The micro-analysis of human burned bones: some remarks

ESTADO DA ARTE The micro-analysis of human burned bones: some remarks David Gonçalves* Centro de Investigação em Antropologia e Saúde – Universidade de Coimbra, Portugal Centro de Ciências Forenses – Instituto Nacional de Medicina Legal, IP, Portugal Laboratório de Arqueociências, IGESPAR, IP and LARC/CIBIO/InBIO, Portugal *davidmiguelgoncalves@gmail.com Artigo recebido a 23 de Março de 2012 e aceite a 09 de Maio de 2012 ABSTRACT The interdisciplinary research of burned bones is focused in this paper by presenting and discussing some methods that can assist the bioanthropologist in the analysis of this kind of remains. In particular, some techniques based on the histological structure of bone and on its molecular composition allow new ways of identifying burned human bone and of determining some aspects of the biological and ontological profile of an individual. A brief summary of those techniques is thus here presented. Keywords: biological anthropology; forensic anthropology; bioarchaeology; burned human bone identification; stable isotopes; radiocarbon dating RESUMO A investigação de ossos queimados baseada numa abordagem interdisciplinar é focada no presente artigo a partir da apresentação e discussão de alguns métodos que podem ser úteis ao bioantropólogo envolvido na análise deste tipo de restos humanos. Em particular, algumas técnicas recentemente desenvolvidas e baseadas na histologia do osso e na sua composição molecular podem contribuir para a identificação de osso humano queimado e para a 32 Gonçalves /Cadernos do GEEvH 1 (1) 2012: 32-40 determinação de alguns aspetos relacionados com o perfil biológico e ontológico do indivíduo. Uma breve descrição dessas técnicas é aqui apresentada. Palavras-chave: antropologia biológica; antropologia forense; bioarqueologia; identificação de ossos humanos queimados; isótopos estáveis; datação por radiocarbono Introduction Identifying burned bone Although some progress has been made in recent years, nowadays burned bones still represent one of the main challenges that bioanthropologists have to face whilst analyzing human skeletons. This is so because of the high fragmentation and other heat-related changes affecting bones that inevitably impair our ability to retrieve information from them. As a result, a critique of the bioanthropological methods that are conventionally used is required whenever burned bones are analyzed. This has been done to some degree in the past (Van Vark et al., 1974; 1975; Buikstra and Swegle, 1989; McKinley, 1989; Duday et al., 2000; Thompson, 2002, 2005; Ubelaker, 2009; Gonçalves et al., 2011a; Gonçalves et al., 2011b; Gonçalves, 2012). However, bioanthropologists are sometimes unaware of the potential of other approaches regarding the analysis of burned bones which can unquestionably increase the amount of information drawn from this sort of human remains. Therefore, a summation of those potentialities is here presented with the aim of assisting the bioanthropologist in his task of, not only analyzing bones, but also of maximizing the retrieval of data from them by resorting to other than bioanthropological analyses. The determination of whether or not the bone is burned is a very important issue. This assessment influences our own ability to identify human bone fragments and sets the bioanthropological methods that are to be used in the analysis. However, assessing if bone was affected by a heat-source is not a straightforward procedure. Although some macroscopic heat-induced changes – colour, fractures, dimension and warping – may assist us in such determination (revised by: Duday et al., 2000; McKinley and Bond, 2001; Silva, 2007), these do not always allow for a conclusive judgment because other taphonomic and pathological factors can mimic them. For instance, colour changes can occur in bone as a result of soil discolouration or sun exposure (Shahack-Gross et al., 1997; Buikstra and Ubelaker, 1994). Fractures can be produced by several agents such as the ones related to bioturbation or to weathering (Buikstra and Ubelaker, 1994). Postdepositional changes in size can also occur (Piepenbrink, 1986), although probably not in such a significant degree as the dimensional alterations witnessed in burned bones. Warped bones can be caused by several pathologies like rickets, osteomalacia, Paget’s disease (Mays, 2008) or congenital syphilis (Ortner, 