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Keywords:

  • bovine enamel;
  • microstructures;
  • Hunter–Schreger bands;
  • decussation;
  • SEM

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. LITERATURE CITED

Bovine teeth have been considered as an excellent substitute for human teeth for dental research, however, the enamel microstructures of bovine incisors that include arrangements of prisms and interprisms, and their spatial relationships have not been well described. The aim of this study was to investigate the detail enamel microstructures of bovine incisors. Eight bovine mandibular incisors were cut into 77 pieces at eight equal intervals either in the longitudinal direction or in the horizontal direction before each piece had been tangentially cut (parallel to enamel–dentin junction) through the middle of the enamel thickness. All the sectioned surfaces were treated 1 M HCl for 10 sec to expose the prisms and interprisms before observation by scanning electron microscopy. The parallel enamel prisms were located in all the outer enamel, the cervical region and the incisal ridge of the bovine incisors. Most labial inner enamel and the cingulum of lingual inner enamel were composed of the Hunter–Schreger bands with the characteristics of decussating groups of prisms and decussating planes between interprisms and prisms. The interprisms were thicker in the inner enamel than in the outer enamel. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. LITERATURE CITED

Several types of nonhuman teeth have been used as substitutes for dental experiments. Bovine mandibular incisors have been considered as an excellent substitute for human teeth because they are easy to obtain (Mellberg, 1992) and have a relatively large flat surface without caries lesions and defects (Rueggeberg, 1991; Zero, 1995; Skene, 2002). Bovine incisors have been successfully used in numerous studies as a substitute for human teeth for many years (Attin et al., 2007; Soares et al., 2010; Yassen et al., 2011).

Bovine enamels possess many similarities with human enamels because they have similar chemical compositions and physicochemical properties (Feagin et al., 1969; Putt et al., 1980). Feagin et al. (1969) found that the demineralization and remineralization characteristics of both bovine enamel and human enamel were similar. No significant differences were found between human and bovine enamel in their carbonate contents, physical properties, polishing ability, luminescence, and refractive indices (Putt et al., 1980). In addition, controversial findings were reported on radiodensity between human and bovine enamel (Fonseca et al., 2004; Tanaka et al., 2008). Fonseca et al. (2004) reported that both enamels shared similar radiodensity, while Tanaka et al. (2008) reported that the radiodensity of bovine enamel was significantly greater than that of human enamel.

Slight differences were found in chemical compositions and physical properties between human enamel and bovine enamel (Davidson et al., 1973; Arends and Jongebloed, 1978). Davidson et al. (1973) reported that the calcium contents of bovine enamel and human enamel by weight were 37.9% and 36.8% respectively, and the calcium distribution was more homogenous in bovine enamel compared with human enamel.

To date, many studies have reported on the microstructures of human enamel. The mineral phase of human enamel consists of hexagonal hydroxyapatite (HAp) crystals, and the reported sizes of human enamel crystals varied with different studies, ranging from 50–100 nm in length and 68–500 nm in diameter (White et al., 2001; Xie et al., 2008; Hannig and Hannig, 2010). The average diameter of human enamel crystals was reported to be smaller than bovine enamel crystals (Arends and Jongebloed, 1978). Generally, these crystals form enamel prisms with 3–8 μm diameter keyhole-like structures (Habelitz et al., 2001; White et al., 2001). The enamel coating on the tooth crown comprises densely and orderly packed prisms, extending from the enamel–dentin junction (EDJ) outward (Habelitz et al., 2001; An, 2012). Hunter–Schreger bands (HSBs) characterized with parazone (longitudinally cut prisms) and diazone (transversally cut prisms) (Osborn, 1965; Hanaizumi et al., 1996; Bechtle et al., 2011) were the feature of human enamel as well as other mammalian enamels (Mary Carol Maas, 1999).

Although mammalian enamels are similar, the specific enamel microstructures appear to vary between classes and species (Mary Carol Maas, 1999). Bovine enamel contains a larger number of fibril-like interprisms, whereas human enamel has a lower quantity of interprisms (Fonseca et al., 2004; Fonseca et al., 2008). In addition, Bechtle et al. (2011) reported that HSBs in bovine enamel consists of groups of decussated prisms with the same orientations and are about 20–30 μm wide. However, the microstructures of bovine enamels of mandibular incisors have not been described in detail, especially in the spatial relationship between prisms/prisms and prisms/interprisms.

