Durham Anthropology Journal
Volume 12(2-3) Copyright © 2005, Su Xian Tao, Masashi Takahashi, Yukio Miyagawa, and Sen Nakahara

Comparison of the histological structure and elemental composition between primordial and degenerative third molar enamels

Su Xian Tao, Masashi Takahashi, Yukio Miyagawa, and Sen Nakahara

The Nippon Dental University School of Dentistry at Niigata, 1-8, Hamaura-cho, Niigata 951-8580, Japan

nakahara@ngt.ndu.ac.jp

Summary : Japanese have the highest rate of congenital absence of third molars, while Thais have the lowest. Moreover, most of Thai samples revealed primordial macroscopic features, while most of Japanese samples showed degenerative ones. We examined the histological structure and elemental composition of third molars and compared differences between degenerative samples of Japanese and primordial ones of Thais. Histological structure was observed utilizing scanning electron microscopy. For elemental composition, quantitative analyses were conducted with an electron probe microanalyzer usingstandard samples.Our findings indicated that the outlines of the enamel rod sections of degenerative Japanese lower third molars were significantly distorted; the rod widths were not uniform compared with those of the primordial Thai samples. The enamel of the primordial Thai teeth had clear-cut Schreger's bands. The primordial Thai teeth had considerable amounts of calcium, phosphorus, and sodium, but lacked oxygen in comparison with the degenerative Japanese, whose enamel histological structure was the most distorted. The distortion level in histological structure had a parallel with elemental composition. We consider that an abundance of calcium, phosphorus and sodium may reduce distortion in the histological structure to achieve a balance, allowing optimization of the elemental composition. In particular, the sodium content might be a deciding factor in the formation of enamel. Our results suggest that tooth quality may be defined according to the amount of sodium-containing substance ingested when teeth are being formed.

Key words: degenerative, primordial, third molar, enamel, Vickers' hardness, histological structure, elemental composition

1. Introduction

1.1. Numerous studies have been undertaken on the histological structure and elemental composition of the enamel of human teeth but none has ever considered ethnic differences prior to our investigations (Shobusawa 1952, Kawai 1955, Meckel et al. 1965, Gwinnett 1966, Boyde 1967, Osborn 1968a,b,c, Boyde 1969, Suga et al. 1971, Osborn 1973, Boyde 1976a,b, Gantt et al. 1983, Imanishi et al. 1981, Boyde and Martin 1982, Gantt 1983, Boyde and Martin 1984a,b, Martin 1985, Beynon and Wood 1987, Deutsch et al. 1987, Ogawa and Suga 1988, Takahashi et al. 1990, Takahashi and Kobayashi 1997). In a previous study, we investigated the congenital absence rates of the third molar and compared the differences among different Asian countries (Nakahara et al. 1997). Our study revealed that modern Thais had the lowest rate of absence for all four third molars (0.0% for men; 4.7% for women). In contrast Japanese had the highest rate of absence for all four third molars in the ethnic groups examined: 24.9% for men and 48.6% for women. Moreover, most of the lower third molars of the Thai samples revealed primordial macroscopic features, while most of Japanese specimens showed degenerative ones.

1.2. Thus, the primary aim of our present study was to examine and compare the histological structure and elemental composition of the most primordial lower third molar enamels as seen in Thai specimens and those with the most degenerative enamel as observed in Japanese specimens.

2. Materials and methods

2.1. The lower third molars examined in the study were fixed with 10% (v/v) neutral formaldehyde immediately after extraction. We observed the macroscopic features of a sample of thirty Thai and one hundred Japanese teeth. Seven teeth containing normal macroscopic features were selected and used for each of the Japanese and Thai groups. All of the teeth examined were completely impacted below the oral environment and as such their compositions were not affected by saliva or dietary irons. All ground sections, which were used for the following procedures, were examined by polarizing microscopy and it was confirmed that they contained no dental caries.

