Structural Changes in Human Teeth after Heating up to 1200 ̊C in Argon Atmosphere

The phase transformation of hydroxyapatite (HAP, Ca10(PO4)6(OH)2) to the beta tricalcium phosphate phase (β-TCP, β-Ca3(PO4)2) at 1100 ̊C is well known. However, in the case of human tooth, the HAP phase transformation is still an open area. For example, the CaO phase has sometimes been reported in the set of phases that make up the teeth. In this study, physical changes of human teeth when subjected to heat treatment in inert atmosphere (argon) were studied. The results were compared with those obtained in air atmosphere, from room temperature (25 ̊C) up to 1200 ̊C. Morphological changes were analyzed by light and scanning electron microscopy (SEM). The HAP to β-TCP phase transformation was followed in powder samples by X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). Heating of teeth results in the removal of organic material and structural water before the HAP to β-TCP phase transformation, the increment in hardness and the induced crystal growth. The percentage of the phases, crystal growth and lattice parameter variations as a function of temperature was quantified by Rietveld analysis. The black color was observed in dentin heated under argon atmosphere. Differences in expansivity produce fractures in dentin at 300 ̊C in argon and at 400 ̊C in air. In dentin, the coexistence of the HAP and β-TCP phases was observed after 800 ̊C in argon and after 600 ̊C in air; in enamel it was observed at 600 ̊C in argon compared with 400 ̊C in air. In general, the role played by the argon atmosphere during the thermal treatment of the teeth is to retard the processes observed in air.


Introduction
Enamel has a prismatic structure composed in 96% by weight of hydroxyapatite How to cite this paper: Vargas-Becerril, N., García-García, R. and Reyes-Gasga, J. (2018) Structural Changes in Human Teeth after Heating up to 1200˚C in Argon At- [4] reported the existence of crystalline and amorphous CaO when they analyzed human tooth enamel by transmission electron microscopy.CaO has also been reported in bovine teeth and bones when they are heat treated [5] [6].The remaining question is related to the phases that would be observed when the teeth are subjected to thermal treatments and the temperature at which these phases appear.
Temperature effect on the structure of human tooth has been analyzed in air by different techniques since long ago.It is well known that when teeth are submitted to temperature in air, structural changes can be evidenced by color changes and fractures [7] [8], as well as changes hardness [9] and electrical conductivity [10].For example, Reyes-Gasga et al. [11] and Tiznado-Orozco et al. [12] studied healthy and carious samples in the range 20˚C to 600˚C and the variations in the HAP hexagonal lattice parameters were related with ion substitution of 2 3 CO − in the OH − and 3 4 PO − sites.Sandholzer et al. [13] heated bulk tooth samples from 400˚C to 900˚C in air and suggested that temperature produces a high degree of perfection in the crystal structure of dentin but limited in enamel.Fereira et al. [14] analyzed bulk tooth samples in the range 20˚C to 1150˚C and observed that teeth gradually heated in air showed minimal damage by fracture, but with fast heating dentin and enamel are abruptly separated at the enamel-dentin junction and the fracture damage is severe.In addition, Reyes-Gasga et al. [15] reported a dielectric-conductive transition in human tooth enamel above 250˚C in air and in vacuum.
The aim of this paper is to observe the phases and to find the differences produced by the heating treatments of the teeth done in argon atmosphere compare the results with those observed in air.Therefore, in this work bulk and powder tooth samples were subjected to temperatures in the range from room temperature (25˚C) to 1200˚C in argon and in air atmospheres.The macro and microstructure, color, and mechanical changes in enamel and dentin were analyzed as a function of temperature.The color, mechanical and structural changes of the bulk samples were analyzed through hardness variation via the micro-indentation, and the light (LM) and scanning electron (SEM) microscopies.The structural and phase changes were also analyzed in powder samples via the X-ray diffraction (XRD), thermogravimetric analysis (TGA) and its differential (dTGA) and Fourier transform infrared spectroscopy (FTIR) analyses which were used to determine lattice parameters of crystal size, phase ratios and other crystallographic aspects using the Rietveld method.
It is worth to mention that similar studies have also been carried out in human and animal bone samples in the range 20˚C to 1200˚C to investigate the HAP structure change by heating to design materials with potential applications in odontology and medicine.For example, Rogers and Daniels [5] observed variation in the elastic modulus for temperatures above 600˚C in human bone bulk samples.Mezahi et al. [16] performed the calcination from 800˚C to 1200˚C of bovine bone and synthetic HAP samples and observed the formation of the β-TCP.Figuereido et al. [6] observed the crystal growth together decrease in porosity and formation of CaO in human, bovine and porcine bones samples.
All these experiments were carried out in air.We are interested in doing the heating treatment of teeth in an inert atmosphere to have an idea of the role played by the environment where the heating takes place.
The effects of heating on the structure of the human tooth, mainly in enamel, have been minimized due to its impractical use in the dental clinic.However, exists several clinical scenarios where the temperature is increased of dental treatments (lasers, dental burrs, etc.) that authorize this study.For example, the use of the hand-piece tool in the treatment of carious teeth involves a temperature close to 200˚C [17].Recently there has been an increase in the use of laser radiation in dental surgeries.It has been estimated, for example, that CO 2 lasers raise the temperature between 200˚C and 800˚C at the application site [18], and erbium lasers produce temperatures above 300˚C [19].In addition, the analysis of the effects of higher temperatures on the human dental piece may play an important role in forensic research [13] [20].

