Age and temperature related changes to the ultrastructure and composition of human bone mineral.

This X-ray diffraction (XRD) investigation of heat-treated human femoral bone showed that the main mineral phase of both unheated bone and bone heated to 600 degrees C resembled that of a poorly crystalline form of hydroxyapatite. The rod-shaped apatite crystals in unheated bone persisted in bone heated up to 400 degrees C. Recrystallization at approximately 600 degrees C, produced larger crystals, which either retained their original morphology or changed to tabular or equidimensional shapes. The size of the apatite crystals in unheated and heated bone specimens was dependent on both temperature and age. When heated above 600 degrees C the crystallinity of the bone mineral increased, and the XRD pattern more closely resembled that of hydroxyapatite. Partial decomposition of the hydroxyapatite phase to calcium oxide above 1000 degrees C, and beta-tricalcium phosphate, alpha-tricalcium phosphate, and calcium oxide phosphate between 1200 degrees C and 1400 degrees C, indicated that the original apatite phase was both calcium deficient and contained carbonate. The relative peak intensities of the thermal decomposition products were related to some extent to the age of the deceased person and reflected the compositional changes that occur during bone aging. Because the thermally induced changes to the composition and ultrastructure of bone mineral were influenced by the age of the individual, this investigation proposed that the heat treatment of bone tissue may offer an alternative way of studying bone aging.

Scanning electron microscope observations of incinerated human femoral bone: a case study.

Fragments of the incinerated remains of a fire victim were studied using scanning electron microscopy and microradiography. These observations were then compared with the heat-induced alterations found in laboratory heat-treated human bone. The incinerated bone fragments exhibited a range of colours, including black, grey and white, concomitant with alterations to the ultrastructure and microstructure of the bone tissue. The colour of the incinerated bone tissue, and the crystal habit and size associated with each region of colour, indicated a gradual decrease in the temperature attained in the bone, as a function of the radial distance from the outer cortical bone surface. A maximum temperature in the range approximately 1000-1200 degrees C had been attained in the outer cortical bone regions that were white in colour. A minimum temperature of 300 degrees C had been attained in the inner cortical bone regions that were black in colour. The period of time over which the fire attained the maximum temperature was inferred to be too short for the bone tissue to have reached an equilibrium with the surrounding environment, as the fire was due to a sudden ignition. However, the minimum temperature recorded was attained for a longer period of time as the organic contents of the Haversian canals and the medullary cavity had been completely combusted. From examinations of the spherical-type crystal size in the grey regions of cortical bone, the habit of the hexagonal-type crystals in the white regions of bone, and the preferred orientation of the mineralised collagen fibrils in the Haversian canals situated in the black regions of cortical bone, it was suggested that the deceased person was a young-to-mature adult, possibly aged 20-40 years.

Scanning electron microscope observations of heat-treated human bone.

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 degrees 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 degrees C and recrystallisation of the bone mineral beginning at 600 degrees C. Recrystallisation produced a range of crystal morphologies: spherical, hexagonal, platelets, rosettes and irregular. Crystal growth occurred at temperatures > 600 degrees C. Sintering led to fusion of crystals at 1000 degrees C. This process continued up to temperatures > 1400 degrees C. At 1600 degrees 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.

The effect on the ultrastructure of dental enamel of excimer-dye, argon-ion and CO2 lasers.