2008). Therefore, the recognition of 33 Gonçalves /Cadernos do GEEvH 1 (1) 2012: 32-40 A different approach has been taken by other researchers who have used light microscopy to observe the histological features of sectioned bone. Hanson and Cain (2007) used the microscopic internal structure to differentiate burned from unburned bones of sheep. Although no differences could be found between unburned bones and bones burned at low temperatures, some changes were documented as being the result of burning at medium/high or at high temperatures. Specifically, cracks extending outwards from the haversian canals were present in the first case while a loss of histological structures was observed in the second one. In an investigation with bone specimens from modern cattle, Harbeck et al. (2011) found that heat-induced changes – composed of small fissures – first appear in bone heated at 200o C, while a more marked deterioration of the histological structure was documented at 500o C. Corroborating the observations made by Hanson and Cain (2007), the structural elements were no longer distinguishable in bone sections heated at 800o C. Unfortunately, these guidelines are not straightforward. Contrastingly, Cattaneo et al. (1999) were still able to discern the haversian systems in human and non-human bone heated at 800-1200o C under the light microscope. This finding had already been documented by Holden et al. (1995) while resorting to the scanning electron microscope (SEM) to analyse human bone. Therefore, although a distinction between burned and unburned bone is feasible by looking at the histological structure, a more specific determination of the maximum temperature at which the burning occurred is bone affected by heat is not always an easy task. Some researchers have turned their attention to alternative ways of identifying burned bones. For this purpose, the potential of the crystallinity index (CI) – or splitting factor – has been investigated intensively in the last years (Stiner et al., 1995; Koon et al., 2003; Munro et al., 2007; Olsen et al., 2008; Thompson et al., 2009). The CI measures the order of the crystal structure and composition within bone. The premise behind its use is that the CI increases as crystals become larger and more ordered (Trueman et al., 2008). This naturally occurs at a gradual rate after death but this process is fastened by some diagenetic pathways (Thompson et al., 2009) and an exponential acceleration is furthermore promoted by weathering and heat (e.g.: Stiner et al., 1995; Olsen et al., 2008). Although taphonomy indeed interferes with the CI values, this approach has nonetheless good potential for the identification of burned bones – as long as fossilized bones and weathered bones are left out of this kind of analyses. This is particularly so if the CI values are interpreted in association with the carbonate to phosphate ratio according to Thompson et al. (2009). Even though the precise temperature at which bone was submitted to cannot be determined, these authors state that a differentiation between low and high temperature burnings can be established. The CI is assessed by X-ray diffraction (XRD), small-angle X-ray scattering (SAXS) or Fourier Transform Infrared Spectroscopy (FTIR) analyses (Shipman et al., 1984; Hiller et al., 2003; Thompson et al., 2009). 34 Gonçalves /Cadernos do GEEvH 1 (1) 2012: 32-40 osteological remains is human or not. Fragmentation may be so extreme that no recognizable features are preserved. Therefore, some alternative methods have been investigated in the last few years. Cuijpers et al. (2006) stated that, at least for the primary diaphyseal bone structure, the difference between humans – which is essentially composed of lamellar bone – and some large mammals – which is composed of fibro-lamellar bone – is useful to make a distinction. The authors argue that, since no significant changes in bone microstructure occur at temperatures up to 800o C, the observation of its features is still achievable. Nonetheless, this statement still needs further validation because their research was carried out only on unburned bones. In addition, the authors state that bone structure alone may be sometimes misleading because fibro-lamellar bone is also present in humans during growing spurts or during fracture repairs so caution is required when using this approach. still problematic. The differentiation between low and high