Experimental Procedure
Dentin and enamel of thirty permanent human molars extracted for orthodontic reasons from adult patients (between 25 and 30 years old) were the working ma- For the crystalline structure analysis, dentin and enamel were carefully mechanically separated, avoiding the zone of the amelodentinal junction to eliminate the mixing of materials.The samples were powdered using a hand dental drill and a Lynx EM-II milling cutter.
The X-ray diffraction characterization of powders was performed on a Bruker equipment model D8-Advanced with monochromatic Cu (Kα) radiation (λ = 0.154 nm).The diffractograms were obtained in the 2θ range from 5 to 60˚ with a step of 0.02˚/s, and 25 s of counting time per step using the Bragg-Brentano geometry.Computer structural analysis of diffractograms was performed by the Rietveld method using the software Profex-BGMN-Bundle-3.6.0.
The TGA analysis of the powders was performed on a TA Instrument SDT Q600 V20.9 Build 20 calorimeter from room temperature (25˚C) to 1200˚C with a heating rate of 10˚C/min, both in argon and in air.FT-IR analysis was done in the mid-infrared range 400 to 4000 cm −1 using a Perkin Elmer infrared spectrometer coupled with a total attenuated reflectance (ATR) of germanium.In dentin, from 25˚C to 250˚C, the loss of adsorbed water and some organic material produces the weight loss of 8% in argon and 10% in air.Between 250˚C to 650˚C in argon and from 250˚C to 550˚C in air, the weight losses of 20% and 22%, respectively, are due to the loss of structural water and the total organic material removal.From 650˚C in argon and from 550˚C in air and up to 1200˚C, the weight loss of 4% in both atmospheres is due to decarbonization and dehydroxylation.

TGA and dTGA
In enamel, from 25˚C to 250˚C, the weight loss of 5% is produced both in argon  and in air by the loss of adsorbed water and some organic material.From 250˚C to 450˚C in argon and from 250˚C to 500˚C in air, the weight losses of 11% and 13% respectively are produced by the elimination of structural water and the total elimination of organic material.The last weight loss registered in air is of 4% from 500˚C to 1200˚C; but in argon there are two losses: one of 6% from 450˚C to 900˚C and other of 2% from 900˚C to 1200˚C.These losses are due to decarbonization and dihydroxylation reactions.