2.2. Buccolingual ground sections that were spread across the distolingual cusp point were prepared and etched for 12 hours with 2.5% (w/v) EDTA, for the purpose of observing the Schreger's bands more clearly. After coating with platinum, their enamel histological structures were then examined under a scanning electron microscope (S-800, Hitachi). The re-ground surfaces of the buccolingual ground sections spread across the mesiobuccal cusp point were prepared at a slight incline to the enamel surface. This was performed so that observation could be made from the dentine to the outermost surface layer of the enamel. After being etched for three minutes with 0.05 N HCl in order to observe the emphasized outline of the enamel rod section, they were coated with platinum. The enamel histological structures of the re-ground surfaces were examined under the scanning electron microscope.

2.3. The contents of seven elements (mass %) were analyzed quantitatively using standard samples with an electron probe microanalyzer/EPMA (JXA-8900, JEOL). Measurements were performed at 20 locations in the deep, middle, and surface layers of the enamel in the re-ground surfaces, which were at a slight incline to the enamel surface, of the buccolingual ground sections spread across the distobuccal cusp point. These specimens were not etched with any acid solution in order to avoid any change in the elemental composition and were coated with gold. The measurements were made under the following conditions: accelerating voltage 15 kV, probe current 5.0 x 10-8 A, and probe diameter 50 m.

3. Results

3.1. 1. The majority of the thirty lower third molars of the Thai sample revealed primordial macroscopic features such as five cusps and longer roots. Most of the one hundred Japanese specimens showed degenerative macroscopic features with four cusps and shorter roots.

3.2. 2. Using EDTA, we etched the buccolingual ground sections that were spread across the distolingual cusp point, and then observed the middle layer of the enamel in the center of the lingual side, with the scanning electron microscope. Primordial Thai samples revealed a clear-cut distinction between the parazone of the Schreger's band and the diazone, compared with the degenerative Japanese samples. Using the same specimens, we examined the parazone of the rod sections in the middle enamel. The images were magnified for observation. The findings showed that the primordial Thai specimen presented a clearer contour of apatite crystal, which constitutes the enamel rod section. The re-ground surfaces of the buccolingual ground sections that were spread across the mesiobuccal cusp point were prepared slightly inclining to the enamel surface in order to observe from the dentine to the outermost surface layer of enamel. These re-ground surfaces were etched with HCl and observed with the scanning electron microscope. Compared with the primordial Thai specimens, degenerative Japanese specimens showed a high degree of distortion in the oval rod sections of the enamel of the inner second zone, as well as an indistinct outline of the enamel rod section (Figs. 1 & 2). The same specimens were examined for the third zone of the inner enamel, where U-shaped enamel rod sections were aligned parallel. Compared with the primordial Thai specimens, degenerative Japanese specimens showed a high degree of distortion in the rod sections, as well as an indistinct outline of the enamel rod sections. We also examined the middle enamel of the same specimens, where rod sections shaped like keyholes were aligned in a stepwise manner. In comparison with the primordial Thai specimens, degenerative Japanese specimens showed a high degree of distortion in the rod sections, as well as inconsistent widths of the enamel rod sections. We made the following observations for the outer enamel of the same specimens, which had distorted rod sections in addition to rodless enamel. Compared with the primordial Thai specimens, degenerative Japanese specimens showed a high degree of distortion in the outline of the rod section, as well as an indistinct outline of the enamel rod sections. In addition, the laminal structure, almost parallel to the enamel surface, was clear-cut (Figs. 3 & 4). No difference was found in regards to dentine between the degenerative Japanese specimens and the primordial Thai specimens.

3.3. The contents of the seven elements analyzed for the primordial Thai specimen with the lowest level of distortion in the histological structure of enamel and the degenerative Japanese specimen with the highest level are shown in Table 1. The calcium content in both the degenerative Japanese and the primordial Thai specimens was highest in the middle layer of the enamel and lowest in the deep layer. Overall, the primordial Thai mandibular third molar had higher calcium contents in all layers in comparison to the degenerative Japanese. According to the t-test results, the differences between the two groups were highly significant in the surface layer (p<0.01) and significant in the middle layer (p<0.05; Fig. 5).