This study aimed to investigate the ultrastructural changes that occur in dental enamel irradiated with pulsed excimer-dye, continuous-wave (CW) argon-ion and CW CO2 lasers. The pulsed excimer-dye laser produced deep craters, rough damaged surfaces with underlying porosity and amorphous vitrified material. The vitrification of the enamel indicated that the temperature in these areas must have been at least in the range 1280 to 1600 degrees C. The CW argon-ion laser irradiation produced a changed non-cratered surface with inter-crystalline porosity and a mixture of small and some large irregularly packed recrystallized enamel crystals. The CW CO2 laser produced shallow craters, surface crazing and lifting off the removal of the surface layer to expose the underlying roughened enamel. The ultrastructure revealed inter- and intra-crystalline porosity, a mixture of small but variable size irregularly packed recrystallized enamel crystals and also well packed large crystals which indicated further grain growth. The porosity in lased enamel was overall very similar to that seen in enamel heated in an electric furnace to a temperature of 600 degrees C. The presence of recrystallized enamel crystals indicated a temperature rise of approximately 1000 degrees C and the grain growth indicated that a temperature > or = 1000 degrees C existed for some time after the laser irradiation. In general the excimer-dye laser produced most surface destruction because of its higher power density and shorter interaction time and the argon-ion laser produced least damage. These results indicated that the lasers used in this study require much more refinement before they can find therapeutic application to dental enamel, and this may well be the case for other lasers being investigated for clinical dental practise.

Ultrastructure of spindles and tufts in human dental enamel.

A transmission electron microscope (TEM) study was made of spindles and tufts as identified in the light microscope, from samples prepared by selected-area argon-ion-beam thinning. Spindles in human dental enamel were found to be continuous with dentinal tubules across the dentin-enamel junction (DEJ) and usually appeared at the DEJ as electron-lucent, empty channels nearly circular in cross-section. The spindles were found to cross prism boundaries, branched rarely, and some were occasionally found to be occluded or partially occluded with small needle-like crystals (approximately 5 nm width and approximately 70 nm length), granular material (approximately 1.5 nm diameter) and/or amorphous material. Near the DEJ, the majority of spindles had a diameter less than 2 microns, while in the prismatic enamel away from the DEJ, spindles with a diameter greater than 2 microns were generally found. Spindle varicosity was characterized by an enlargement of their diameter. Tufts started at the DEJ and were not associated with dentinal tubules. Two types of ultrastructure were observed in the TEM: (i) disrupted regions of enamel incorporating large voids (up to approximately 0.1 microns in diameter), or, more commonly, (ii) channels within the enamel occluded or partially occluded, with small needle-like crystals and granular and/or amorphous material similar to that found in the enamel spindles. It was concluded that spindles and tufts represent areas of hypomineralization with increased void volume and partial remineralization.

Effects of continuous-wave CO2 laser on the ultrastructure of human dental enamel.

Laser-induced changes in plano-parallel sections were examined by light microscopy (LM) and scanning electron microscopy (SEM), and correlated with ultrastructural changes as observed by transmission electron microscopy (TEM). LM and SEM revealed two different changes--extensive crazing, and crazing and cratering. Rough exposed enamel was commonly found, resulting from lifting off and removal of the top layer of crazed, or crazed and cratered, enamel. The type of induced change was mainly dependent on the energy density used (range approximately 0.8 to approximately 200 J cm-2) and on enamel prism orientation. Lased enamel was also softer than unlased enamel. TEM of both crazed enamel and rough exposed enamel revealed that most crystals generally resembled those of unlased enamel in size and shape, but that inter- and intra-crystalline voids were present in some areas. The crazed and cratered enamel had significant ultrastructural changes: new homogeneous and inhomogeneous crystals of apatite with a different shape and larger size than those of the original, and a loss of prismatic structure. The lack of uniformity of the laser effect on crazed and cratered enamel was shown by variation in crystal packing (from good to poor), variations in crystal size from area to area, and the presence of pockets of poorly packed homogeneous crystals alongside pockets of well-packed inhomogeneous crystals. The crazing, crazing and cratering, rough exposed enamel and the greater number of voids, as well as the relative softness of lased enamel do not indicate an overall ultrastructural improvement. However, the larger apatite crystal size and loss of prismatic structure in crazed and cratered areas may partly explain previous observations of reduced rates of subsurface demineralization in lased enamel.

The polarized light microscopy and ultrastructure of Polynesian pigmented tooth enamel.