Color Changes
The color changes in enamel are in function of temperature.Figure 2 shows the images of the dental piece after heating in the argon atmosphere.In this case, the changes of color in dentin start from 100˚C, where it is light brown.Enamel conserves its natural color.At 200˚C, dentin changes to yellow-orange, while the enamel is bluish white.At 300˚C, dentin is gray and begins to show cracks.The enamel is light gray.At 400˚C, dentin is dark (!).Enamel is still light gray and begins to fracture, although much less than in dentin, and dentin is separated from enamel.At 600˚C, at the amelodentinal junction, dentin changes to dark gray.At 800˚C, dentin and enamel are light gray.At 900˚C, dentin is white, al- though close to the amelodentinal junction, dentin and enamel are olive-colored.
Enamel is mostly white.At 1000˚C, enamel shows large olive areas at the amelodentinal junction.At 1200˚C both enamel and dentin are white.For comparison, Figure 3 shows the light microscopy images of the dental piece after the heat treatments in air.At 100˚C, the color and surface structure of enamel and dentin still present their natural color.At 200˚C, enamel does not change in color, but dentin is light-brown.At 300˚C dentin is brown with some areas in olive-green color.Enamel presents areas in light-brown color.At 400˚C, the amelodentinal junction shows the olive-green color, which spreads towards enamel, while dentin becomes light gray.In addition, some cracks appear in dentin, and enamel and dentin begin to separate.At 600˚C, dentin presents fractures and its color is dark gray.Enamel is dark gray, which changes to light gray towards the outside.At 800˚C, all enamel is dark gray, while dentin is light gray.At 900˚C both dentin and enamel show the same color, light gray.At 1000˚C, enamel and dentin are white.Finally, at 1200˚C, both structures display a gray white color.
Therefore, enamel follows the color sequence: In air, the dark gray in enamel is observed at 800˚C, while in argon it is observed at around 300˚C.The darkening of the color is due to the removal of the organic material that occurs between 200˚C and 600˚C.
The color changes in dentin in air as a function of temperature follow the sequence: Dentin is observed in black color only in argon atmosphere, at around 300˚C.
This sequence is consistent with that reported in the literature under air atmosphere [13].

Hardness
Table 1 shows the Vickers microhardness values registered in enamel and dentin as a function of temperature, in argon and in air atmospheres.The graphical presentation of the data is shown in Figure 4.The hardness in enamel lies in the range from 220 to 490 HVN, whereas in dentin it is between 65 and 530 HVN, surpassing in air the hardness recorded in the enamel at 1200˚C.Note that the standard deviation of these values is higher in enamel than in dentin.As shown in Figure 4, the hardness in argon of enamel is quite different than in air.In air, it shows two increments: at 200˚C, when the organic material is carbonized, and at 800˚C, when the β-TCP phase is present (as it will be commented below).The hardness in argon and in air is almost similar when the organic material is eliminated.
In dentin, the increment in hardness is observed after 200˚C in argon while it decreases in air.This is, after the elimination of the organic material.After 800˚C, the hardness increases both in air and in argon.Moreover, the hardness in dentin in air is higher than in enamel at 1200˚C.Mezahi et al. [16] obtained similar results in bovine bones and synthetic HAP samples heated up to 1200˚C in air.produced by the smaller crystal size of HAP (see Table 2) [4].Above 800˚C, the diffraction peaks are thinner, which indicates an increment in crystal size.

XRD
The presence of the β-TCP phase was observed after the elimination of water and organic material, and after the crystal growth of HAP.The coexistence of HAP and β-TCP, in enamel was registered at 400˚C in air and at 600˚C in argon.
In dentin, it was observed at 600˚C in air and at 800˚C in argon.