The aim of this study was to describe the microstructures of bovine enamel of mandibular incisors, particularly the prism and interprism arrangements and relationships by scanning electron microscopy (SEM).

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. LITERATURE CITED

Specimen Preparations

Eight freshly extracted bovine mandibular incisors stored in 1‰ thymol solution at 4°C were used within 1 month after extraction. This research protocol was performed in accordance with the international Ethical Guideline and Declaration of Helsinki and approved by the ethics committee of Zhejiang University School of Stomatology (World Health Organization, 2002; World Medical Association, 2008). Roots were cut off 1 mm under the cemento–enamel junction with a low speed diamond saw (Isomet, Buehler, Lake Bluff). After the incisors were cut longitudinally into three fragments (mesial, middle, and distal thirds) at the labiolingual direction, each fragment was horizontally cut into three smaller fragments (occlusal, middle, and cervical thirds) (Fig. 1a). Each fragment was further trisected as shown in Fig. 1b before each piece was cut into two through the middle of the enamel parallel to EDJ (Fig. 1c).

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Figure 1. Diagram of specimen preparations of bovine incisors. Initially, the bovine incisors were prepared into nine fragments as following lines: Line 1 indicates the line of mesial/middle one-third; Line 2 indicates the line of middle/distal one-third; Line 3 indicates the line of occlusal/middle one-third; Line 4 indicates the line of middle/cervical one-third(a) Subsequently, each fragment had been equally trisected(b), tangential sections were obtained by cutting through the middle of enamel thickness(c).L and H indicate the longitudinal and horizontal direction of the bovine incisors, respectively. T indicates the tangential surfaces of bovine enamel that parallel to the enamel surface. EDJ: Enamel–dentin junction. OES: Outer enamel surface.

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Scanning Electron Microscopy

After the longitudinal section surfaces of each small fragment had been wet-ground with a series of silicon carbide papers (#500, #800, and #1,200; HERMES, Germany), the specimens were treated with 1 mol/L hydrochloric acid for 10 sec and rinsed with distilled water. The specimens were dehydrated in ascending concentrations of ethanol (25, 50, 75, and 100% for 20 min, each time), air-dried, and then sputtered with platinum coat about 5–6 nm thickness. They were then observed with an SEM (Zeiss Ultra 55, Germany). The SEM was operated at 4.5–6 KV with a working distance of 5–8.5 mm in the secondary electron mode. Subsequently, the other sectioned enamel surfaces on each fragment were prepared and observed with SEM as described above to view the horizontally and tangentially sectioned enamel surfaces.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. LITERATURE CITED

The arrangements and the distributions of enamel prisms and interprisms are shown in Figs. 2–9. The SEM findings revealed decussating prisms (Figs. 4–6) and parallel prisms in the bovine enamel (Figs. 7 and 8). Decussating prisms appeared with characteristics of HSBs (Fig. 2). HSBs were found in the inner enamel of lingual cingulum and in most of the areas of the labial inner enamel (Fig. 2). Parallel prisms were found in the outer enamel, the incisal ridge, the cervical region, and the most lingual enamel except the inner enamel of the lingual cingulum (Fig. 2).

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Figure 2. Longitudinal section of bovine incisors schematically revealing the orientations of enamel prisms and interprisms of labial and lingual enamels. Most labial inner enamel and the cingulum of lingual inner enamel are characterized with HSBs, consisting of parazones (longitudinally cut prisms) and diazones (transversally cut prisms). Parallel prisms are located in all the outer enamel, cervical region, and incisor ridge. D: diazone; P: parazone; IE: the inner enamel; OE: the outer enamel; PP: parallel prism.

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Figure 3. SEM micrograph of longitudinal section of bovine enamel. (a) Image of enamel close to dentin shows decussation between the prisms and interprisms. An angle between prism and EDJ is about 45–55 degrees. (×2,500, bar = 10 μm). In the middle of enamel, the longitudinally cut prisms (parazones (P)) and transversely cut prisms (diazones (D) alternate at intervals. They were demarcated by two dotted lines. Prism/prism decussating plane (solid line with two arrows) is almost perpendicular to the prism/interprism decussating plane (hollow line with two arrows). The shapes of the cross-sectioned prisms are not same in a band due to the gradual change of orientations of prisms. (×2,500, bar = 10 μm). Higher magnification image of diazone shows that a row of transversally cut enamel prism (dotted circles) alternates with interprism layer by layer. A row of prisms consist of 8–10 prisms. The bifurcations of interprisms are indicated with a symbol (inclined, inverted V). (×5,000, bar = 2 μm).

