archives of oral biology 60 (2015) 768–775

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Comparison of chemical composition of enamel and dentine in human, bovine, porcine and ovine teeth Juan de Dios Teruel a, Alberto Alcolea b, Ana Herna´ndez a, Antonio Jose´ Ortiz Ruiz a,* a

Department of Integral Pediatric Dentistry, Faculty of Medicine, University of Murcia, Hospital Morales Meseguer, 2 planta, C/Marque´s de los Ve´lez s/n, 30008 Murcia, Spain b Servicio de Apoyo a la Investigacio´n Tecnolo´gica, Universidad Polite´cnica de Cartagena, 30202 Cartagena, Murcia, Spain

article info

abstract

Article history:

Objective: The aim of this paper was to compare the chemical composition of human teeth

Accepted 31 January 2015

with other mammal species that are likely candidates for replacing them in studies that test

Keywords:

Design: Dentine and enamel fragments extracted from 400 sound human, bovine, porcine

C/N analysis

and ovine – 100 teeth per species – incisors and molars were mechanically ground up to a

dental material.

TG–MS

final particle size of less than 100 mm. C/N analysis, thermogravimetric analysis coupled to

WDXRF

mass spectrometry (TG–MS), and wavelength dispersive X-ray fluorescence (WDXRF) were

Enamel

used to analyse the samples’ composition.

Dentine

Results: Elemental analysis showed more organic carbon and nitrogen in dentine than in enamel. Human enamel was the most highly mineralised, with C and N values close to hydroxyapatite. Bovine dentine and enamel were the most similar to human. TG–MS: in all species, enamel contained less carbon and organic matter than dentine. Thermal decomposition of human enamel showed great similarity to synthetic hydroxyapatite, and large differences from bovine, ovine and porcine enamel. Thermal decomposition showed the greatest similarity between human and bovine dentine. WDXRF: Dentine contained larger quantities of Mg, S, Sr and Zn than enamel. Enamel contained larger quantities of P, Ca, Cl, Cu, K and Ca/P ratio than dentine. Human enamel and dentine contained a higher Ca/P ratio, larger quantities of Cl and Cu and lower quantities of Mg, S, Zn than the animal species. Conclusions: Elemental analysis, TG–MS and WDXRF have shown that human and bovine enamel and dentine show the greatest similarity among the species analysed. # 2015 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: +34 868 88 85 81; fax: +34 868 88 85 76. E-mail address: [email protected] (A.J.O. Ruiz). http://dx.doi.org/10.1016/j.archoralbio.2015.01.014 0003–9969/# 2015 Elsevier Ltd. All rights reserved.

archives of oral biology 60 (2015) 768–775

1.

Introduction

The majority of in vitro testing of dental materials is performed on extracted human teeth, which would appear to be the perfect candidates for these studies. However, the use of human teeth suffers several limitations: they are often difficult to obtain in sufficient quantity and with adequate quality, since many are extracted due to extensive caries lesions and other defects; it is hard to establish sample homogeneity because it is difficult to control the source and age of the teeth; finally, increasing awareness of the infection hazard and other ethical issues have led to increased restrictions on their use. As a result, alternative substrates have been proposed and have entered into use in dental research. Several types of non-human teeth, such as primate, bovine, swine, ovine and equine teeth, have been used as substrates for in vitro dental experiments. The main criteria for the choice of animal teeth are that the physico-chemical, structural, and biological characteristics should be similar to human teeth.1–3 Bovine teeth have been the most widely used substitute for human teeth in dental studies. However, their chemistry and structure are not identical. Indeed, it has been shown that acid etching of bovine enamel causes the formation of a rougher surface and the hydroxyapatite crystals are oval shaped and narrow, in contrast to the round shape observed with human enamel.4 The mineral phase of vertebrate teeth contains one or more types of phosphate minerals, predominantly calcium phosphates. The most abundant mineral in human teeth is a basic calcium phosphate idealised as calcium hydroxyapatite [Ca10(PO4)6(OH)2]. Other calcium phosphates and magnesium phosphates have been identified with or without association with apatite: brushite (CaHPO42H2O), octacalcium phosphate (Ca8H2[PO4]65H2O), tricalcium phosphate or whitlockite (b-TCP, b-Ca3[PO4]2), calcium pyrophosphate dehydrate (Ca2P2O7), and amorphous calcium phosphates, struvite (MgNH4PO46H2O), newberyite (MgHPO43H2O), and amorphous calcium magnesium pyrophosphates.5 Enamel and dentine are predominately composed of hydroxyapatite crystals. Enamel consists of an inorganic matrix (96%, w/w) and organic constituents (i.e. proteins and lipids) and water (4%, w/w), which occupy the gaps among the apatite crystals in the enamel. Hydroxyapatite crystals in enamel are hexagonal and bundled to form approximately 4 mm diameter rods. Mature dentine is about 70% mineral, 20% organic matrix, and 10% water by weight. The hydroxyapatite crystals in dentine are in the form of flattened plates with approximate dimensions of 60– 70 nm length, 20–30 nm width, and 3–4 nm thickness. The calcium and phosphorus (as phosphate) content of the teeth range 34–39% and 16–18% by weight, respectively. Various cations and anions are incorporated into cationic (Ca2+) and anionic centres (OH , PO43 ) of the hydroxyapatite matrix. Sodium (Na+), potassium (K+), and magnesium (Mg2+) can substitute in the calcium position, fluoride (F ) and chloride (Cl ) in the hydroxyl position and carbonate (CO32 ) in the hydroxyl and phosphate positions. Close to 40 elements have been reported to be present, ranging from 1000 ppm