3.4. The phosphorus content in both the degenerative Japanese and the primordial Thai specimens was highest in the surface layer and very low in the deep layer. The primordial Thai specimen had significantly more phosphorus than the degenerative Japanese in the middle and surface layers. The two groups had similar phosphorus contents in the deep layer (Fig. 6).

3.5. The oxygen content in both the degenerative Japanese and the primordial Thai specimens was highest in the middle layer and considerably low in the surface layer. Overall, the degenerative Japanese specimen had higher oxygen contents in all layers than the primordial Thai specimen. The differences between the two groups were highly significant in the middle and surface layers and significant in the deep layer (Fig. 7). The carbon content in both the degenerative Japanese and the primordial Thai specimens was the highest in the deep layer and the lowest in the middle layer, and the content in the surface layer was intermediate. No significant differences were found between the two groups in any layers (Fig. 8).

3.6. The sodium content in both the degenerative Japanese and the primordial Thai specimens was the highest in the middle layer and remarkably low in the surface layer. Overall, the primordial Thai specimen had higher sodium contents in all layers than the degenerative Japanese. The differences between the two groups were highly significant in the middle and deep layers and significant in the surface layer (Fig. 9). The magnesium content of both the degenerative Japanese and the primordial Thai specimens was the highest in the deep layer and the lowest in the surface layer. For the surface layer, the degenerative Japanese specimen had significantly more magnesium than the primordial Thai specimen. Both groups, however, had similar magnesium contents in the middle and deep layers (Fig. 10). The fluorine content in both the degenerative Japanese and the primordial Thai specimens was highest in the surface layer and lowest in the middle layer. Significant differences between the two groups were not found in any layers (Fig. 11).

4. Discussion

4.1. The acid etching method of the enamel was examined for observation with SEM (Boyde et al. 1978, Li and Risnes 2004). In this study, however, the enamel was etched for 12 hours with 2.5% (w/v) EDTA, for the purpose of observing the Schreger's bands more clearly. The enamel was etched for three minutes with 0.05 N HCl in order to observe the emphasized outline of the enamel rod sections.

4.2. The Japanese mandibular third molars were more degenerative in macroscopic features than those from Thais, and their enamel rod sections had more strongly distorted outlines and inconsistent rod widths. Schreger's bands and the contours of apatite crystal, which constitute the enamel rod sections, were more distinctive in the primordial Thai enamel than in that of the degenerative Japanese teeth. Therefore, we propose that there may be a parallel among the congenital absence rate of the third molars, the degeneration level in their macroscopic features, and the distortion level of the histological structure of enamel. The etching of enamel with an acid solution might change the enamel's elemental composition (Risnes, 1990). Therefore, to analyze the elemental composition accurately, specimens were used that had not been etched with an acid solution. The total amounts of the raw concentration data of the seven elements were within 100 % ± 2 % at almost all of the locations measured. This result demonstrates that the elemental analysis of enamel by EPMA was performed suitably. The average measurement error was considered to be less than two percent of the analyzed value. The practical elemental composition of enamel does not agree with the theoretical elemental composition based on the chemical formula of pure hydroxyapatite crystal. This is because the dental enamel contains a small amount of organic matter and other inorganic materials besides pure hydroxyapatite crystals. The following differences from the theoretical elemental composition of hydroxyapatite crystal, however slight, could be very significant findings in our present study.

4.3. The calcium content in both the degenerative Japanese and the primordial Thai specimens in the middle and surface layers of enamel ranged from 34% to 35%, approximately 5% lower than 39.9% of the theoretical rate based on the chemical formula of hydroxyapatite. The presumption is that this results from circumstances where calcium ions inside the crystal lattice are substituted by other metallic ions. The primordial Thai specimens had higher calcium contents than the degenerative Japanese ones, and the calcium content was highest in the middle layer where the histological structure was the least distorted. From these findings, we surmise that calcium content is an opposite to the distortion level of the histological structure.

