Histomorphologic and Gravimetric Changes of Teeth Exposed to High Temperature - In vitro Study

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Introduction
Unidentified human remains can be accurately identified using dental identification. In addition, it is commonly accepted in evidence in the trial. Dental evidence typically survives much better than soft tissue evidence such as facial characteristics or fingerprints. The human tooth is the hardest substance in the body, more rigid than bone. Since they are calcified, they are resistant to environmental conditions that destroy soft tissue evidence. As a result, teeth cannot be damaged by water immersion, putrefaction, or degradation. Human disaster associated with fire accidents is a common scenario encountered in forensic investigation. Varying sources of fire such as fuel explosion, bomb blast, plane accidents, etc., expose the human body to a high temperature causing mutilation of the soft tissues, thereby making human identification difficult 1 . Teeth can, however, be destroyed by heat in rare cases when temperatures exceed 1000°C and the teeth are not protected by the soft tissues of the cheeks and lips. The most crucial task a pathologist may accomplish during an autopsy is determining the cause of death. In terms of legality, law enforcement must demonstrate beyond a reasonable doubt that the deceased died of causes other than natural causes.

Aim of the Study
The study aims to examine macroscopic, stereo microscopical, histological, and gravimetric changes in teeth exposed to heat at different temperatures ranging from 100°C to 900°C.

Materials and Methodology
The study was conducted on thirty-six mandibular premolar teeth extracted for orthodontic purposes, with an age range of 15 to 25 years. Four groups of nine premolar teeth were studied (Group 1 -morphological analysis, Group 2 -stereomicroscopy, Group 3 -ground section, and Group 4 -decalcified section). The samples were exposed to controlled temperatures ranging from 100°C to 900°C in a burnout furnace. Stereomicroscopy, ground sections, and decalcification were performed on the samples. Magnus MLX stereomicroscope was used to examine the samples. Images were captured. Using a Leica semiautomatic microtome, sections 3μm thick were prepared of each sample after decalcification with osteomol (10% HCL). Hematoxylin-eosin was used to stain the sections. Using a Labomed SP-Achro microscope and a photomicrograph, the sections were analyzed. The samples were used for the ground section, sectioned by using Arkansas stone, thin sections of 2 μm were prepared and fixed on microscope slides. Images were captured with a photomicrograph.

Results
At 100°C, crown showed the mottled appearance of enamel, cervical band/discoloration of the crown, an alteration in the scalloping nature of dentino-enamel junction and roughness on the tip of the root. At 200°C, the crown appeared slight greyish white patchy. There were micro-cracks on crown and root, roughness in the cervical area, radicular dentinal tubules structure and pattern distorted. The apical 3 rd of radicular dentin showed changes in dentinal tubular pattern. At 300°C, the crown showed band like brownish-orange, and the root appears brownish orange. The crown showed loss of enamel in the cervical margin and surface irregularities. Root began to deform, producing micro-cracks from cervical margin to root tip and scorched appearance. Radicular dentinal tubules were coalescing, dentinal tubule structure deformed at the apical region and dilated. Loss of normal architecture and dentinal tubules showed the vapor bubble appearance in decalcified section. At 400°C, crown began to appear metallic blackish bronze, intact and vertical crack. The root appeared charcoal black, intact and grainy on apical 1/3 rd and cervical line. Enamel and dentinal surface showed fur-like appearance. Teeth disintegrated completely during decalcification. At 500°C, the crown was seen as glistening greyish black with patchy blackish areas, crown-enamel shell cracking off, pitting defects, pit and grooves. The root has appeared greyish black with an apical 1/3 rd patchy blackish area and a portion of dentin was lost. Peeling of root surface layers and layer of dentin structure lost and amorphous blackish with irregular margins. At 600°C, Gray in color, entire crown fracture and surface roughness on apical 1/3 rd . Enamel could come out like a cap; micro-cracks were prominent, more crack lines appeared, bands like crack on the cervical area were seen and cementum was lost. Sand cracking appearance was noticeable in-ground section. At 700°C, outer surface appeared grayish-blue in color. Inner surface enamel was greyish and dentin was black apical 1/3 rd yellowish white. (Root tip of tooth ivory) Fractured at coronal 1/3 rd of the root. At 800°C outer surface of the crown was greyish blue in color and the inner surface was bluish greyish white. The root appeared outer surface yellowish-white, inner surface-bluish, deep cracks on crown portion. Tooth fractured with fragile crown and root-greyish black amorphous irregular margins. At 900°C, outer and inner surface chalky white crown was fractured into fragments with patchy roughness, thin apical 1/3 rd of the root. Root showed irregular surface. (Table 1, 2, 3, 4, 5) (Figure 1, 2, 3, 4) On gravimetric analysis, there was a consistent reduction in the weight of teeth above 300°C, with a steep decline from 400 to 900°C.