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(i.e., Zn, Sr, Fe, Al, B, Ba, Pb, etc.) to 100 ppb (i.e., Ni, Li, Ag, As, Se, Nb, Hg, etc.).6,7 The presence of these trace elements will determine different physico-chemical and biological behaviour; in this way, Scholfield et al.8 observed a high correlation between tooth hardness and zinc content. Various analytical methods have been applied for identifying trace elements in teeth. They include atomic absorption spectrophotometry,9 proton induced X-ray emission,10 electrothermal vaporisation inductively coupled plasma-mass spectrometry,11 laser ablation ICP-MS,12 and energy-dispersive X-ray elemental analysis.13 In order to determine which will be the best substrate for replacing human teeth, the elemental composition of enamel and dentine in each species must be known. Although several studies have compared animal with human teeth, none have conclusively warranted the use of animal teeth in laboratory tests. The aim of this study was to determine the chemical composition of the enamel and dentine in human, bovine, porcine and ovine teeth, using C/N analysis, thermogravimetric analysis linked to mass spectrometry (TG–MS) and wavelength dispersive X-ray fluorescence spectrometry (WDXRF).

2.

Materials and methods

2.1.

Experimental groups. Enamel and dentine samples

The dental substrates studied were bovine enamel, bovine dentine, ovine enamel, ovine dentine, porcine enamel, porcine dentine, human enamel and human dentine; hydroxyapatite powder (Reference #04238, Sigma–Aldrich, St. Louis, MO, USA) was also analysed as a control substrate. The study used 400 incisors and molars freshly extracted and free from enamel cracks, caries, and fillings: 100 human, 100 bovine, 100 porcine, and 100 ovine. The teeth were washed in water to remove any traces of blood and then stored in distilled water, which was changed daily to avoid deterioration. In no case was a tooth stored for more than a month after extraction. The teeth were sectioned, removing enamel and dentine, with a water-cooled diamond saw (Horico, Berlin, Germany). To be sure that the selection of fragments had been performed correctly, they were observed with a stereoscopic microscope (NIKON SMZ-U, Yokohama, Japan). Samples were hand-milled with an agate mortar and any pieces larger than 100 mm were ground with a mill (Disc mil HSM 100H, Herzog, Maschinenfabrik, Osnabrick, Germany). Every disc-milling process was refrigerated with 2 mL of hexane (n-hexane 95%, Panreac, Barcelona, Spain) in order to avoid structural changes in the specimens. The resulting dust was sieved in order to ensure a controlled comminution. Samples were dried at 60 8C for 24 h in a furnace (UFP 500, Memmert, Nuremberg, Germany), in order to release most of the free water, and then ground by hand-milling with an agate mortar (NAHITA, Navarra, Spain) and with a disc mill for 1 min, to give a final particle size of less than 100 mm. Lastly, 5 g of powder samples were pelletised using a semiautomatic

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hydraulic press (Nannetti Mignon-SS, Faenza, Italy) without any binder additive. The specimens obtained were distributed between the different test techniques as follows: 0.2 g per powder sample for elemental analysis; 10 mg of powder for TG–MS, and a pellet of 5 g for WDXRF.