temperature burnings outlined by Hanson and Cain (2007) seems to be a more conservative and reliable approach while using this method. Shipman et al. (1984) and Nicholson (1993) also analysed microscopic features but focussed on the morphology of bone surface. This was done through SEM analysis. Although neither one have included unburned bone in their analyses, both have presented descriptions for various heating stages. In sum, an undulating surface with observable vascular canals was present at temperatures lower than 200o C; at approximately 300o C, bone surface presented a glassy appearance; then, bone acquired a frothy appearance when heated at 400-700o C; finally, melting and coalescence of particles into larger structures with very variable shapes occurred at temperatures above 800o C. The observations of both authors seem to be somewhat uniform thus possibly having good potential to infer more specific temperature determinations. However, weathering and fossilization are once again misleading agents because they can mimic heat-induced changes (Nicholson, 1993; Hanson and Cain, 2007). As a result, it also seems safer in this case to limit ourselves to distinguish bones heated at lower temperatures from bones heated at higher temperatures. Cattaneo et al. (2009) also used the histological structure to differentiate humans from non-humans. They chose to analyse osteons both metrically and morphologically in order to assess if these approaches were of some use in this matter. Indeed, the metric analysis allowed for a correct classification of all specimens by using discriminant function analysis specifically developed for this purpose, although the predicted correct classification was calculated to be of only 79%. As for the morphological assessment – in which any bone section presenting irregularly shaped osteons set in parallel rows and the presence Identifying human burned bone Heat-induced changes sometimes lead to the impossibility of determining macroscopically if an assemblage of 35 Gonçalves /Cadernos do GEEvH 1 (1) 2012: 32-40 of plexiform bone was determined to be nonhuman – this procedure was not as successful. Two to four cases were misclassified by two observers. taken prior to cremation. The contamination problems surrounding this kind of procedure were therefore highlighted. Harbeck et al. (2011) stated that the analyses of remains from modern crematoria and from archaeological context are not advisable because the handling of such remains can lead to contamination. Nonetheless, they were able to retrieve DNA from modern cattle tibiae subjected to temperatures up to 700o C. The authors stated that the duration of heat exposure has an important role in the preservation of genetic material. DNA analysis is then one more alternative to consider despite the limitations abovementioned. Another approach was taken by Cattaneo et al. (1994) who have attempted the immunological detection of human albumin in 31 archaeological cremations. Their success rate was of 26% and the authors concluded at this point that albumin can survive to cremation around 300o C on account of occasional incomplete cremation or thanks to the insulating effect of soft tissues on bones. In another investigation, Cattaneo et al. (2009) were still successful at detecting this protein in burned remains subjected to temperatures ranging from 800 to 1200o C, but this was so in only 4 out of 9 cases. This demonstrated that although burning events are very destructive of human albumin, it may still be preserved in some cases. Beckett et al. (2011) have proposed another method of identifying human bones. This one is based on the lattice parameters of bone mineral crystals. According to them, these present significant inter-species variation and can therefore be used to distinguish human from non-human bone through X-ray diffraction analysis. The investigation indicated that this method can be adopted when dealing with bones heated to temperatures up to 600o C and 1400o C. Nonetheless, these results were obtained on modern specimens and its value for archaeological materials is still unknown since diagenetic changes may interfere with the lattice parameters (Hedges, 2002). In addition, further validation of this method is required to confirm its reliability. Although having its own problems, DNA analysis of burned bones also seems to be of some value. Besides allowing for the identification of human