4.4. The phosphorus content in both the degenerative Japanese and the primordial Thai specimens in the middle and surface layers of the enamel ranged from 22% to 23%, roughly 4% higher than 18.6% of the theoretical rate based on the chemical formula of hydroxyapatite. The extra amount of phosphorus can be explained as existing as ions out of the crystal lattice. The phosphorus content is higher in the surface layer than in the middle layer, while the calcium content is higher in the middle layer than in the surface layer. The greater amount of phosphorus in the surface layer can be explained by its existence as ions out of the crystal lattice.

4.5. The oxygen content in both the degenerative Japanese and the primordial Thai specimens in all layers ranged from 37% to 39%, 2.5%-4.5% lower than 41.5%, which is the theoretical rate based on the chemical formula of hydroxyapatite. The deficiency can be explained as stemming from the relatively low oxygen content due a portion of phosphorus, which constitutes phosphoric acid, existing as ions out of the crystal lattice. The carbon content in both the degenerative Japanese and the primordial Thai specimens was the highest in the deep layer. Therefore, the amount of carbon is considered to reflect organic content.

4.6. In 1971, it was reported that the sodium content was lowest in the outermost surface layer and highest in the deepest layer (Suga et al. 1971). However, the results of our study indicated that the sodium content was highest in the middle layer in both the degenerative Japanese and the primordial Thai specimens. Both studies showed that the sodium content was lowest in the surface layer. The low sodium content can be explained as a parallel with the decline in calcification in the surface layer. The primordial Thai specimens had higher sodium contents than the degenerative Japanese ones in all layers; thus, primordial Thai mandibular third molars may demonstrate a higher degree of calcification in all layers than in the degenerative Japanese.

4.7. The magnesium content in both the degenerative Japanese and the primordial Thai specimens was highest in the deepest layer. Thus, the amount of magnesium is surmised to reflect the content of magnesian whitlockite, which is a type of crystal that is primarily comprised of calcium phosphate, like apatite.

4.8. Some studies have reported that the fluorine content increased in the outermost surface layer as teeth aged (Brudevold et al. 1956, Ogawa et al. 1988). However, our findings suggest that the fluorine content in the surface layer is initially high during the period of enamel formation and continues to increase as teeth age. The reason is that our results showed that impacted teeth, which are not affected by oral cavity milieu, had the highest fluorine content in the surface layer.

4.9. The Thai specimens with primordial features had a low distortion level in the histological structure of enamel, higher content of calcium, phosphorus and sodium, but a lower content of oxygen. Thus, we conclude that the distortion level in the histological structure of enamel may be parallel with its elemental composition.

4.10. Higher contents of calcium, phosphorus and sodium may reduce distortion in the histological structure to achieve a balance, allowing optimization of the elemental composition. Our findings suggested that, among other elements, the sodium content might be a deciding factor in the formation of the enamel. Further study is required to investigate the possibility that the ingestion of sodium-containing substances such as sodium chloride during tooth formation has the potential to promote the strengthening of teeth.

Acknowledgments

We are grateful to Prof. Hideo Ogura, Dr. Shinichi Goto, Dr. Mikio Ishiyama, and Dr. Zac Morse of The Nippon Dental University School of Dentistry at Niigata, for their advice and cooperation.

References

Beynon AD, Wood BA (1987) Patterns and rates of enamel growth in the molar teeth of early hominoids. Nature 326: 493-496

Boyde A (1967) The development of enamel structure. Proc Roy Soc Med 60: 923-928

Boyde A (1969) Correlation of ameloblast size with enamel prism pattern, use of scanning electron microscope to make surface area measurements. Z Zellforsch 93: 583-593

Boyde A (1976a) Enamel structure and cavity margins. Operative Dentistry 1: 13-28

Boyde A (1976b) Amelogenesis and the structure of enamel. In Cohen B, Kramer IRH (Ed) Scientific foundations of dentistry, pp. 335-352. William Heineman Medical Books Ltd: London

Boyde A, Martin L (1982) Enamel microstructure determination in hominoid and cercopithecoid Primates. Anatomy & Embryol 165: 193-212

Boyde A, Martin L (1984a) The microstructure of primate dental enamel. In Chivers DJ, Wood BA, Bilsborough A (Ed) Food acquisition and processing in primates, pp. 341-367. Plenum Press: New York