2.2.

Elemental analysis (C/N analysis)

Carbon and nitrogen determination by combustion is a common technique for analysing all types of chemical matrices. In this technique, light elements, such as C, N, and H, are oxidised and detected by infrared spectrometry (C and H) and thermal conductivity (N). This analysis was performed with the automatic elemental analyser LECO TRUSPEC CN (LECO, St. Joseph, MI, USA). Two 0.1 g preweighed and encapsulated samples per group were placed in the instrument’s loader. Samples were transferred to the instrument’s purge chamber, directly above the furnace. All atmospheric gases were removed during the transfer process. A separate, optimised, non-dispersive infrared (NDIR) cell was used to detect CO2, ensuring a short analysis time. The NOx gases were passed through a reduction tube filled with copper to produce N2, which was detected using a thermal conductivity cell. In order to differentiate the organic and inorganic fractions of carbon, a drop-by-drop acid attack, with 2 N HCl, was carried out on a sample from each group until the decomposition of the carbonates had taken place. Afterwards, samples were heated for 24 h at 110 8C, to remove H2O. In order to determine total carbon and total nitrogen, samples were not subjected to acid pre-treatment.

2.3.

TG–MS

Samples of powder (mass 10 mg) were heated in a thermogravimetric analyser (Model TGA/DSC 1 HT, Mettler-Toledo, Schwerzenbach, Switzerland). These were enclosed in 70 ml aluminium oxide crucibles and heated at a rate of 10 8C/min, from 30 8C to 1100 8C, in a stream of oxygen (50 mL/min) to provide an oxidizing environment. An empty crucible was heated under the same conditions as the samples. The resulting thermograms were subtracted from those obtained from the samples. A detailed mathematical analysis of the thermal decomposition of each sample was performed to assess thermogravimetric curves (Fig. 1). The entire temperature range was distributed in three sigmoids whose height matched the loss of the gases released during each interval (Fig. 1a). The derivative of the thermogravimetric curve (DTG) was used to establish the intermediate limits of each of the decompositions (Fig. 1b). For assessment of the gases that had evolved during combustion, the TG analyser was linked to a Balzers ThermoStar Mass Spectrometer (model QMS 300M3). m/z relationships for the species analysed were: H2O (m/z = 18), CO2 (m/z = 44), HCl (m/z = 36) and SO2 (m/z = 64). The dwell time for each ion was 10 s and the cathode voltage in the ion source was 65 V. This analysis identified the gases released and displayed a profile over time, providing information on the decomposition mechanism (Fig. 2).

2.4.

WDXRF

Sample size was optimised to 5 g on the basis of a previous study of a standard hydroxyapatite (Reference #04238,

Fig. 1 – TG–MS. Results for human dentine: (a) TG curve. (b) DTG curve. Three types of human dentine components are shown sequentially. The first loss of mass is produced at temperatures up to 200 8C and corresponds to more volatile components (water); from 200 8C to 560 8C combustion of the organic fraction takes place to release CO2, H2O and NOx; over 560 8C a gradual loss of gases linked to inorganic matter is produced (CO2). The residual fraction corresponds to calcium phosphate.

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Fig. 2 – TG–MS. Spectrometric curves relative to the masses chosen for the analysis in the thermobalance of gases separated. The results shown in Fig. 2 are for human dentine. The four lines shown correspond to water (H2O), carbon dioxide (CO2), hydrogen chloride (HCl) and sulphur dioxide (SO2). H2O line: the initial tendency to descend refers to free H2O and the peak to the decomposition of CO2 and H2O in organic matter. CO2 line: the difference between organic and inorganic carbon can be observed. Both decomposed in two stages. A single peak was obtained in the HA sample. SO2 line: both peaks are linked to proteins. They are more intense in dentine than enamel. There is no sulphur in the HA sample. HCl line: the horizontal line expresses the absence of Cl linked to organic matter.