Transmitted and polarized light microscopy of unerupted and erupted teeth affected by a pigmented anomaly found in two geographically isolated Polynesian populations, the New Zealand Maori and the French Polynesian Marquesas Islander, showed similar histological characteristics. Mounted in water, the pigmented areas were positively birefringent and covered with a thin negatively birefringent surface layer 200-250 microns thick. Cervical areas were negatively birefringent. Transmission electron microscopy of argon-ion-beam thinned specimens of affected enamel revealed large voids, disruption in the packing of crystals and spacing at prism boundaries. In the surface layer of enamel from erupted and unerupted teeth, the intra- and interprismatic spaces were occluded by fine crystals or amorphous material. A well-defined prism structure and close crystal packing were found in cervical enamel. The ultrastructure of these pigmented enamels was similar to and consistent with a hypomaturation type of amelogenesis imperfecta.

Electron microscopic investigation relating the occlusal morphology to the underlying enamel structure of molar teeth of the wombat (Vombatus ursinus).

This investigation relates the occlusal morphology of the continuously growing molars of common wombats (Vombatus ursinus) to the underlying enamel ultrastructure that was investigated using the techniques of light microscopy, scanning electron microscopy, and transmission electron microscopy. The main feature of the occlusal enamel was a prominent ridge, which followed the contour of the dentine-enamel junction (DEJ). It was found that the occlusal morphology depended upon the orientation of the dentinal and enamel tissues, variations in prism orientation, Hunter-Schreger bands (HSB), and presence or absence of cleavage. Cleavage of enamel promoted by sheets of parallel prisms occurred along the face between the DEJ and the ridge, whereas on the face between the ridge and the cementum-enamel junction (CEJ) cleavage was inhibited by HSB. The slope of the latter face was mainly due to a decrease in wear resistance going from the ridge, where prisms were intercepted transversely, toward the CEJ, where they were intercepted obliquely. Occasionally small surface undulations were observed on the face between the ridge and the CEJ. These undulations were found to correspond to gradually decussating enamel regions. The pronounced cleavage of enamel parallel to the face between the DEJ and the ridge played an important role in conferring on the continuously growing molars a distinct property to develop and maintain a self-sharpening ridge throughout the life of the tooth.

The ultrastructure of human dental enamel heat-treated in the temperature range 200 degrees C to 600 degrees C.

Heating enamel in the temperature range 200 degrees C to 600 degrees C resulted in poor crystal packing due to void formation, permanent change in the sign of its birefringence (from negative to positive) in some areas, and an altered crystal morphology. Transmission electron microscopy of enamel heated in the temperature range 200 degrees C to 400 degrees C revealed that the distinction between the positively birefringent regions and the negatively birefringent regions (which were present up to 350 degrees C and occasionally up to 400 degrees C) at the tooth's surface was due to the greater volume of intra- and inter-crystalline voids within the positively birefringent regions. There was a significant increase in void volume at 400 degrees C, and above this all of the enamel was positively birefringent and opaque. Large remineralized crystals of beta-tricalcium phosphate (beta-TCP) phase (whitlockite) were initially formed at 400 degrees C, and their size and number increased at 500 degrees C and above. Both the greater solubility of beta-TCP crystals and the increased surface area due to the presence of voids would increase the rate of demineralization of heat-treated enamel.

Laminated zones in carious human dental enamel.

Laminated zones within the body of carious lesions were studied by polarized light microscopy and transmission electron microscopy. Areas from within and surrounding the laminated zones, precisely selected using light microscopy, were argon-ion-beam thinned and examined by transmission electron microscopy. Laminated zones were present in approximately 7% of the samples studied. Polarized light microscopy showed variation in mineralization from zone to zone and the enamel surrounding the zones in the body of the lesion. Laminated zones whose central region showed approximately 1% of space when examined in air and whose boundaries showed approximately 2-4% of space when imbibed in quinoline were selected for ultrastructural studies. Electron microscopy showed the laminated zone to be less demineralized than the surrounding enamel in the body of the lesion. The ultrastructure of their central regions was similar to healthy enamel but their boundaries showed demineralization which increased into the body of the lesion. Within the central region of lamination there was greater evidence of resistance to demineralization rather than the presence of remineralization.