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Figure 4. SEM micrograph of the horizontal section of bovine enamel. (a) Enamel prisms and interprisms near to EDJ are mixed and cannot make a distinction from each other. Parallel interprism and prism are indicated with white arrows. (×2,500, bar = 10 μm). (b) Wide parazones (P) and narrow diazones (D) alternate in inner enamel. EDJ: enamel–dentin junction. (×2,500, bar = 10 μm).

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Figure 5. SEM micrograph of tangential section of bovine enamel. (a) Low magnification image shows arrangements of enamel prisms in two directions. (×1,000, bar = 10 μm). (b) A row of enamel prisms are alternate with a thin layer of interprisms layer by layer. (×5,000, bar = 2 μm).

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Figure 6. SEM micrograph of parallel prism in longitudinal section of bovine enamel. (a) In inner enamel, all prisms parallel to each other, and are perpendicular to interprisms. Prisms and interprisms are indicated with white arrow. (×2,500, bar =10 μm). (b) In outer enamel, enamel prisms are arranged in a parallel pattern and interprisms alternated with them at a small angel about 30 degree. (×2,500, bar=10 m).

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Figure 7. SEM micrograph of parallel prism in the horizontal section of bovine enamel. A typical honeycomb pattern is visible in the outer enamel. (×10,000, bar=1 μm).

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Figure 8. SEM micrograph of parallel prism in tangential section of bovine enamel. (a) Low magnification image shows parallel prisms in an undulation. (×1,000, bar = 10 μm). (b) High magnification of (a) shows decussation of prisms and interprisms. (×10,000, bar = 2 μm).

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Figure 9. High magnification SEM micrograph showing arrangement of hydroxylapatite (HAp) crystals in the prism and interprisms. The prism is composed of several parallel fibers of HAp crystals in longwise arrangements (dotted circle with a hollow arrow). HAp crystals are densely parked between prisms as interprisms. (×50,000, bar = 200 nm).

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Hunter–Schreger Bands

The SEM findings in this study demonstrated that HSBs of bovine enamel were characterized as alternating parazone and diazone in the inner half to two-thirds of the enamel thickness, consisting of two decussating groups of enamel prisms (Figs. 3–5). In the longitudinal sections of bovine enamel, prisms went outward from EDJ at an angle of about 45–55 degrees, and interprisms appeared as plate-like structures (Fig. 3a). Interprisms inclined cervically from EDJ but they were almost perpendicular to prisms (Fig. 3a). Two decussating groups of prisms alternated at intervals in the inner enamel (Fig. 3b). Prisms within a group were in a general direction, but not highly unified (Fig. 3b). From the inner enamel outward, the angle between two decussating prism groups gradually decreased to zero in the outer enamel where prisms were parallel to each other (Fig. 6b). Abundant interprisms alternated with rows of enamel prisms (Fig. 3c). From the inner enamel outward, the intersecting angles between prisms and interprisms at right angles were gradually reduced to about 30 degree in the outer enamel (Figs. 3b and 6b).

In horizontal sections of bovine enamel, most prisms were transversally cut (Fig. 4) due to their incisal inclination. Prisms and interprisms were not distinct about 20–30 μm distant from the EDJ and parallel rows of interprisms were nearly perpendicular to the EDJ (Fig. 4a). In the inner enamel, two groups of prisms were decussated and interprisms went through them perpendicularly at intervals (Fig. 4b).

In tangential sections of bovine enamel, decussation of adjacent prism groups and alternating arrangements of prisms and interprisms are shown in Fig. 5.