Sigma–Aldrich, St. Louis, MO, USA). The samples were analysed using a spectrometer (Bruker S4 Pioneer, Karlsruhe, Germany) equipped with an Rh anticathode X-ray tube (20– 60 kV, 5–150 mA, and 4 kW maximum), five analyser crystals (LiF200, LiF220, Ge, PET, and XS-55), a sealed proportional counter for detection of light elements, and a scintillation counter for heavy elements. This wavelength dispersive X-ray fluorescence spectrometer works sequentially, adapting the optical settings for each spectral line. The energy resolution and efficiency for each analytical line were determined by both the collimator aperture and the analyser crystal. Analyses were performed in vacuum mode to avoid signal losses by air absorption, allowing the detection of low-Z elements. The recorded spectra were evaluated by the fundamental parameters method, using SpectraPLUS software linked to the equipment (specifically EVA 1.7, a commercial package from Bruker-AXS and Socabim, Bruker AXS GmbH, Karlsruhe, Germany, 2006). A standardless method was used owing to the lack of satisfactory certified reference materials with elemental concentrations in the same range as the inorganic materials analysed in this study. The use of standardless procedures in the fundamental parameters method has been described by Rousseau.14 Analytes were assessed in the form of oxides, as is usual in mineral samples. The stoichometric Ca/P ratios were calculated using the following formula, taking into account the respective atomic masses: Ca (mol)/P (mol) = [Ca (wt%)/40.08 (g/mol)]/[P (wt%)/ 30.97 (g/mol)].

3.

Results

3.1.

Elemental analysis (Table 1)

For all the species analysed, the concentration of organic carbon was higher in dentine than in enamels; ovine (11.50 g/ 100 g) and porcine (11.10 g/100 g) dentine showed the highest concentrations. Human (1.60 g/100 g) and bovine (4.40 g/100 g) enamel showed the lowest concentrations of organic carbon. Among all the substrates analysed, ovine enamel contained the highest concentration of inorganic carbon (3.44 g/ 100 g) and porcine enamel the lowest (0.41 g/100 g).

Table 1 – Elemental analysis results: N: nitrogen; iC: inorganic carbon; oC: organic carbon; tC: total carbon.

Bovine enamel Bovine dentine Ovine enamel Ovine dentine Porcine enamel Porcine dentine Human enamel Human dentine Hydoxyapatite

N (g/100 g)

iC (g/100 g)

oC (g/100 g)

tC (g/100 g)

1.68 2.72 2.54 2.90 2.29 2.93 0.55 2.64 0.07

1.73 1.85 3.44 1.1 0.41 0.9 0.76 0.93 0.07

4.40 8.35 6.34 11.50 7.95 11.10 1.60 9.97 0.07

6.13 10.20 9.78 12.60 8.36 12.00 2.36 10.90 0.14

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Fig. 3 – TG–MS. The figure illustrates three well-differentiated thermal decomposition behaviours (in an oxidizing atmosphere). HA, being of geological origin, shows hardly any thermogravimetric losses. The enamel samples show a medium level of decomposition (HE < BE < OE < PE), due to their high degree of mineralisation. Among these, human enamel shows great similarity to synthetic hydroxyapatite. Dentine samples show high levels of decomposition (HD < BD < OD < PD) due to the greater presence of organic matter in their composition, human and bovine dentine showing greater similarity of behaviour. HA: hydroxyapatite; HE: human enamel; BE: bovine enamel; OE: ovine enamel; PE: porcine enamel; HD: human dentine; BD: bovine dentine; OD: ovine dentine; PD: porcine dentine.

Concentrations of nitrogen were higher in dentine than enamels. The lowest concentrations were found in bovine (1.68 g/100 g) and human enamel (0.55 g/100 g).

3.2.

TG–MS

Thermal decomposition curves in an oxidizing atmosphere showed three well-differentiated patterns (Fig. 3). The thermal decomposition of human enamel displayed great similarity to synthetic hydroxyapatite, but was very different from bovine, ovine and porcine enamels. Thermal decomposition curves of organic matter for dentine was characterised by a dramatic step; the greatest similarity in behaviour was seen between human and bovine dentine. Total thermogravimetric weight loss (Table 2) for HA was 5.04% over the whole temperature range. Dentine showed high

Table 2 – TG–MS. Total thermogravimetric losses: percentage of water, organic material, carbonate (inorganic CO2). Moisture Organic Carbonate Total (%) matter (%) (%) losses (%) Bovine enamel Bovine dentine Ovine enamel Ovine dentine Porcine enamel Porcine dentine Human enamel Human dentine Hydoxyapatite

