of hydroxyapatite derived from human and animal bone. Ceramics international, 36(8), 2383-2393.
13. Haberko, K., Bućko, M. M., Brzezińska-Miecznik, J., Haberko, M., Mozgawa, W., Panz, T., Pyda, A. & Zarębski, J. (2006). Natural hydroxyapatite—its behaviour during heat treatment. Journal
of the European Ceramic Society, 26(4-5), 537-542.
14. Holden, J. L., Clement, J. G., & Phakey, P. P. (1995). Age and temperature related changes to the ultrastructure and composition of human bone mineral. Journal of Bone and Mineral
Research, 10(9), 1400-1409.
15. Holden, J. L., Phakey, P. P., & Clement, J. G. (1995). Scanning electron microscope observations of heat-treated human bone. Forensic Science International, 74(1-2), 29-45.
16. Jackson, A. R., & Jackson, J. M. (2008). Forensic science. Pearson Education.
17. Krap, T., Nota, K., Wilk, L. S., van de Goot, F. R., Ruijter, J. M., Duijst, W., & Oostra, R. J. (2017). Luminescence of thermally altered human skeletal remains. International journal of legal
medicine, 131(4), 1165-1177.
18. LeGeros, R. Z., & LeGeros, J. P. (1984). Phosphate minerals in human tissues. In Phosphate
minerals (pp. 351-385). Springer, Berlin, Heidelberg.
19. Mamede, A. P., Gonçalves, D., Marques, M. P. M., & Batista de Carvalho, L. A. (2018). Burned bones tell their own stories: A review of methodological approaches to assess heat-induced diagenesis. Applied Spectroscopy Reviews, 53(8), 603-635.
20. Marques, M. P. M., Mamede, A. P., Vassalo, A. R., Makhoul, C., Cunha, E., Gonçalves, D., Parker, S. F. & Batista de Carvalho, L. A. E. (2018). Heat-induced Bone Diagenesis Probed by Vibrational Spectroscopy. Scientific reports, 8(1), 15935.
22. Memmler, R. L., & Rada, R. B. (1970). The human body in health and disease.
23. Reidsma, F. H., van Hoesel, A., van Os, B. J., Megens, L., & Braadbaart, F. (2016). Charred bone: Physical and chemical changes during laboratory simulated heating under reducing conditions and its relevance for the study of fire use in archaeology. Journal of Archaeological
Science: Reports, 10, 282-292.
24. Rogers, K. D., & Daniels, P. (2002). An X-ray diffraction study of the effects of heat treatment on bone mineral microstructure. Biomaterials, 23(12), 2577-2585.
25. Shipman, P., Foster, G., & Schoeninger, M. (1984). Burnt bones and teeth: an experimental study of color, morphology, crystal structure and shrinkage. Journal of archaeological science,
11(4), 307-325.
26. Snoeck, C., Lee-Thorp, J. A., & Schulting, R. J. (2014). From bone to ash: Compositional and structural changes in burned modern and archaeological bone. Palaeogeography,
palaeoclimatology, palaeoecology, 416, 55-68.
27. Verkerk, G. (2013). Binas havo/vwo (6e editie). Noordhof.
Appendix 1: Search Strategy
Provided by T. Krap:
Castillo, R. F., Ubelaker, D. H., Acosta, J. A. L., & De la Fuente, G. A. C. (2013). Effects of temperature on bone tissue. Histological study of the changes in the bone matrix. Forensic science international,
226(1-3), 33-37.
Castillo, R. F., Ubelaker, D. H., Acosta, J. A. L., De la Rosa, R. J. E., & Garcia, I. G. (2013). Effect of temperature on bone tissue: histological changes. Journal of forensic sciences, 58(3), 578-582. Ellingham, S. T., Thompson, T. J., Islam, M., & Taylor, G. (2015). Estimating temperature exposure of burnt bone—a methodological review. Science & Justice, 55(3), 181-188.
Krap, T., Nota, K., Wilk, L. S., van de Goot, F. R., Ruijter, J. M., Duijst, W., & Oostra, R. J. (2017). Luminescence of thermally altered human skeletal remains. International journal of legal medicine,
131(4), 1165-1177.
