Scanning electron microscope observations of heat-treated human bone

Forensic Science International 74 (1995) 29-45

Forensic Science Internatiial

Scanning electron microscope observations of heat-treated human bone J.L. Holden*a, P.P. Phakey”, J.G. Clementalb “Department of Physics, Monash University, Clayton, Victoria, Australia bSchool of Dental Science. University of Melbourne, Parkville, Victoria, Australia

Received 13 October 1993:revision received 17 December 1993;accepted 31 May 1994

Abstract

This report describes the heat-induced alterations in human bone tissue observed using scanning electron microscopy and microradiography. Femoral bone samples were taken from persons varying in age from 1 year to 97 years at the time of their death. The bone was heated at selected temperatures in the range 200-1600°C for periods of 2, 12, 18 and 24 h. Macroscopically, changes in colour occurred, together with some shrinkage, fracturing and distortion. However, dramatic changes occurred at the ultrastructural level. These changes included the progressive combustion of the organic portion of the bone tissue up to 400°C and recrystallisation of the bone mineral beginning at 600°C. Recrystallisation produced a range of crystal morphologies: spherical, hexagonal, platelets, rosettes and irregular. Crystal growth occurred at temperatures >6OO”C. Sintering led to fusion of crystals at 1000°C. This process continued up to temperatures > 1400°C. At 1600°C the bone mineral melted. On heating, the morphology of crystals formed, and the ultrastructural changes which occurred, were found to be related to the age of the deceased, the temperature to which the bone had been heated and the duration of heating. These results are of importance to forensic scientists, arson investigators and paleoarcheologists in their investigation of cremated human bones, particularly when only fragments of bone are available, in order to determine something of the life history of the deceased and the circumstances surrounding the death. Keywords: Human bone; Heat-treatment;

Ultrastructure;

Archaeology

* Corresponding author. 0379-0738/95/509.50 0 1995Elsevier Science Ireland Ltd. All rights reserved SSDl 0379-0738(95)01735-2

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1. Introduction Investigations into the effects of heat on the human body and skeleton have been of interest in the forensic [l-5] and anthropological [6- 131fields. Destruction of the human body by tire poses many difficulties which limit accurate determinations of the identity of the deceasedperson. Only in the last 30 years have ancient cremated bone finds been considered to be of value: prior to this cremated remains were considered to be impossible to interpret and analyse. In modem day situations, a fire destroys physical evidence such as clothing, documents, tattoos, fingerprints and hair which otherwise would aid the identification process[2]. Stevens[ 1,2] in a study of fatal civil aircraft accidents claimed that in the aftermath of a post-crash tire the most useful means of identifying the incinerated remains is through fire-resistant personal items such asjewellery or metal surgical implants if present, and X-rays and dental records if available. The effect of fire on the soft tissues of the human body is reported to be similar in appearanceto mechanical injuries [ 11,the depth of a burn being dependent on the degreeof heat, the duration of exposure and the region of body in contact with the heat [4]. For an average adult body subjected to a fire at a temperature of 680°C skeletonisation of the face and arms occurs after a period of 15 min, the ribs and skull appear after 20 min, and the thigh and shin bones appear after 35 min [3]. The amount of fat stored on the body influences the combustibility and rate of destruction of the flesh and soft tissues [3,7]. Accurate identification of fire victims and of historic cremated remains is therefore dependent on the degree of destruction and fragmentation, which is proportional in turn to the heat of the fire [3,10,13]. Fragmentation, when it occurs, can either be a direct result of the heat of the tire, which causesfracturing and splitting of the bone cortex [ 151, particularly along the contours of the ossification centres [ 131;or a result of an impact, such as from falling debris or an explosion; or in the caseof ancient cremations, a result of the deliberate crushing of the skeletal remains in order to place them in a burial vessel[6,7,8,10,13]. Age determination of deceasedpersons can either be carried out on a macroscopic scale by examining anatomical features of the human skeleton including both bone and teeth [ 141,or by examining the relationships between microscopic features such as osteons, interstitial lamella and circumferential lamella [ 151.Anatomical means of age determination in children include examining the degree of epiphyseal union and tooth development, and measurementsof the transverse diameter of the long bones [ 131.In adults the degreeof dental attrition, and the degreeof suture closure, can provide estimatesof age. Shrinkage, distortion and fragmentation of bone tissue, however, are limiting factors when applying macroscopic methods for age estimation. In addition, when exposed to heat the enamel crowns of teeth crack and flake away from the bulk of the underlying tooth [7,8,13]. The exposed dentine is then weakenedby exposure to tire, often fractures and is then lost. Therefore, the crowns of burnt erupted teeth are often missing, but unerupted teeth are often found intact as they are protected from burning by their position inside the jaw [7,8]. The degree of thermal shrinkage is an important factor when using microscopic techniques of age determination, as a greater number of bone microstructures will be viewed in a