4.63 8.16 5.54 8.17 5.83 7.87 2.03 6.46 2.16

10.90 19.20 12.89 23.23 13.07 21.35 5.70 19.19 1.79

3.65 2.24 2.83 2.05 2.64 2.03 2.91 2.59 1.09

19.18 29.60 21.26 33.45 21.54 31.25 10.64 28.24 5.04

levels of thermal decomposition (ranging 28.24–33.45%). Enamel showed medium levels of thermal decomposition, above HA but below dentine (ranging 10.64–21.54%). Enamel presented a lower percentage of water release and organic matter than dentine. Water released from human enamel (2.03%) was similar to hydroxyapatite (2.16%). Organic matter in bovine dentine (19.20%) was similar to human dentine (19.19%). Enamel showed higher concentrations of carbonates (inorganic CO2) than dentine. The highest concentration of carbonate was seen in bovine enamel (3.65%).

3.3.

WDXRF

Detailed information on the chemical composition of these calcified tissues is compiled in Table 3. In general, all dentine contained larger quantities of Mg, S, Sr and Zn than enamel, with the exception of human enamel, which contained smaller quantities of Zn and Sr than human dentine. All enamel contained greater quantities of P, Ca, Cl, Cu, K than dentine, as well as a higher Ca/P ratio. Human enamel and dentine contained higher quantities of P and Ca and a higher Ca/P ratio, greater quantities of Cl and Cu, and smaller quantities of Mg, S, Zn than the other species. Ca/P ratios are shown in Table 3.

4.

Discussion

The present study employed three analytic techniques: elemental analysis (C/N analysis), thermogravimetric analysis

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Table 3 – Chemical analysis (wt%) of the different specimens in terms of elements as measured by WDXRF. Moisture, organic matter, and inorganic CO2 were determined by TG–MS. Composition (%) Moisture Organic matter Inorganic CO2 Stoichiometric O Na Mg Al Si P S Cl K Ca Mn Fe Cu Zn Sr Ba Ca/P (mol/mol)

BE

BD

OE

OD

PE

PD

HE

HD

HA

4.63 10.90 3.65 32.8804 0.7160 0.536 – 0.0150 15.2900 0.0444 0.1550 0.0335 31,1100 – 0.0138 0.0017 0.0073 0.0169 – 1.57

8.16 19.20 2.24 28.9800 0.5960 1.0360 – – 13.5800 0.0710 0.0312 0.0245 26,0300 – 0.0048 0.0013 0.0121 0.0191 0.0140 1.48

5.54 12.89 2.83 32.2504 0.7380 0.6969 – 0.0072 15.1800 0.0524 0.1330 0.0336 29,6100 – 0.0052 0.0018 0.0097 0.0218 – 1.51

8.17 23.23 2.05 27.5346 0.5720 1.0680 – – 13.0300 0.0663 0.0250 0.0157 24.1800 – 0.0049 0.0013 0.0138 0.0264 0.0120 1.43

5.83 13.07 2.64 32.1989 0.7190 0.9117 – – 15.1900 0.0425 0.1400 0.0352 29.1900 – 0.0051 0.0016 0.0098 0.0162 – 1.48

7.87 21.35 2.03 28.4269 0.5870 1.0990 – 0.0065 13.4400 0.0629 0.0405 0.0244 25.0200 – 0.0065 0.0012 0.0161 0.0190 – 1.44

2.03 5.70 2.91 36.1166 0.8110 0.2910 – 0.0130 16.8000 0.0209 0.3170 0.0282 34.9100 – 0.0061 0.0023 0.0183 0.0256 – 1.61

6.46 19.19 2.59 29.3280 0.9020 0.7158 – 0.0180 13.6400 0.0697 0.0593 0.0257 26.9600 – 0.0055 0.0013 0.0132 0.0215 – 1.53

2.16 1.79 1.09 38.5695 0.1140 0.2860 0.0190 0.0350 17.8500 0.0181 0.0160 0.0150 37.9900 0.0084 0.0214 0.0020 – 0.0156 – 1.64

BE: bovine enamel; BD: bovine dentine; OE: ovine enamel; OD: ovine dentine; PE: porcine enamel; PD: porcine dentine; HE: human enamel; HD: human dentine; HA: hydroxyapatite. In this table only appear the elements whose final concentrations surpassed 3 times the signal to noise ratio. Values were blank-corrected for the following elements: Fe 0.0024% (part of the Fe signal comes from the sample holder) and Cu 0.0011% (part of the Cu signal comes from de collimator).