Discussion
In this study, the morphologic, stereo microscopical, histologic, and gravimetric changes of teeth exposed to high temperatures were assessed, keeping in mind that teeth are still intact following a fire accident to serve as a tool of identification. Further, for different fire accidents the temperatures may vary. For example, some of the sources for fire accidents such as house fire reach temperatures of 649°C, combustion of kerosene 65°C to 220°C, combustion of gas cylinder 100°C to 200°C, car crash 220°C to 990°C, incinerator 850°C to1093°C, combustion of petrol 800°C to1100°C, cremation 871°C to 1009.3°C and aircraft 1000°C to 3000°C but chemical fires can exceed several thousand degrees 2 . Based on Andersen Classification the fire injuries to teeth and jaws are classified into six grades: no injury, injury to an anterior tooth, Unilateral injury to anterior and posterior teeth, Bilateral injury to anterior and posterior teeth, fragments of jaw bones including teeth or roots, and no dental remains 11,12 .
The morphological appearance is not same for teeth at different temperatures. Loss of translucency of enamel has been attributed to loss of water. Our study observed and recorded various macroscopic, stereomicroscopic, histologic characteristics and weight differences in teeth when exposed to different temperatures. On macroscopic examination, specific color changes were noted with increasing temperature varying from yellowish orange to charcoal black.
The most obvious observation and characteristic for each range of temperature was color change. The study's macroscopic heat-induced color changes resemble prior observations of unrestored human teeth 13,14 . The stereomicroscope revealed an intact tooth at 100°C and a mottled appearance at 200°C. The color changes following   thermal exposure are similar for a given temperature. Similar results have been reported in previous studies as well [5][6][7] . Invisible carbonization was attributed to the loss of glossiness on the surface of crown and root after 400°C by Rotzscher et al., 8 . A pinkish discoloration is present on the root surface after exposed to 1000°C. Previous researchers reported similar observations, although the reason for discoloration was unknown 10 . Structural alterations such as micro cracks, fragmentation, and loss of tooth integrity were noted with temperatures ranging from 300°C to 900°C. According to Hughes and White, teeth are dehydrated, causing dentin materials to become brittle and dentin-enamel junctions to weaken. Intertubular tensile stress is responsible for the origin of cracks near dental pulp cavities, which can cause cracks to modify through structurally modified enamel and dentin 11,15 . In human enamel, water exists as adsorbed water, which is lost continuously and reversibly from 20°C to 200°C, and lattice water, with irreversible loss at 250°C to 300°C. Translucency continued to deteriorate with an increase in temperature. Delattre claims that the teeth of a burnt victim remain intact, have superficial discoloration, become charred, burned, and burst apart 3, 4 .  Table 3. Histological analysis (Ground section) Table 4. Histological analysis (Decalified section) Table 5. Weight of teeth before and after temperature exposure According to Rafael Fernandez's research, when bone is exposed to temperatures of 100-200°C, longitudinal fractures occur in cortical and trabecular bone, while crystallization occurs from 200°C higher. At 300°C and 400°C, organic material disappears, fractures become more evident, and connective bone tissues distort. Similarly, at this temperature, crystalline formation expands in size. In the collagen and extracellular matrix, crystalline linear macromolecular polymers develop between 500°C and 600°C. The number of crystalline formation increases between 600°C and 800°C. The bone structure also changes from a laminar pattern with a homogeneous structure to a more crystalline structure. The structure becomes entirely crystalline at 900°C, however, it remains amorphous and granular in shape 13 .
Ground and decalcified sections showed an altered histological pattern of dentinal tubules with amorphous changes at increased temperatures. Decalcification fluid disintegrates the tissue with higher temperatures, as collagen frameworks are destroyed, and collagen structures are worn out. There was a consistent reduction in weight of teeth above 300°c, with a steep decline from 400 to 900°c.

Conclusion
In our study, we were able to identify structural changes at varying degrees of temperature on human teeth, thereby providing valuable information about the thermal exposure when dental evidence remains. The distinctive characteristics of teeth exposed to different temperatures provide a clue to the source of the fire and serve as significant scientific evidence in forensic analysis.