remains, DNA may sometimes be the only way to achieve the identification of individuals or can also help on the biological and demographic profiling of paleo-populations. DNA retrieval is hardly achievable for burned bones and teeth because genetic material is very sensitive to heat thus preserving badly (Ye et al., 2004). Nonetheless, some researchers have been successful in doing so (Brown et al., 1995; Sweet and Sweet, 1995; Williams et al., 2004; Ye et al., 2004). Wurmb-Schwark et al. (2004) found out that the DNA retrieved from burned remains did not match a buccal swab Documenting the ontological profile Stable isotopic analyses may provide with important information regarding the 36 Gonçalves /Cadernos do GEEvH 1 (1) 2012: 32-40 burned bones. If the 200o C hurdle is indeed confirmed as an indicator of the usefulness of these analyses, then this means that many of the burned skeletal remains handled by bioanthropologists cannot be subject to such examinations. This is especially true for archaeological cremations since most remains present charring or calcination. ontological profile of an individual. For that purpose, carbon (C) and nitrogen (N) light elements give us some indication of the diet while oxygen (O) of bone apatite is useful to help determining the geographic origin of someone. In addition, strontium (Sr) gives clues about the geographic origin and the migrant movements. Harbeck et al. (2011) concluded that the latter is unaltered even at temperatures of 1000o C, so it can be examined in burned skeletal remains. This had already been demonstrated by Grupe and Hummel (1991). In contrast, the remaining elements stay only unchanged at temperatures lower than 200o C (Harbeck et al., 2011) which somewhat corroborates the previous conclusions presented by Deniro et al. (1985). Harbeck et al. (2011) thus state that no reliable biological signal should therefore be expected for specimens heated at higher temperatures and that bone colourations including black, grey or white are indicative of material that is unfit for these kinds of analyses. Besides knowing where an individual lived in, it is also important to know when that occurred. The dating of burned bones is nowadays a reality by using the structural carbonate from the mineral fraction of bone instead of using the collagen fraction. This is possible because carbonate ions are incorporated into the inorganic bone matrix in living organisms as a substitution for phosphate in the crystal lattice (Lanting et al., 2001; Olsen et al., 2008). Lanting et al. (2001) found that this structural carbonate could be used to successfully date burned bones by resorting to AMS dating techniques which require small amounts of bone (2 g). The success of such procedure however, is influenced by temperature and by recrystallization of the mineral matrix as was demonstrated by Olsen et al. (2008). These authors found a difference of approximately 160 14C years (sd = 34) between charred and calcined bones from the same individual of Late-Neolithic provenance. The bones heated at the lower temperatures – the charred ones – yielded slightly younger ages. Therefore, sampling should preferentially focus on calcined white bone. A negative correlation between 13 temperature and the δ C values was recorded by Harbeck et al. (2011). This was also observed by Deniro et al. (1985) but nothing of the sort was documented by Schurr et al. (2008). As for the δ15N, both of the latter have found an enrichment progression according to increasing temperature which is in contrast to what Harbeck et al. (2011) have found. Differences in sampling and experimental conditions may eventually explain these contrasting results. With the mentioned exception of Sr, the potential of stable isotopic analyses is apparently very limited for the inspection of 37 Gonçalves /Cadernos do GEEvH 1 (1) 2012: 32-40 Conclusion References The bioanthropological analysis of burned human skeletal remains may sometimes lead to a very limited amount of information due to the fragmentation and heat-induced changes that this kind of material often displays. A wider approach that does not rely only on the gross observation of bones may allow obtaining a better knowledge of the targeted individual. Beckett, S.; Rogers, K. D.; Clement, J. G. 2011. Inter-species variation in bone mineral behavior upon heating. Journal of Forensic Sciences, 56 (3): 571-579. Brown, K. A.; O'Donoghue, K.; Brown, T. A. 