coupled to mass spectrometry (TG–MS) and wavelength dispersive X-ray fluorescence (WDXRF). WDXRF evaluates each element on the basis of oxides, which is the most suitable method for evaluating mineralogical samples, such as a hydroxyapatite matrix. This spectrometric technique measures heavy elements accurately but do not measure light elements such as C, H, O or N as these elements do not emit sufficient radiation to allow effective detection by this method. For this reason, it was necessary to adopt other techniques (C/N analysis and TG–MS) to evaluate these elements. Both additional techniques are based on combustion reactions, the difference between the two being that elemental analysis obtains a quantitative result by means of a calibration with a standard for each element, while TG–MS might be considered a semi-quantitative technique, as there is no reference pattern.15 The results obtained by the three techniques correlated and detailed information on the chemical composition of these calcified tissues is compiled in Table 3. The inorganic fraction ranges from 81 to 92% for enamels, 69–74% for dentins, and 96% for the hydroxyapatite standard, human teeth being the most mineralised tooth and bovine the closest to the human, in both enamel and dentine. Although WDXRF data were evaluated on the basis of oxides, which is usual when assessing mineral samples, the data obtained were eventually listed as elements, adding up the stoichiometric oxygen from each oxide in one amount. Inorganic CO2 is released by the partial decomposition of the inorganic fraction, mainly carbonated apatite.16 The presence of carbonate disrupts the crystal lattice, making the carbonated-apatite chemically weaker than hydroxyapatite. All enamels are more carbonated than the corresponding dentine; human values are characterised through high carbonate content, and similarities were found between human and ovine enamel, and between human and bovine dentine.

For all the species studied, the percentage of organic carbon and nitrogen, evaluated by C/N analysis, was found to be higher in dentine than in enamel (Table 1). This is an indication of the higher percentage (by weight) of organic material in dentine than in enamel – a well-known fact.17 Human enamel was the most highly mineralised of all, with C and N values the closest to pure hydroxyapatite. Bovine dentine and enamel presented the most similar C and N values to human dentine and enamel. The most abundant chemicals elements in enamels and dentine of the four species were calcium and phosphorus. The Ca/P molar ratio was approximately 1.67 for pure hydroxyapatite.18 The rest of biological apatites appear to be ‘calciumdeficient’. The Ca/P molar ratio was higher in enamel than dentine, as expected. In human enamel it was 1.61, the nearest to pure hydroxyapatite. Bovine enamel was the closest to human (1.57), followed by ovine (1.51) and porcine (1.48). In dentine, the Ca/P relationship was higher in human (1.53) followed by bovine (1.48), porcine (1.44) and ovine (1.43). In this way, human enamel and dentine showed the highest degree of mineralisation, and again, bovine showed the most similar ratios to human teeth. Hydroxyapatite can present numerous substitutions; metal cations such as K+, Na+, Mn2+, Ni2+, Cu2+, Mg2+ or Zn2+ can occupy the Ca2+ position, anionic complexes such as AsO43 , SO42 , CO32 or SiO44 can replace PO43 and anions such as Cl or F can replace OH in the crystal structure. These ions, found in trace quantities, proceed from dental pulp capillaries and, during the pre-eruptive period, are incorporated into dentine or into the enamel layers close to the amelodentinal junction.19 They may also proceed from the environment surrounding the tooth, mainly from saliva, and are then incorporated post-eruption to a depth of 150 mm into the enamel surface.10,20 The incorporation of trace elements into