Mamede, A. P., Gonçalves, D., Marques, M. P. M., & Batista de Carvalho, L. A. (2018). Burned bones tell their own stories: A review of methodological approaches to assess heat-induced diagenesis.
Applied Spectroscopy Reviews, 53(8), 603-635.
Marques, M. P. M., Mamede, A. P., Vassalo, A. R., Makhoul, C., Cunha, E., Gonçalves, D., Parker, S. F. & Batista de Carvalho, L. A. E. (2018). Heat-induced Bone Diagenesis Probed by Vibrational Spectroscopy. Scientific reports, 8(1), 15935.
Thompson, T. (2004). Recent advances in the study of burned bone and their implications for forensic anthropology. Forensic Science International, 146, S203-S205.
Ubelaker, D. H. (2009). The forensic evaluation of burned skeletal remains: a synthesis. Forensic
science international, 183(1-3), 1-5.
To obtain knowledge about the structure and components of bones, I used the textbooks from my bachelor/master:
Martini, F. (2006). Anatomy and Physiology'2007 Ed. Rex Bookstore, Inc.. Memmler, R. L., & Rada, R. B. (1970). The human body in health and disease. White, T. D., & Folkens, P. A. (2005). The human bone manual. Elsevier. Jackson, A. R., & Jackson, J. M. (2008). Forensic science. Pearson Education.
To find the melting and boiling point of iron:
Verkerk, G. (2013). Binas havo/vwo (6e editie). Noordhof.
'Fire bone mineral': Correia, P. M. (1997). Fire modification of bone: a review of the literature.
Forensic taphonomy: the postmortem fate of human remains, 275-293.
'Bone physiology': Clarke, B. (2008). Normal bone anatomy and physiology. Clinical journal of
the American Society of Nephrology, 3(Supplement 3), S131-S139.
'Calcium oxide bone heat'
Holden, J. L., Clement, J. G., & Phakey, P. P. (1995). Age and temperature related changes to the ultrastructure and composition of human bone mineral. Journal of Bone and Mineral Research, 10(9), 1400-1409.
Rogers, K. D., & Daniels, P. (2002). An X-ray diffraction study of the effects of heat treatment on bone mineral microstructure. Biomaterials, 23(12), 2577-2585.
'Collagen bone fire':
Reidsma, F. H., van Hoesel, A., van Os, B. J., Megens, L., & Braadbaart, F. (2016). Charred bone: Physical and chemical changes during laboratory simulated heating under reducing conditions and its relevance for the study of fire use in archaeology. Journal of Archaeological Science: Reports, 10, 282-292.
'Temperature cremation accident':
Alunni, V., Grevin, G., Buchet, L., & Quatrehomme, G. (2014). Forensic aspect of cremations on wooden pyre. Forensic science international, 241, 167-172.
'Hydroxyapatite heat bone':
Haberko, K., Bućko, M. M., Brzezińska-Miecznik, J., Haberko, M., Mozgawa, W., Panz, T., Pyda, A. & Zarębski, J. (2006). Natural hydroxyapatite—its behaviour during heat treatment. Journal of the European Ceramic Society,
26(4-5), 537-542.
Holden, J. L., Phakey, P. P., & Clement, J. G. (1995). Scanning electron microscope observations of heat-treated human bone. Forensic Science
International, 74(1-2), 29-45.
Figueiredo, M., Fernando, A., Martins, G., Freitas, J., Judas, F., & Figueiredo, H. (2010). Effect of the calcination temperature on the composition and microstructure of hydroxyapatite derived from human and animal bone.
Ceramics international, 36(8), 2383-2393.
‘Amount collagen bone’:
Dequeker, J., & Merlevede, W. (1971). Collagen content and collagen extractability pattern of adult human trabecular bone according to age, sex and amount of bone mass. Biochimica et Biophysica Acta (BBA)-General Subjects, 244(2), 410-420.
Found in reference list of other used articles: Ubelaker, D. H. (2009). The forensic evaluation of burned skeletal remains: a synthesis.