1995. DNA in cremated bones from an early bronze age cemetery cairn. International Journal of Anthropology, 5(2): 181-187. Buikstra, J.; Swegle, M. 1989. Bone modification due to burning: Experimental evidence. In: Bonnichsen, R.; Sorg, M. H. (Eds.) Bone modification. Orono, M.E., Center for the Study of the First Americans: 247-258. Buikstra, J.; Ubelaker, D. 1994. Standards for data collection from human skeletal remains: Proceedings of a seminar at the Field Museum of Natural History. Arkansas Archaeological Survey Report, 44. Some of the procedures that have been described may be expensive or hard to access to. Light microscopes or SEM are now more common, but the other equipments mentioned in this paper are not always as easy to reach. Nonetheless, such analytical approaches may prove priceless in some cases. Cattaneo, C.; Dimartino, S.; Scali, S.; Craig, O. E.; Grandi, M.; Sokol, R. J. 1999. Determining the human origin of fragments of burnt bone: A comparative study of histological, immunological and DNA techniques. Forensic Science International, 102(2-3): 181-191. Cattaneo, C.; Gelsthorpe, K.; Sokol, R. J.; Phillips, P. 1994. Immunological detection of albumin in ancient human cremations using elisa and monoclonal antibodies. Journal of Archaeological Science, 21: 565-571. The first concern when dealing with burned bones is to determine if these are indeed burned – and to what degree – and if they are actually human. Other assessments regarding the biological and ontological profiles should be achieved only after that confirmation is carried out because the selection of analytical methods depends on it. Cattaneo, C.; Porta, D.; Gibelli, D.; Gamba, C. 2009. Histological determination of the human origin of bone fragments. Journal of Forensic Sciences, 54 (3): 531-533. Cuijpers, A. 2006. Histological identification of bone fragments in archaeology: Telling humans apart from horses and cattle. International Journal of Osteoarchaeology, 16 (6): 465-480. Deniro, M. J.; Schoeninger, M. J.; Astorf, C. A. 1985. Effect of heating on the stable carbon and nitrogen isotope ratios of bone collagen. Journal of Archaeological Science, 12(1): 1-7. Duday, H.; Depierre, G.; Janin, T. 2000. Validation des paramètres de quantification, protocoles et stratégies dans l'étude anthropologique des sépultures secondaires à incinération. L'exemple des nécropoles protohistoriques du midi de la france. In: Dedet, B.; Gruat, P.; Marchand, G.; Py, M. ; Schwaller, M. (Eds.) Archéologie de la mort, archéologie de la tombe au premier âge du -fer. Lattes, UMR: 7-29. Acknowledgements The author would like to thank the anonymous reviewer for the suggestions and alterations made to the paper. Gonçalves, D. 2011a. The reliability of osteometric techniques for the sex determination of burned human skeletal remains. Homo - Journal of Comparative Human Biology, 62(5): 351-358. Gonçalves, D.; Thompson, T. J. U.; Cunha, E. 2011b. Implications of heat-induced changes in bone on the 38 Gonçalves /Cadernos do GEEvH 1 (1) 2012: 32-40 Olsen, J.; Heinemeier, J.; Bennike, P.; Krause, C.; Hornstrup, K. M.; Thrane, H. 2008. Characterization and blind testing of radiocarbon dating of cremated bone. Journal of Archaeological Science, 35(3): 791-800. interpretation of funerary behaviour and practice. Journal of Archaeological Science, 38(6): 1308-1313. Gonçalves, D. 2012. Cremains: The value of quantitative analysis for the bioanthropological research of burned human skeletal remains. PhD thesis in Biological Anthropology, Coimbra, University of Coimbra. Ortner, D. 2008. Differential diagnosis of skeletal lesions in infectious disease. In: Pinhasi, R.; Mays, S. (Eds.) Advances in Human Palaeopathology. Chichester, John Wiley & Sons Ltd.: 190-214. Grupe, G.; Hummel, S. 1991. Trace element studies on experimentally cremated bone. I. Alteration of the chemical composition at high temperatures. Journal of Archaeological Science, 18(2): 177-186. Piepenbrink, H. 1986. Two examples of biogenous dead bone decomposition and their consequences for taphonomic interpretation. Journal of Archaeological Science, 13(5): 417430. Hanson, M.; Cain, C. R. 2007. Examining histology to identify burned bone. Journal of Archaeological Science, 34(11): 1902-1913. Schurr, M. R.; Hayes, R. G.; Cook, D. C. 2008. Thermally induced changes in the stable carbon and nitrogen isotope ratios of charred bones. In: Schmidt, C. W.; Symes, S. A. (Eds.) The