Ultrastructure of the intact surface zone of white spot and brown spot carious lesions in human enamel.

Electron microscopy of the intact surface zone of white spot and brown spot carious lesions showed that in general their ultrastructure was similar. Their outermost crystalline surface consisted of small crystals similar to those in healthy enamel, crystals with central core dissolution, and rounded crystals. Below this, surface demineralization of enamel was observed as the enlargement of micropores, the central core dissolution of crystals, the formation of channels and the enlargement of spaces at prism boundaries. Remineralization of enamel was observed as the partial occlusion of voids, the rounding and enlargement of crystals, and some new needle-shaped crystals. Some other features indicated combined demineralization and remineralization. The occlusion of spaces at prism boundaries was a more common feature in brown spot lesions, whereas the pockets of rounded crystals were more common in white spot lesions. A relatively uniform distribution of needle-shaped crystals throughout the intact surface zone was a feature of some brown spot lesions only.

On the nature of the opaque and translucent enamel regions of some macropodinae (Macropus giganteus, Wallabia bicolor and Peradorcas concinna).

Teeth of three macropod species, M. giganteus, W. bicolor and P. concinna, have been studied using the techniques of light microscopy, scanning- and transmission-electron microscopy and hardness measurement. Light microscope observations showed that the teeth of these species had a translucent enamel region close to the dentine and an outer opaque enamel region at the tooth's surface. These regions were not related to the presence or absence of tubules which are a characteristic feature of marsupial enamel. Hardness tests showed that the opaque enamel was softer than the translucent enamel. Scanning electron microscope observations revealed that there was no correlation between any particular prism packing or orientation and the opaque and translucent enamel regions. Transmission electron microscope observations showed that the translucent enamel region consisted of well defined prisms and well packed, lath-like crystals, whereas the opaque enamel was disrupted by voids (which ranged in size from enlarged micropores to about 2 microns in diameter in extreme cases) between crystals and some randomly oriented, loosely packed crystals. This disruption within the opaque enamel region was more common at prism boundaries but pockets of disrupted enamel were also found within prisms and interprismatic regions. The opacity of the enamel was caused by scattering of light from the voids. The ultrastructure of the opaque enamel region indicated that this region was hypomineralized; hardness tests and polarized light microscope observations were consistent with these results.

Electron-microscope study of the dentine-enamel junction of kangaroo (Macropus giganteus) teeth using selected-area argon-ion-beam thinning.

Transmission electron microscopy of selected-area argon-ion-beam thinned kangaroo (Macropus giganteus) enamel revealed a complex ultrastructure in the region of the dentine-enamel junction (DEJ). Characteristic features were multiple branching of dentinal tubules, rejoining of enamel tubules, elongated defects, extended protrusions of dentine into enamel, two types (A and B) of hypomineralized enamel and a continuity between dentinal and enamel tubules. In the intertubular regions of the DEJ a complex intermingling of finer enamel and dentine crystals, similar to that found in human enamel, was observed. The varicosities observed in the light microscope were a combined optical effect caused by the hypomineralized (type A) enamel and the branching and rejoining of the enamel tubules.

Ultrastructural changes in the translucent and dark zones of early enamel caries.

Selected areas of early enamel caries ('White spot' lesion) in human teeth were argon-ion-beam thinned and examined by transmission electron microscopy. A systematic examination of areas which were histologically defined as the translucent zone and the dark zone showed that the pattern of early caries was consistent with that of demineralization of the tissue commencing with a widening of the micropores and of the inter-rod spaces and gradually progressing to a severe destruction of the original enamel crystals, with possibly some remineralization. Observations indicate that the intercrystalline micropores and inter-rod spaces are the pathways by which acidic agents reach the crystals causing carious dissolution.