Phase Transformation
Table 3 indicates the relative percentage of the HAP and β-TCP phases obtained by the Rietveld method from Figure 7.For dentin, the coexistence of the two phases is observed after 600˚C in air and after 800˚C in argon.At 1200˚C, for dentin in air the percentage is 84% HAP and 15% β-TCP, while in argon it is 72% HAP and 27% β-TCP.For enamel, the coexistence of these phases is observed after 400˚C in air and after 600˚C in the argon.At 1200˚C, the percentage is 94% HAP and 5% β-TCP in air, and 91% HAP and 8% β-TCP in argon.

Crystal Size
The crystal-size values were also obtained by the Rietveld analysis.Table 2 shows the crystal size for the HAP and β-TCP phases.In general, above 400˚C in enamel and above 600˚C in dentin and up to 1000˚C, the crystal size of both phases grows in a directly proportional relationship with temperature.At 1200˚C, in dentin in air and enamel in argon, the HAP crystals keep growing.In contrary, the crystal size decreases in dentin in argon and enamel in air.In all cases, after 1000˚C the β-TCP crystals decrease.The biggest decrement of the HAP and β-TCP crystals is observed in enamel in air.

Lattice Parameters
The lattice parameters were also obtained by the Rietveld analysis.Table 4 shows the percentage of variation for the HAP (PDF card 09-0432) and β-TCP (PDF card 70-2065) phases as a function of the temperature.This percentage was obtained by: where the "reported values" (nominal values) were a = 0.94 nm and c = 0.688 nm for HAP and a = 10.43 nm and c = 37.375 nm for β-TCP; the "calculated value" is the one calculated by Rietveld.In dentin, for HAP in argon, a ∆ and c ∆ are highly expanded at 25˚C.
They slowly decrease after 200˚C reaching 0.1% at 1000˚C.For β-TCP in dentin and in argon, a ∆ and c ∆ are expanded (around 1.0%) at 600˚C, and after 1000˚C both parameter present contraction (0.6%).
In dentin in air, for HAP a ∆ is highly expanded (5.9%) at 25˚C.It rapidly decreases after 200˚C reaching −0.1% at 600˚C, and it is expanded again but only 0.2% at 1200˚C.For β-TCP in dentin in argon, a ∆ presents a high variation in contraction from 5.6% at 600˚C to 0.9% at 800˚C.After this, remains in contraction until 0.5% at 1200˚C.c ∆ is expanded from −0.6% at 600˚C to 0.8% at 1000˚C.
Enamel in argon, the variations from their nominal values are very small.For HAP in enamel and in argon, a ∆ , varies from −0.1% (contraction) at 25˚C to 0.0% at 600˚C and remains there till 1200˚C.c ∆ varies from 0.2% (expansion) to 0.0% (contraction) at 1200˚C.For β-TCP in enamel and in argon, a ∆ and c ∆ are expanded (around 0.9%) at 600˚C, and after 1000˚C both parameter present contraction.
Therefore, in dentin, the lattice parameters of HAP and β-TCP phases show contraction during heating but in argon these variations are slower than in air.
In enamel, the lattice parameters show very small variations during heating, and they are very similar in argon and in air.Both materials reach their nominal lattice parameter values during heating.5 shows the positions of the bands and the corresponding mode of vibration.In dentin, the biggest differences are observed after 800˚C, when the β-TCP phase appears, mainly in the band at 1120 cm −1 (the asymmetric P-Oν 3 stretching mode of the β-TCP phase); it is not observed in argon.In enamel, this band, and the band at 946 cm −1 , they do not appear both in argon and in air.