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given microscopic field. If their packing density is normally used as a indicator of age, this will affect the age estimations. Thermally induced carbon colouration of bone tissue has been reported in association with various temperature transitions [8- 10,12,13],and indicates that the cremation of the bone tissue is ‘incomplete’ [9]. Herrmann [9] has reported that cremation is ‘complete’ when the temperature is raised above a critical level of 700-800°C. Temperatures > 1000°C have been reported in modem-day domestic house fires [ 161,and temperatures r 900°C in ancient cremation pyres [7,13], which implies that the complete cremation of human skeletal remains is not unusual. Above the critical temperature level the organic matter is completely combusted and shrinkage occurs to a greater extent [9,12]. Simultaneously the bone crystals becomemore crystalline, merge into larger crystals and fuse together [9,12,13]. Investigations of the heat-induced effects on the organic portion of bone have found that soft, non-mineralised collagen melts and forms gelatine when heated to - 60°C whereasmineralised collagen remains intact at this temperature. Bonar and Glimcher [ 171suggestedthat bone mineral is incorporated within the collagen fibrils and acts as a mechanical restraint, thereby stabilising the collagen against thermal denaturation and shrinkage. It is also known that the biological age of the bone tissue influences the degreeof thermal shrinkage, due to the increasedcross-linking of collagen with age, thus providing resistance to movement [18]. The present investigation involved a study of the heat-induced changes to the ultrastructure of human bone and encompassedalterations inherent in both the organic and mineral components in the interior of the bone. Age- and temperaturerelated changes on the ultrastructural level were also compared with those on the microstructural level. The ultrastructure of normal and pathological bone tissue is well known and documented by Boyde and Jones [ 18,191.This ultrastructure was accepted as that of unheated bone, for comparison with our heat-treated femoral bone samples. 2. Materials and methods Full width, mid-shaft, transverse sections of human femoral bone were collected from cadavers at autopsy. Full ethical clearance was obtained for this study at the Victorian Institute of Forensic Pathology, where the specimenswere collected, and at Monash University where the experimental work was conducted. Thirty-one samples were taken from deceasedpersonswho had enjoyed good health prior to sudden death. The samplesof bone representedvarious age groups, ranging from infant (1 year) to old age (97 years), and representedboth sexes.Specimens- 5 cm thick were cleaned using a small brush and distilled water. The periosteum and the marrow were removed mechanically and the bone was then stored in 70% ethanol. Thin transverse sections of bone, -2-3 mm in thickness, were cut using a rotating, water-cooled, diamond saw. Each of thesecross-sectionswas then cut into a number of sectors of roughly equal size. The number of sectors of bone tissue obtained from each section was dependent on the size of the shaft of the original