Boyde A, Martin L (1984b) A non-destructive survey of prism packing patterns in primate enamel. In Fearnhead RW, Suga S (Ed) Tooth enamel IV, pp. 417-421. Elsevier Science Publishers: Amsterdam

Boyde A, Jones SJ, Reynolds PS (1978) Quantitative and qualitative studies of enamel etching with acid and EDTA. Scan Electron Microsc 2: 991-1002

Deutsch D, Ogawa M, Suga S (1987) Mineral distribution from enamel surface to enamel-dentin junction in developing human deciduous teeth. Odontology 75: 106-116

Gantt DG (1983) The enamel of Neogene hominoids, structure and phyletic implications. In Ciochon RL, Corruccini RS (Ed) New interpretations of ape and human ancestry, pp. 249-298. Plenum Press: New York

Gantt DG, Pilbeam D, Steward GP (1977) Hominoid enamel prism patterns. Science 198: 1155-1157

Gwinnett AJ (1966) Ultrastructure of the "prismless" enamel of deciduous teeth. Arch Oral Biol 11: 1109-1115

Imanishi I, Hosoi M, Akisaka T, Nishimori T, Suemune S (1981) A whorled arrangement of the enamel rods in human teeth. J Hiroshima Univ Dent Soc 13: 324-328

Kawai N (1955) Comparative anatomy of the bands of Schreger. Okajima Folia Anat Jap 27: 115-131

Li C, Risnes S (2004) SEM observations of Retzius lines and prism cross-striations in human dental enamel after different acid etching regimes. Arch Oral Biol 49: 45-52

Martin L (1985) Significance of enamel thickness in hominoid evolution. Nature 314: 260-263

Meckel A, Griebstein WJ, Neal RJ (1965) Ultrastructure of fully calcified human dental enamel. In: Stack MV, Fearnhead RW (Ed) Tooth enamel, pp. 160-162. John Wright and Sons Ltd: Bristol

Nakahara S, Tao SX, Kee CD, Chang Y, Lee Y, Yee JN, Wang D, Zheng GY, Sun DL, Lo CS, Chou MY, Gervasio NC, Amante A, Boncan R, Rivera R, Chearapongse N, Chearapong P (1997) Ethnic differences concerning the congenital absence of third molars: a comparison of modern people in six Asian countries. Odontology 84: 551-559

Ogawa M, Suga S (1988) Fluoride distribution in the enamel caries lesions. Jpn J Oral Biol 30: 247-276

Osborn JW (1968a) Evaluation of previous assessments of prisms direction in human enamel. J Dent Res 47: 217-222

Osborn JW (1968b) Directions and interrelationships of enamel prisms from the sides of human teeth. J Dent Res 47: 223-232

Osborn JW (1968c) Directions and interrelationships of prisms in cuspal and cervical enamel of human teeth. J Dent Res 47: 395-402

Osborn JW (1973) Variations in structure and development of enamel. Oral Science Reviews 3: 16-72

Risnes S (1990) Shark tooth morphogenesis. An SEM and EDX analysis of enameloid and dentin development in various shark species. J Biol Buccale 18: 237-248

Shobusawa M (1952) Vergleichende Untersuchungen uber die Form der Schmelzprismen der Saugetiere. Okajima Folia Anat Jap 24: 371-392

Suga S, Kondo M, Onodera A, Kubota Y, Ohtsuka M (1971) Electron microprobe analyses on the distributions of Cl, Mg and Na, in the enamels of various animals. Jpn J Oral Biol 13: 85-94

Takahashi M, Kobayashi K (1997) Histological studies on the cervical enamels of each surface in human permanent teeth. In Motta PN (Ed) Rec Adv Micro Cell Tiss Org., pp. 353-356. Antonio Delfino Editore: Rome

Takahashi M, Asami Y, Kobayashi K (1990) Histological studies on enamels of human deciduous and permanent teeth based on an evolutional viewpoint. Odontology 78: 743-754