FTIR
The carbonate CO − ions may occupy two sites within the apatite structure.When they occupy the hydroxyl groups OH − sites, HAP is a type-A carbonated apatite and an expansion in the a-axis and the contraction in the c-axis are registered [21].The bands at 876 -880 cm −1 and at 1444, 1456 -1460 and 1546 cm −1 are observed [22] [23].When they occupy the tetrahedral 3 4 PO − sites, HAP is a carbonated apatite type-B, and the a-axis contracts and the c-axis expands [21].
Then, the band at ~875 cm −1 , corresponding to the ν 2 vibration, and the bands at  In dentin, amount of CO − eliminated in air is bigger than in argon.Table 5 indicates that in dentin the  PO − ion, no simple ion substitution mechanism can consider the magnitude and direction of the observed parameter changes, especially when the movement of organic material and water is also present.
After the combustion of the organic components, removal of water and carbonates, the increment of hardness in dentin must have to do with the crystal growth.The particle size is reduced by the nucleation and growth of the β-TCP phase, producing a kind of composite material increasing hardness.Note that CaO was not observed.Analyzing the decomposition of HAP, Savino et al. [30] indicated that processes of dehydroxylation in Ca-deficient apatites start at about 500˚C, while Shi et al. [31] ensured that in dental enamel the β-TCP phase appears above 700˚C.They also indicated that the β-TCP phase appears because of dehydration and decarbonization, producing stoichiometric HAP in dental enamel.
The chemical reactions in air for the Ca-deficient HAP by dehydroxylation are:  The content of β-TCP phase is higher in dentin.This could be since the crystal size in dentin is smaller than in enamel, resulting in the increase in surface energy which favors the decrease of the activation energy that the formation of this phase needs, even when this transformation starts after 600˚C.
The temperature effect on the crystal size and morphology of the enamel and dentin grains during the heat treatment of human tooth will be analyzed by electron transmission microscopy.We are working on it.

Conclusion
Heating of teeth in air and in argon atmospheres presents significant structural and chemical changes; the CaO was not observed.The heating analysis becomes simple if the process is divided in two parts: those occurring before and after the water and organic removal (600˚C, approximately).During the removal of the organic material and structural water the air atmosphere play an important role in the reaction of the combustion products.The appearance of the β-TCP phase depends on the temperature, the atmosphere and the chemical composition.
During the phase transformation to β-TCP, the air atmosphere is important in the heat dissipation process, both in dentin and in enamel.In addition to this, the removal of organic material and of the structural water content modifies significantly the lattice parameters, mainly in the dentin.The lattice parameter variations are slower in argon than in air.The phase transformation induces hardness, mainly in dentin, and the hardness increment is accompanied by crystal growth.Therefore, there is difference between the results obtained in the heating treatment of the teeth done in air than those obtained from the heating done in argon atmosphere.In general, the argon atmosphere delays the process observed in air.Because oxidation is important, similar behavior as those in argon can be expected in vacuum.

Figure 1
Figure1shows the TGA and dTGA graphs of dentin in air (Figure1(a)) and in argon atmosphere (Figure1(b)), and of enamel in air (Figure1(c)) and in argon (Figure1(d)).The analysis indicates the highest percentage of weight loss occurs between 250˚C and 650˚C in dentin, and between 250˚C to 500˚C in enamel.The mean weight loss observed in dentin (34%) was greater than in enamel (23%), but the weight loss velocity is slower in argon.

Figure 1 .
Figure 1.TGA and dTGA graphs of human tooth dentin and enamel from room temperature (25˚C) to 1200˚C.(a) Dentin in air; (b) Dentin in argon; (c) Enamel in air; (d) Enamel in argon.

Figure 2 .
Figure 2. Light microscopy images of the dental piece after heating in argon at different temperatures.Note the color changes in each case and the occurrence of fractures in dentin after 400˚C.

Figure 3 .
Figure 3.Light microscopy images of the dental piece after heating in air at different temperatures.Note the color changes in each case and the occurrence of fractures in the dentin after 400˚C.

Figure 4 .
Figure 4. Vickers microhardness graph of the values presented in Table1for enamel and dentin at different temperatures in air (E and D) and argon (EAr and DAr).Note the variation and the increment of the hardness in enamel and dentin respectively.