Enamel with Parallel Prisms

In the longitudinal sections of bovine enamel, interprisms were nearly perpendicular to prisms in the inner enamel (Fig. 6a), and at a small angle to prisms in the outer enamel (Fig. 6b). In the horizontal sections, each prism was surrounded by interprisms as shown in Fig. 7. In the tangential sections, parallel prisms were arranged in an undulation pattern from the inner enamel outward (Fig. 8a), and alternated with a thin layers of interprisms (Fig. 8b). The SEM findings in this study revealed that the interprisms of the inner enamel were much thicker than those of the outer enamel (Figs. 3a,b, 4a,b, 6a,b, 7, and 8b).

Crystal Arrangements

An enamel prism was composed of a bundle of HAp fiber-like crystals in a lengthwise arrangement (Fig. 9). The HAp crystals of prisms and interprisms were perpendicular to each other (Fig. 9). The crystals of bovine enamel were about 600 nm in length and 80 nm in diameter (Fig. 9).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. LITERATURE CITED

Bovine Enamel and Human Enamel

The findings in this study revealed two main characteristics of the microstructure of bovine enamel that possessed a complicated spatial-relationship between prisms and interprisms (Figs. 3–5) and a larger size of fiber-like enamel crystals (Fig. 9). The interprisms of bovine enamel were arranged into continuous plate-like structures between rows of enamel prisms (Fig. 3b,c). The orientations of the interprism crystals were quite different from the orientations of prism crystals at the prism/interprism decussating planes, which were perpendicular to the planes of decussating prism groups as shown in Fig. 3b. In contrast, the interprism crystals of human enamel only slightly deviated from the enamel prism crystals, and appeared to “lock” the prisms in place (Popowics et al., 2004). Therefore, there were no prism/interprism decussating planes in human enamel (Popowics et al., 2004).

Bovine enamel and human enamel possess HSBs consisting of decussating prism groups, however, the arrangements of prisms through the HSBs were in a different pattern (Popowics et al., 2004) (Fig. 6). Popowics et al. (2004) found that the groups of the cross-sectioned prisms of HSBs in the human enamel were highly unified in orientation within a band, and the prisms of diazones were nearly perpendicular to the prisms of parazones. However, bovine enamel prisms in a band were not unified in orientation (Fig. 3c), and prism orientations of two adjacent bands slightly deviated from each other (Fig. 3b).

The different arrangements of prisms and interprisms between bovine enamel and human enamel might result in some different outcomes in certain studies using bovine enamel (Cadwell and Johannessen, 1971; Barkmeier and Erickson, 1994; Oesterle et al., 1998; Saleh and Taymour, 2003; Abuabara et al., 2004; Bechtle et al., 2010). Previous research indicated that bovine enamel possessed a lower fracture-resistant value than human enamel (Bechtle et al., 2010). This might help to explain the easy propagation of cracks within the bovine enamel interprisms. In bovine enamel, the crack only followed a single interprism/prism decussating plane, whereas in the human enamel, the crack should follow curvatures of the prism/interprism boundaries (Rasmussen et al., 1976; Xu et al., 1998). Furthermore, a greater difference in the orientations of decussating enamel prisms of human enamel means more loss of energy in redirecting the crack path, thus impeding the progression of the crack (Bajaj and Arola, 2009). Therefore, the enamel of bovine incisors would not be a suitable substitute for human enamel used to study enamel crack propagation.

Moreover, prisms parallel to the adhesive interfaces may result in lower bond strengths than prisms perpendicular to the adhesive interfaces (Ikeda et al., 2002; Gamborgi et al., 2007). Therefore, a large number of plate-like interprisms of the bovine enamel parallel to the adhesive interfaces might be one of the reasons why bovine enamel possesses lower bond strengths than the human enamel (Cadwell and Johannessen, 1971; Barkmeier and Erickson, 1994; Oesterle et al., 1998; Saleh and Taymour, 2003).