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the crystalline structure modifies the physico-chemical, and mechanical properties and the solubility of hydroxyapatite crystals. In this way, a high correlation between tooth hardness and its zinc content has been shown, while the presence of magnesium makes the substrata more porous and fluoride makes the tooth more resistant to acid attack.8 Using WDXRF, the present study detected chemical elements as detailed in Table 3. Cl, which indicates the quantity of salt and water in each sample, showed greater concentrations in enamels than in dentine for all four species; human teeth showed the highest concentrations. Gutie´rrezSalazar and Reyes-Gasga21 determined higher concentrations in the surface layers using energy dispersive X-ray spectroscopy but did not find any Cl in human dentine. As for zinc, non-human teeth showed a higher concentration in dentine than enamel, while the opposite occurred in human teeth. Strontium (Sr) showed the same behaviour; it can replace calcium ions within the apatite crystal lattice, one strontium ion replacing one out of 10 calcium ions.22 Sulfur (S) was not relevant in human enamel but was identified in the other animal species, showing a higher concentration in dentine than in enamel as it is related to protein content. Copper (Cu) showed the opposite behaviour, with higher concentrations in enamel than dentine, and higher concentrations in humans than animals. Magnesium shows similar behaviour to zinc and strontium, with higher concentrations in dentine than enamel in all the species analysed, as observed in other studies.21 The greater presence of these three elements in dentine explains why the Ca/P ratio is lower in dentine, as these usually substitute Ca directly in the crystal structure. Although some authors have described fixed and ordered concentrations of trace elements as a habitual characteristic of dental structures (Sr > Mg > Zn > Pb > Fe > Cu),19 these elements and the order by concentration have been calculated as mean values of each in the whole tooth (including enamel, dentine, pulp). According to Reitznerova´ et al.20 calcium, phosphorus, magnesium, and sodium constitute the most abundant and basic metal ions that form the structure of mineralised tooth tissues. Indeed, these four elements were found to be more abundant in the enamel and dentine of the four species analysed in the present study. However, the order of concentrations of the other elements does not coincide with that described by Curzon and Featherstone,19 and furthermore, concentrations were different between enamel and dentine in the four species analysed. In fact, concentrations of trace elements vary between studies10,20,21,23 and it has been observed that these vary with age, gender, the type of tooth (incisor, premolar or molar), nutrition, environmental pollution and whether the tooth is deciduous or permanent.9,23 Furthermore, in a single tooth, concentrations vary in relation to the depth within layers. Reitznerova´ et al.20 – using FAAS, ETA AAS, ICP-AES and ICP-MS – determined the presence of seven elements (Cu, Fe, Mg, Mn, Pb, Sr and Zn) in the whole enamel layer and close to the surface of extracted non-carious human teeth. With the exception of Sr and Mg, all elements showed significantly higher concentrations in the surface layers. A tidal effect occurs, whereby the enamel crystallites are dissolved and then recrystallised and the composition of

trace elements in the enamel layer can be affected by saliva, in the de- and re-mineralisation process, up to 100–150 mm from the surface. Fluorine presence was not reported in this study due to the lack of sensitivity of the instrumental technique chosen to explain teeth composition (WDXRF). Ion chromatography could be used to determine total fluoride by microwave digestion of the specimens in nitric acid. In this case, digestion and later 1:100 dilution should not preclude a good analysis of the ion, due to the low limit of detection of this technique for the analyte, close to 1 ppb. Furthermore, an extract of free fluoride could be assessed by 1:10 (w/v) leaching of the solid samples in water for 24 h, according to the leaching test DIN 38414-S4. Although the aim of our study was to determine the elemental composition of enamel and dentine of four species, the knowledge of its macrostructure and microstructure is essential to understand the excellent physicochemical properties and mechanical behaviour of teeth.7 Thus, although a high mineral content gives the tooth mechanical hardness and high resistance to wear,24 Kinney et al.25 that the Young’s modulus, tensile and compressive strength, and fracture toughness are properties that depend on both the composition and microstructure.

5.

Conclusions

Regarding to thermal decomposition, enamel showed similar behaviour to that of synthetic hydroxyapatite; but dentine, with a higher organic content (organic carbon and nitrogen), showed a significantly different thermal behaviour. All the substrates studied were low in calcium; the Ca/P ration was lower than hydroxyapatite. Among all the species, human enamel and dentine were the most mineralised. Elemental analysis, TG–MS, and XRF showed that human enamel and dentine showed the greatest similarity to bovine enamel and dentine. Therefore, on the basis of their chemical composition, bovine teeth should be the first choice as substitutes for human teeth in research. Additionally, a wide range of mechanical tests should be performed to monitor the degree of adequacy of alternative dental tissues as a substitute for a human tooth.

Funding The authors have conducted this investigation with own funding.

Competing interests None declared.

Ethical approval To perform this investigation the authors not needed ethical approval.

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Comparison of chemical composition of enamel and dentine in human, bovine, porcine and ovine teeth.

The aim of this paper was to compare the chemical composition of human teeth with other mammal species that are likely candidates for replacing them i...
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