Figure 5
Figure 5 shows the SEM images of dentin at different temperatures in air (Figure 5(a)) and in argon (Figure 5(b)).Note that in air, HAP dentin crystals coalesce at 200˚C, during the water and organic material losses.Coalescence of these crystals in argon is slower than in air.It begins at 200˚C; but at 600˚C the dentinal conducts are still present.After elimination of the organic material, at 800˚C, the dentinal conducts disappear.Above 1000˚C, an equiaxial granular structure is formed.

Figure 6
Figure6shows the corresponding SEM images for enamel in air (Figure6(a)) and in argon (Figure6(b)).The elongated HAP enamel crystals coalesce at 200˚C in argon (at 600˚C in air).After elimination of the organic material, at 600˚C, both structures show similar contrast.Above 600˚C in argon, the crystals form a molten structure, initially porous (1000˚C) and then they grow (1200˚C).

Figure 5 .
Figure 5. SEM images of dentin at different temperatures in air (A) and in argon (B).

Figure 6 .
Figure 6.SEM images of enamel at different temperatures in air (A) and in argon (B).

Figure 7
Figure 7 shows the XRD diffractograms of dentin (Figure 7(a) and Figure 7(b)) and enamel (Figure 7(c) and Figure 7(d)) at different temperatures in air (Figure 7(a) and Figure 7(c)) and in argon (Figure 7(b) and Figure 7(d)).For the identification of the HAP and β-TCP phases, the PDF cards No. 09-0432 and 70-2065, respectively, were used.At room temperature, HAP is the phase identified, although in dentin it shows an "amorphous-type" spectrum when the temperature is below 400˚C

Figure 7 .
Figure 7. XRD diffractograms of dentin (a) (b) and enamel (c) (d) at different temperatures in air (a) and (c) and in argon (b) and (d).

Figure 8 .
Figure 8. FTIR spectra of dentin (a) (b) and enamel (c) (d) at different temperatures in air (a) and (c) and in argon (b) and (d).

2 3 COFor the phosphate group 3 4 PO
− ions in the type-B apatite disappear around 400˚C in air and argon, while the type-A remains after 800˚C.In enamel, the type-A and the type-B apatites remains in argon at 1000˚C, but in air the type-B disappear after 800˚C.− , the bands at 1010 and 1026 cm −1 and at 1080 and 1088 cm −1 , corresponding to the ν 3 triple asymmetric degeneration vibration mode, and at 958 to 968 cm −1 , corresponding to the ν 1 symmetric vibration mode, are always observed both in detin and enamel and both air and argon.Also, the degenerate double bond of the ν 4 mode is in the bands at 598 and 601 N. Vargas-Becerril et al.DOI: 10.4236/msa.2018.97046651 Materials Sciences and Applications The argon atmosphere affects the temperature at which the β-TCP phase is observed because in air, by being an oxidizing atmosphere, it interacts with the material forming oxides and releasing the β-TCP phase.The decomposition N. Vargas-Becerril et al.DOI: 10.4236/msa.2018.97046653 Materials Sciences and Applications reactions need a greater amount of heat.

Table 1 .
Vickers microhardness in enamel and dentin at different temperatures in air and argon atmospheres.Note the increase of hardness in dentin, and the variation of values in enamel.

Table 2 .
Crystal size of the HAP and β-TCP phases obtained by Rietveld analysis from the XRD diffractograms shown in Figure7using the (110) and (002) for HAP and the (002) for β-TCP.

Table 3 .
Relative percentage of the HAP and β-TCP phases obtained by the Rietveld method from the XRD diffractograms shown in Figure7.The % RWP is also indicated for each case.

Table 4 .
Percentage variation of the HAP and β-TCP lattice parameters obtained from the XRD diffractograms shown in Figure7by Rietveld analysis.

Table 5 .
Band position assignment for the human dental tissues, enamel and dentin, from 25˚C to 1000˚C in air and in argon atmosphere.D indicates the main difference between the bands observed in the argon and air atmospheres.