Forensic science international, 183(1-3), 1-5:
Shipman, P., Foster, G., & Schoeninger, M. (1984). Burnt bones and teeth: an experimental study of color, morphology, crystal structure and shrinkage. Journal of archaeological science,
11(4), 307-325.
Correia, P. M. (1997). Fire modification of bone: a review of the literature. Forensic
taphonomy: the postmortem fate of human remains, 275-293.:
Bonucci, E., & Graziani, G. (1975). Comparative thermogravimetric, X-ray diffraction and electron microscope investigations of burnt bones from recent, ancient and prehistoric age. Atti della
Accademia Nazionale dei Lincei. Classe di Scienze Fisiche, Matematiche e Naturali. Rendiconti, 59(5), 517-532.
Civjan, S., Selting, W. J., De Simon, L. B., Battistone, G. C., & Grower, M. F. (1972). Characterization of osseous tissues by thermogravimetric and physical techniques.
Journal of dental research, 51(2), 539-542.
Ellingham, S. T., Thompson, T. J., Islam, M., & Taylor, G. (2015). Estimating temperature exposure of burnt bone—a methodological review. Science & Justice, 55(3), 181-188:
Castillo, R. F., Ubelaker, D. H., Acosta, J. A. L., & de la Fuente, G. A. C. (2013). Effects of temperature on bone tissue. Histological study of the changes in the bone matrix. Forensic science international,
226(1-3), 33-37.
Correia, P. M. (1997). Fire modification of bone: a review of the literature. Forensic
taphonomy: the postmortem fate of human remains, 275-293.
Bonucci, E., & Graziani, G. (1975). Comparative thermogravimetric, X-ray diffraction and electron microscope investigations of burnt bones from recent, ancient and prehistoric age. Atti della
Accademia Nazionale dei Lincei. Classe di Scienze Fisiche, Matematiche e Naturali. Rendiconti, 59(5), 517-532.
Civjan, S., Selting, W. J., De Simon, L. B., Battistone, G. C., & Grower, M. F. (1972). Characterization of osseous tissues by thermogravimetric and physical techniques.
Journal of dental research, 51(2), 539-542.
Holden, J. L., Clement, J. G., & Phakey, P. P. (1995). Age and temperature related changes to the ultrastructure and composition of human bone mineral. Journal of Bone and
Mineral Research, 10(9), 1400-1409.
LeGeros, R. Z., & LeGeros, J. P. (1984). Phosphate minerals in human tissues. In Phosphate minerals (pp. 351-385). Springer, Berlin, Heidelberg.
Reidsma, F. H., van Hoesel, A., van Os, B. J., Megens, L., & Braadbaart, F. (2016). Charred bone: Physical and chemical changes during laboratory simulated heating under reducing conditions and its relevance for the study of fire use in archaeology. Journal of
Archaeological Science: Reports, 10, 282-292.
Braadbaart, F., Wright, P. J., Van der Horst, J., & Boon, J. J. (2007). A laboratory simulation of the carbonization of sunflower achenes and seeds.
Journal of Analytical and Applied Pyrolysis, 78(2),
316-327.
Figueiredo, M., Fernando, A., Martins, G., Freitas, J., Judas, F., & Figueiredo, H. (2010).
Barrère, F., van Blitterswijk, C. A., & de Groot, K. (2006). Bone regeneration: molecular and cellular
Effect of the calcination temperature on the composition and microstructure of hydroxyapatite derived from human and animal bone. Ceramics international, 36(8), 2383-2393.
interactions with calcium phosphate ceramics.
International journal of nanomedicine, 1(3), 317.
Mamede, A. P., Gonçalves, D., Marques, M. P. M., & Batista de Carvalho, L. A. (2018). Burned bones tell their own stories: A review of methodological approaches to assess heat-induced diagenesis. Applied Spectroscopy
Reviews, 53(8), 603-635.
Snoeck, C., Lee-Thorp, J. A., & Schulting, R. J. (2014). From bone to ash: Compositional and structural changes in burned modern and
archaeological bone. Palaeogeography,