The SEM finding in this study indicated that bovine enamel crystals have a thickness and length of approximately 80 and 600 nm, respectively (Fig. 9). They are larger, in both thickness and length, than reported for human enamel crystals (Xie et al., 2008; Hannig and Hannig, 2010). The larger crystals of the bovine enamel might be the result of a more rapid development during tooth formation (Fridell et al., 1988). Bovine enamel is more porous than human enamel which may be attributable to the larger crystals found in bovine enamel (Arends and Jongebloed, 1978). This could help to explain why demineralization of bovine enamel progresses about three times faster than human enamel (Flim and Arends, 1977; Featherstone and Mellberg, 1981). In addition, the calcium and phosphorus contents of human enamel are slightly higher than those of bovine enamel. However, bovine enamel has a significantly higher microhardness value than reported for human enamel does (Davidson et al., 1973; Souza-Gabriel et al., 2010). Attin et al. (2007) found that losses of enamel in human teeth were significant less than that of bovine teeth after exposure to erosion and erosion-abrasion challenges. Bovine enamel is still used as a substitute for evaluating demineralization and remineralization of enamel because bovine enamel and human enamel react similarly to acidic challenges and remineralization conditions (Mellberg, 1992). In addition, some previous research demonstrated that bovine enamel produced more extensive microleakage (Abuabara et al., 2004). Abuabara et al. (2004) found that the microleakage pattern could be affected by the bovine enamel substrate, allowing a higher marginal leakage than a human enamel substrate. This might be attributable to the greater porosity and larger crystal size of bovine enamel compared to human enamel (Fig. 9; Arends and Jongebloed, 1978; Abuabara et al., 2004; Xie et al., 2008; Hannig and Hannig, 2010). Thus, bovine enamel may not be suitable for microleakage studies when used as a substitute for human enamel.

Microstructure and Mechanical Properties

Enamel with parallel prisms in the incisal ridge could provide an efficient edge for incising hard or tough foods like plants and roots (Macho and Berner, 1993). Parallel prisms in the outer enamel of the incisal ridge ensure suitable abrasion to keep enamel edges sharp, because parallel prisms are less resistant to abrasion when compared with the HSBs (Bechtle et al., 2010). Prisms become more resistant when they intersect the occlusal surface perpendicularly (Bechtle et al., 2010). In the inner enamel of the incisal ridge, the parallel prisms that have an undulating structure from the EDJ to the outer enamel (Fig. 8a) could resist the occlusal loads effectively (Macho and Berner, 1993). In addition, parallel prisms in the outer enamel could ensure a suitable structure of the bovine teeth (Bechtle et al., 2010).

The HSBs of the bovine enamel provide structural support and biomechanical advantages in the teeth (Shimizu and Macho, 2008; Bajaj and Arola, 2009; Chai et al., 2009). The directions of the bovine enamel prisms changed from the EDJ to the OES as the prisms passed through the HSBs (Figs. 3 and 4). Thus, the prisms could intersect the enamel surface at a suitable angle to offer the greatest resistance to abrasion (Xie et al., 2009).

Mammalian HSBs are reported to be species specific (Osborn, 1965; Hanaizumi et al., 1998; Lyngstadaas et al., 1998; Lucas et al., 2009). A band of HSBs in the bovine enamel was composed of rows of the prism groups, each having 8–10 prisms and plate-like interprisms at intervals (Fig. 3c). The HSBs of bovine enamel are less developed than those of human enamel because of the existence of interprism/prism decussating planes (Popowics et al., 2004). Because the planes result in a weak combination of prisms, bovine enamel becomes less fracture-resistant (Rensberger and Pfretzschner, 1992; Bechtle et al., 2010). Furthermore, the gradual change of prism orientations in the adjacent bands makes bovine enamel more prone to breakage than human enamel. This is likely because less energy during propagation of a crack would be dissipated throughout the continuous interprism planes between prisms in bovine enamel.

The prism/interprism decussating planes also exist in the pig and the warthog (Koenigswald Wv, 1991; Pfretzschner, 1992). The combined decussation of enamel prisms and interprisms of swine teeth fortify the enamel against crack propagation in multiple directions (Popowics et al., 2004). Therefore, it could be inferred that interprism/prism decussating planes of the bovine enamel might enhance the fracture resistance of the bovine enamel in multiple directions.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. LITERATURE CITED

This study demonstrated that the parallel prisms of enamel from bovine incisors were located in all the outer enamel, the cervical region and the incisal ridge. Most labial inner enamel and the cingulum of lingual inner enamel were composed of the HSBs with their characteristic decussating groups of prisms and decussating planes between interprisms and prisms. The interprisms were thicker in the inner enamel than in the outer enamel. The enamels of bovine mandibular incisors are still considered as an excellent substitute for human enamel in numerous dental experiments although bovine enamels may not be suitable for enamel microleakage and crack propagation studies when used as a substitute for human enamel.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. LITERATURE CITED
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