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bone organ. Hence, only six sectorsof usable size could be obtained from the infant specimens,and up to 12 sectors could be obtained from the adult specimens. The bone sectorswere placed in an alumina crucible and heat-treated in a Kanthal rapid high temperature furnace in air and at atmospheric pressure, at various selectedtemperatures, over the range 200-16OO’C for various time periods (2, 12, 18 and 24 h). The sampleswere heated at a constant rate of 50°C min-’ until the selectedtemperature was reached and this was then maintained for the selectedtime period at f 1°C. At the end of the heating period the specimenswere cooled at 10°C min- ’ back to room temperature. A total of 527 bone samplesrepresenting various combinations of age, temperature and duration of heat-treatment was studied. After heat-treatment, to determine the degreeof uniformity of the observed heat-induced alterations to the bone tissue, each samplewas fractured a number of times, in planes both parallel and transverse to the long axis of the femur. The fractured surfacesrevealed structural features of the bone that had not been damaged by handling and sample preparation. These were sputter coated with platinum to facilitate their examination in the scanning electron microscope. An Hitachi S/570 scanning electron microscope was utilised to study the ultrastructural changes.The microscope was operated in secondary electron mode, using accelerating voltages of 20-30 kV. High resolution examinations of samples were performed at a working distance of l-3 mm, with a maximum magnification of 100 000 times. Measurementsof crystals formed as a result of the heat-treatment were made from the scanning electron micrographs (SEMs). When the new crystals formed were approximately spherical, their diameters were measured from 25 examples in each micrograph. For comparing the size of the hexagonal crystals we measured the averagedistance between opposite apices of a crystal face, for 25 exampleson each micrograph. Thin sections of unheated and heat-treated bone, taken from a lZyear-old and a 78-year-old, were cut perpendicular to the long axis of the femur using a Leitz 1600 sawing microtome and then lapped to 100 f 5 pm in thickness. Contact microradiographs of these sampleswere made using a Matchett Laboratories OEG X-ray tube, with a copper target, operated at 25 kV and 10 mA tube current. Images were recorded on Kodak SO-343 Pelicula film, and examined in transmitted white light using an Olympus BHA-P light microscope, at magnifications of loo-250 times. A comparison of the unheated and heat-treated bone sampleswas made. 3. Results temperature (200~600°C) heat-treatmentfor 2 h In this temperature range, distinct and consistent colour changesof the specimens from orange (200°C) through to black (300°C) and grey (600°C) were observed. Scanning electron microscopy revealed that the main original structural features of the mineralised bone tissue were unaltered when the specimenswere heat-treated at any temperature in the range 200-600°C. For example, the osteon systems,lamellae

3.1. Low

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Fig. 1. Scanning electron micrographs (SEMs) of bone samples from a 21-year-old person, heat-treated at 400°C for 2 h. Two patterns of disintegration of the endosteal membrane (A) by flaking and lifting, and (B) by shrinkage and splitting, can be seen.

and osteocytelacunae remained intact. Bundles of mineralised collagen tibres could be observed on the walls of the Haversian canals and in the osteocyte lacunae, and in many samplesindividual mineralised collagen fibres could be distinguished. The most noticeable heat-induced change in this temperature range was observed within the Haversian canal, where the endosteum was found to begin to disintegrate at - 200°C. Two patterns of disintegration of the endosteal membrane were found to occur. These were a flaking or lifting of the membrane (Fig. 1A), and shrinkage which was evident from the appearanceof the holes in the membrane (Fig. 1B). The extent of the disintegration of the endosteal membrane was found to increase with the age of the deceasedand the temperature of heating. When the bone samples were heat-treated at a temperature of 600°C the endosteum was found to be completely destroyed, exposing the underlying mineralised skeleton of the collagen tibres. The overall structure of the mineralised collagen fibres was generally more oriented in samples from older persons (Fig. 2A,B), although there were some variations within individual samples. At high magnifications (30-100 000) new crystals could be seento have formed throughout all bone samplesheat-treated at a temperature of 600°C, indicating that somerecrystallisation had already occurred at this temperature. These crystals were uniform in size within an individual sample, and the morphology of the crystals was approximately spherical. The crystals observedon surfacesexposedby fracturing the samples after their heat-treatment were less densely packed (Fig. 2C) than those crystals on naturally-occurring free surfacessuch as the canal walls, where they were tightly packed and protruded from the mineralised residue of the collagen tibres

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Fig. 2. SEMs of bone samples heat-treated at a temperature of 600°C for 2 h, showing mineralised collagen tibres in bone samplestaken from (A) a l-year-old, and (B) a 93-year-old person, and recrystallisation observed (C) on a fractured surface of bone from a 60-year-old person, and (D) on the mineralised collagen tibres in an Haversian canal, in bone from a ‘IO-year-oldperson.

(Fig. 2D). The diameters of the spherical-type crystals were significantly smaller in those specimenstaken from persons ranging in age from mature adult to old aged, as compared with those aged between infancy through to young adult (Fig. 3). The averagediameter of the spherical-type crystals for all bone samplesheated at 600°C was measured as 0.064 f 0.040 pm. 3.2. High temperature (800-1600°C) heat-treatmentfor 2 h When heat-treated at temperatures between 800°C and 1400°Ca number of heat-

J.L. Holden et al. /Forensic Science International Spherical

crystal

diameter

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vs Age.