analysis of burned human remains. London, Academic Press: 95-108. Harbeck, M.; Schleuder, R.; Schneider, J.; Wiechmann, I.; Schmahl, W. W.; Grupe, G. 2011. Research potential and limitations of trace analyses of cremated remains. Forensic Science International, 204(1-3): 191-200. Shahack-Gross, R.; Bar-Yosef, O.; Weiner, S. 1997. Blackcolored bones in hayorium cave, israel: Differentiating between burning and oxide staining. Journal of Archaeological Science, 24 (5): 439-446. Hedges, R. E. M. 2002. Bone diagenesis: An overview of processes. Archaeometry, 44 (3): 319-328. Holden, J. L.; Phakey, P. P.; Clement, J. G. 1995. Scanning electron microscope observations of human femoral bone: A case study. Forensic Science International, 74(1): 17-28. Shipman, P.; Foster, G.; Schoeninger, M. 1984. Burnt bones and teeth: An experimental study of colour, morphology, crystal structure and shrinkage. Journal of Archaeological Science, 11 (4): 307-325. Koon, H. E. C.; Nicholson, R. A.; Collins, M. J. 2003. A practical approach to the identification of low temperature heated bone using TEM. Journal of Archaeological Science, 30(11): 1393-1399. Silva, F. C. 2007. Abordagem ao ritual funerário da cremação através da análise dos restos ósseos. Al-madan, 15 (II série): 40-48. Lanting, J. N.; Aerts-Bijma, A. T.; Plicht, J. V. D. 2001. Dating of cremated bones. Radiocarbon, 43 (2A): 249-254. Mays, S. 2008. Metabolic bone disease. In: Pinhasi, R.; Mays, S. (Eds.) Advances in Human Paleopathology. Chichester, John Wiley & Sons Ltd.: 215-251. Stiner, M. C.; Kuhn, S. L.; Weiner, S.; Bar-Yosef, O. 1995. Differential burning, recrystallization and fragmentation of archaeological bone. Journal of Archaeological Science, 22(2): 223-237. Mckinley, J. 1989. Cremations: Expectations, methodologies and realities. In: Roberts, C. A.; Lee, F.; Bintliffe, J. (Eds.) Burial archaeology. 65-78. Sweet, D. J.; Sweet, C. H. 1995. DNA analysis to dental pulp to link incinerated remains to homicide victim in crime scene. Journal of Forensic Sciences, 40 (2): 310-314. Mckinley, J.; Bond, J. M. 2001. Cremated bone. In: Brothwell, D. R.; Pollard, A. M. (Eds.) Handbook of Archaeological Sciences. Chichester, John Wiley and Sons: 281-292. Thompson, T. J. U. 2002. The assessment of sex in cremated individuals: Some cautionary notes. Canadian Society for Forensic Sciences, 35 (2): 49-56. Munro, L. E.; Longstaffe, F. J.; White, C. D. 2007. Burning and boiling of modern deer bone: Effects on crystallinity and oxygen isotope composition of bioapatite phosphate. Palaeogeography, Palaeoclimatology, Palaeoecology, 249 (12): 90-102. Thompson, T. J. U. 2005. Heat-induced dimensional changes in bone and their consequences for forensic anthropology. Journal of Forensic Sciences, 50 (5): 185-193. Thompson, T. J. U.; Gauthier, M. ; Islam, M. 2009. The application of a new method of Fourier transform infrared spectroscopy to the analysis of burned bone. Journal of Archaeological Science, 36(3): 910-914. Nicholson, R. A. 1993. A morphological investigation of burnt animal bone and an evaluation of its utility in archaeology. Journal of Archaeological Science, 20(4): 411-428. Trueman, C. N.; Privat, K.; Field, J. 2008. Why do crystallinity values fail to predict the extent of diagenetic alteration of 39 Gonçalves /Cadernos do GEEvH 1 (1) 2012: 32-40 bone mineral? Palaeogeography, Palaeoecology, 266(3-4): 160-167. Williams, D.; Lewis, M.; Franzen, T.; Lissett, V.; Adams, C.; Whittaker, D.; Tysoe, C.; Butler, R. 2004. Sex determination by PCR analysis of DNA extracted from incinerated, deciduous teeth. Science and Justice, 44 (2): 89-94. Palaeoclimatology, Ubelaker, D. H. 2009. The forensic evaluation of burned skeletal remains: A synthesis. Forensic Science International, 183(1-3): 1-5. Wurmb-Schwark, N. V.; Simeoni, E.; Ringleb, A.; Oehmichen, M. 2004. Genetic investigation of modern burned corpses. International Congress Series, 1261(1): 50-52. Van Vark, G. N. 1974. The investigation of human cremated skeletal material by multivariate statistical methods i. Methodology. Ossa, 1: 63-95. Ye, J.; Ji, A.; Parra, E.; Zheng, X.; Jiang, C.; Zhao, X.; Hu, L.; Tu, Z. 2004. A simple and efficient method for extracting DNA from old and burned bone. Journal of Forensic Sciences, 49 (4): 754-759. Van Vark, G. N. 1975. The investigation of human cremated skeletal material by multivariate statistical methods ii. Measures. Ossa, 2: 47-68. 40