.20

10

20 30

40

50 60

70 60

90 100

Age (years) Fig. 3. Spherical-type crystal diameters as a function of the age of the person from whom the bone sample was taken, at 600°C. (Error = f lo).

induced changeswere observed in the remaining white mineral portion of the bone tissue. In this temperature range two crystals morphologies were observed. Firstly, crystals with an approximately spherical morphology similar to those seenat a temperature of 600°C were present, and secondly, new crystals with an hexagonal morphology and prismatic habit were also found (Fig. 4A-D). The hexagonal type crystals were found to increasein size with temperature, ranging from 0.30 f 0.05 pm at 8OO”C,to 1.2 f 0.10 pm at 1200°C. The average diameter of the spherical crystals situated at the boundaries between the hexagonal prisms was also found to increasewith temperature, ranging from 0.070 f 0.010 pm at 800°C to 0.200 f 0.050 pm at 1200°C.At temperatures in the range 800-1200°C neither of the two crystal morphologies appeared to exhibit any significant variations in crystal size that correlated with the age of the person from whom the specimen had been obtained. However, the hexagonal-type morphology was found to improve with the age of the person from whom the sample had been taken (Fig. 4A,B) and also with an increasing temperature of heat-treatment, concomitant with crystal growth (Fig. 4B,C). Heat-treatment at temperatures of lOOO-1400°Cresulted in the fusion of the hexagonal crystals in localised areas (Fig. 4C,D). Localised fusion was found both on the Haversian canal walls and on the fractured surfaces. This fusion, which was

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Fig. 4. SEMs of fractured surfacesof heat-treated bone exhibiting hexagonal type crystals in bone samples taken from (A) a 90-year-old person and (B-D) a Cyear-old person; (A) showsgood hexagonal morphology at 800°C and (B) shows poor hexagonal morphology at 800°C. Localised fusion (F) of the hexagonal crystals at 12OOT (C) on a fractured surface and (D) in a high density region of bone mineral at low magnification.

probably due to sintering, was consistently more pronounced in regions of the osteon at some distance from the central Haversian canal (Fig. 4D), and in all bone tissue obtained from persons aged l- 15 years. The overall degree of fusion was enhanced by increasing the temperature of heat-treatment above 1000°C and as a consequenceit was not possible to determine the average size of the hexagonal prisms in bone samples heat-treated at 1400°C. Occasionally, voids could be

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Fig. 5. SEMs of bone samples displaying a range of crystal morphologies including (A) rhombohedral crystals (bone from a IIPyear-old person at 12OO”C),(B) rosette-like crystals (bone from a 93-year-old person at lOOOT), (C)crystals with an irregular morphology (bone from a 21-year-old person at 12OO”C), and (D) platelet-type crystals (bone from a 30-year-old person at 1200°C).

observedin these altered regions of bone, suggestingthat bubbles of gas, formed as a result of heating, might have become entrapped on cooling. When heat-treated in the temperature range lOOO-14OO”C,new crystals with a distinctly rhombohedral morphology, and with a diagonal length in the range 0.300-6.0 pm, were a characteristic feature of the majority of the samples(Fig. 5A). The quality of the habit of rhombohedral-type crystals varied from single, perfectly formed crystals to clusters of poorly formed crystals.

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In the temperature range lOOO-1400°Csome crystals with other morphologies were also found to be randomly dispersed within many individual samples.These crystals fell into three main categories according to their morphology: a rosette-like morphology (Fig. 5B), an irregular morphology (Fig. 5C) and those with a platelet morphology (Fig. 5D). All crystals exhibited a range of sizesand were found either as single crystals or as clusters (Fig. 5B-D). A few bone specimensdid not exhibit any of these extra crystal morphologies and only contained the crystals with either the spherical or hexagonal prism morphology previously described. The Haversian canals and osteocyte lacunae retained their integrity up to a temperature of 14OO”C,whereas the lamellar structure was lost at - 800°C due to rapid crystal growth. When heat-treated at 16OO”C,all structural features were completely destroyed due to the total melting and subsequentrecrystallisation of the bone mineral on cooling (Fig. 6A). Most fractured surfaces of melted and resolidified bone appearedvirtually featurelessand glass-like, but someareasrecrystallised in a crosshatch pattern reminiscent of the original collagen fibre structure (Fig. 6B). 3.3. Prolonged heat-treatment (12-24 h) For comparison with the observed changesdetected in the bone samplesafter the 2-h heat-treatment, some samples were heated at selected temperatures for prolonged periods of 12-24 h. Prolonged heat-treatment of 12 h at 400°C removed most of the endosteum. No endosteal tissue was found to remain after a duration of 24 h at this temperature. The overall preferred orientation of the underlying mineralised collagen residue was clearly disrupted by prolonged heat-treatment, with individual fibres fraying

Fig. 6. SEMs of bone samples, (A) from a 31-year-old and (B) from a 39-year-old person, heat-treated at 1600°Cfor 2 h.

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Fig. 7. SEM showing the effect of heat-treatment at 1200°C for 12 h on a bone sample from a 39-year-old person.

away from the main fibre bundles. The amount of fraying decreasedwith the age of the person from whom the bone had been taken. Prolonged heating of bone above 600°C resulted in an improvement in the hexagonal crystal morphology but with no significant additional increase in the size

Fig. 8. Microradiographs of bone samples taken from a 12-year-old person and heat-treated for 2 h at (A) 1000°C and (B) 1400°C. The tertiary structure of the bone is still virtually intact.

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of the spherical and hexagonal crystal types. Along the Haversian canals, the quality of somecrystal morphology improved in directions both parallel and perpendicular to the wall. On a fractured surface the crystals were seento be increasingly closely packed, corresponding to both an improvement in crystal morphology, and an overall shrinkage of the bone samples. Fine lines were observed on the surface of somecrystals producing a tiered effect (Fig. 7). The degree of fusion above 1000°C was also found to increase slightly with the duration of heat-treatment. 3.4. Microradiograph

observations

Microradiographs showedvery little changein the structure of the bone with heattreatment up to 1400°C(Fig. 8). The osteocyte lacunae, seenas dark flecks oriented parallel to the lamellae surrounding the Haversian canals, were observed up to 1400°C. The dark and light regions of bone, indicating the low and more highly mineralised osteons, respectively, were clearly present up to a temperature of 1200°C. Above 12OO”C,it became more difficult to distinguish between light and dark osteons. Fractures due to the shrinkage and distortion of the bone tissue, and possibly also due to thermal shock on cooling, were clearly visible in the light microscopein bone samplesheat-treated above 1000°C.These cracks becamelonger and more numerous with increasing temperature. 4. Discussion Herrmann [9] has proposed that cremation is ‘complete’ at temperatures above 700-900°C when the organic matter has beencompletely cornbusted and the crystals of bone have fused. Below this critical temperature bone is ‘incompletely’ cremated and ‘For histological examination incompletely cremated bones are of the same value as fresh bone’ [9]. In this study of heat-treatments in the range 200-600°C little effect on the macro- and ultrastructure of the bone was observed, in agreement with Herrmann’s observations. Colour changes,which are associatedwith the combustion of the organic matter, were virtually identical to those found in heat-treated tooth enamel [20] and ancient, cremated bone [ 131.However, Shipman, Foster and Schoeninger [12] have reported colour changes that are not consistent with our observations. For example, in the temperature range 525-645°C the colours reported were neutral black with medium blue and reddish yellow [ 121,as compared with our observation of a consistently grey coloured bone at 600°C. These apparent colour changesand the eventual disintegration of the endosteal membrane indicate the breakdown and removal of the collagen tibres, proteins and fats in the bone tissue. This processwas complete when bone specimenswere heat-treated to a temperature of -600°C. Vincentelli [21] in a study of fresh human tibia1 bone, found that the majority of newly synthesised collagen Iibres in mature adult bone were circularly oriented around the longitudinal axis of the osteon, whereas those in a young bone were oriented nearly parallel to the longitudinal axis. Observations of collagen tibre orientation in this study in the temperature range 200-600°C are, however, complicated as a result of heat-treatment. The density of cross-links betweenthe collagen

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chains is proportional to the degreeof mineralisation and is known to increasewith the biological age of the osteon [22]. Since the individual cross-links do not possess equal energies, they will not break simultaneously when heat-treated, thus causing differing amounts of fraying. As a result, variations in fibre orientation and the amount of fraying are dependent on the biological age of individual osteons (or the degreeof mineralisation) and also the temperature of heat-treatment. Although it is recognised that the osteons present within a single sample of bone exhibit a range of biological ages,lessthan or equal to the chronological age of the deceased,in this study the majority of osteonsin the young samples(l-22 years) did appear to exhibit more random orientation of the mineralised collagen fibres, and an increaseddegree of fraying overall than when compared to mature samples (22-60 years) and oldaged samples (2 60 years). Above 6OO”C,crystal growth inhibited any qualitative observations of the mineralised collagen fibres. Over the temperature range 600- 16OO”C,heat-induced ultrastructural alterations observed in bone tissue were mainly due to changesin the size and morphology of the crystals. Holck (131 has reported that ancient human bone that has been powdered and then heated at -200°C contains more rounded particles compared with bone powder that has been heated at a higher ‘grade’ of temperature, where the bone particles are more angular and crystalline. In this study of the contempory bone tissue, once the organic matter had been completely combusted, small spherical-type crystals were observed at a higher temperature of 600°C. In addition, Shipman, Foster and Schoeninger [12] have reported a fleecy, highly particulate appearanceto the outer surface of sheepand goat bone tissue which had beenheated between 440°C and 800°C. This latter reported heat-induced feature could possibly be a low magnification observation of the spherical crystals observed here, on the fractured surfaces.The significantly larger, spherical-type crystals in the young bone samples(l-22 years), as compared to the mature bone samples(L 22 years), suggest that either recrystallisation has taken place at a lower temperature, or recrystallisation has occurred at 600°C accompanied by an enhancedcrystal grain growth. Both explanations imply that bone tissue taken from persons aged between infancy and young adulthood is thermodynamically more unstable than mature bone. Driessens,Van Dijk and Borggreven [25] have reported that the mineral constituent of the most active or unstable areasof bone are also the least crystallised, and X-ray diffraction studies have shown that the bone mineral structure of newly synthesisedbone mineral is amorphous in nature [26,27]. Becauseyoung bone samples are likely to contain a larger proportion of newly synthesisedbone in comparison to adult bone, the younger specimenswould initially be more active with respectto heat-treatment. At -800°C the differences in the size of crystals due to age was found to decrease,becoming nearly constant for all age groups. At this temperature, the bone samplesprobably attained a more stable state due to recrystallisation, and variations in crystal size within individual bone samplesbecamedependent on other factors, such as the presenceof impurities and variations in the composition of the calcium phosphate phases. Heat-treatment of hydroxyapatite above - 600°C is known to produce pyrophosphate as the acid phosphate groups in the apatite decompose [28]. The amount of

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pyrophosphate formed in bone mineral depends to some extent on the age and diet of the speciesconcerned [29,30]. It is also recorded that under physiological conditions the presenceof pyrophosphate in bone suppressescrystal growth [31]. It is possible therefore that the smaller spherical-type crystal size in bone taken from a mature person (2 22 years) could be attributed to the inhibiting effect of any pyrophosphate present in the bone mineral at 600°C. However, the amount of pyrophosphate liberated on heating is inversely proportional to the Ca/P molar ratio [30,32] which increaseswith age [33]. As a result the spherical crystal size should be expected to increasein size with age, which is contrary to our observations. Thus, variations in crystal size with age in bone heat-treated at 600°C is not the result of the presence of pyrophosphate. An accelerated crystal growth and increased crystallinity above 800°C has previously been reported during the sintering of synthetic hydroxyapatite [23,24], and in cremated human bone [9]. It is well known that stoichiometric hydroxyapatite, which has a higher Ca/P molar ratio than Ca-deficient hydroxyapatite phase of bone mineral, has an hexagonal prism habit. In addition, the Ca/P molar ratio of bone hydroxyapatite is reported to increase with the degree of mineralisation or biological ageof the bone mineral [33]. Thus the improvement of the hexagonal-type crystal morphology in our samples with increased age could be associated with a higher Ca/P ratio and an increasing degreeof mineralisation in the older bone tissue. The improvement of the hexagonal-type crystal morphology with the temperature of heat-treatment was simply due to the additional thermal energy available for atom diffusion. In contrast with Herrmann’s [9] microscopic observations of ancient cremated bone, and observations by Shipman, Foster and Schoeninger [12] of heat-treated sheepand goat bone, fusion of the bone mineral was not observed on the ultrastructural level at a temperature of 800°C. In this study the onset of sintering was found to occur in small localised areas at - 1000°C.These regions of fusion becamemore prominent as the temperature was increased up to 1400°C. Over the temperature range at which sintering was observed (lOOO-14OO”C),younger bone samples(l- 15 years) exhibited an increaseddegreeof fusion compared with the older bone samples (L 15),which can be associatedwith their greater thermodynamic activity. However, in bone samples of all ages, the biologically older, highly mineralised bone in the outer regions of the osteons also displayed a high degree of fusion. Fusion in these areaswas probably due to the original high density of the bone mineral at these sites [341.

Crystals with other morphologies such as platelet, rosette and irregular were a feature above 12OO”C,but their presencecould not be related to age, temperature or duration of heating. Thesevarious crystal morphologies may be associatedwith new heat-induced phases such as /3-tricalcium phosphate or cr-tricalciumphosphate [23,35-371. Asada, Oukami, Nakamura and Takahashi [24] have also proposed that the crystal morphology of hydroxyapatite is dependent on the Ca/P ratio. Thus, the crystal habits in different regions of bones may be a reflection of the biological age of the tissue at that site. A further suggestion is that the range of crystal morphologies observed is dependent on the type and concentration of the impurities present in the bone tissue.

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A comparison of the heat-induced changeson bone surfacesfractured transversely and longitudinally to the long axis of the femur did not reveal any ultrastructural differencesdue to the orientation of the exposed surface. Also, although both male and female sampleswere studied, no significant sex-related, heat-induced changesto the ultrastructure of the bone tissue were observed. It is interesting to note, however, that in examining both male and female ancient cremated remains, Holck [ 131found that the female remains had generally been burned at a higher ‘grade’ or temperature, compared with the male remains. Holck inferred that this was a consequence of the greater distribution of stored fat on a female body. Bradtmiller and Buikstra [l l] report that at 600°C bone tissue retains all structures necessaryfor microscopic ageing, and that bone shrinkage does not appear to have any significant effect on age estimations using this technique. In this study, shrinkage was found to occur to a much greater extent above 600°C and the appearanceof the bone changed from a lamella pattern to a more homogeneoustexture as the bone mineral becamemore crystalline. The distribution of bone mineral remained unchanged up to - 1200°C.At 2 1200°Cmicroradiographic studies found that the dark and light regions of low and more highly mineralised osteons became more difficult to distinguish as fusion of the bone mineral crystals becamemore prominent. As a consequenceof shrinkage and fusion at temperatures >6OO”C, this study concludesthat agedeterminations using microscopic methods which rely upon measuring the packing densities of structural subunits would not be appropriate. 5. Conclusion To conclude, the most important results of this study are the observation and quantification of profound changesto the ultrastructure of the bone tissue, as compared with the obvious lack of heat-induced changesto its tertiary structure. At low magnifications there is very little structural difference between an unheated sample and one heated over a temperature range of 200-14OO”C,except for the occurrence of shrinkage and fractures. At higher magnifications, the heat-induced effects are much more dramatic, to the eventual extent that the ultrastructural features observed are quite specific to the associatedtemperature transitions and age of the deceasedperson. From these studies it is possible to define the ‘grade’ or temperature attained in the bone tissue to within 200°C and to estimate the age at death of a fire victim as being young (l-22 years), adult (22-60 years) or old (160 years). Even though these three age categories are broad, when only fragments of incinerated bone are being examined this information can be invaluable. Further investigations of the physical and chemical heat-induced changesare currently being pursued, which may be able to provide more information to aid in the identification of burned bones. Acknowledgments We wish to thank the scientists and technicians of the forensic technical services division within the Victorian Institute of Forensic Pathology for collection of the femoral bone specimens.We also wish to thank S. Morton and A. Dyer of the Faculty of SciencePhotographic Services, Monash University.

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