Characterization of Enamel Incremental Markings and Crown Growth Parameters in Minipig Molars

Authors


ABSTRACT

We studied the structure and periodicity of regular incremental markings in third molar enamel of minipigs. Light microscopy of ground sections revealed the presence of incremental markings matching the description of laminations. Their number within the section planes closely paralleled crown formation time (CFT) in days reported for minipig third molars, thereby indicating the daily nature of laminations. Spacing of consecutive laminations increased from lowest values in the inner to highest values in the outer enamel, where mean daily secretion rates of about 20 µm were recorded. Mean enamel extension rates determined for deciles along the enamel-dentin junction varied between highest values (155 µm/day) in the most cuspally located and lowest values (19 µm/day) in cervical enamel. Backscattered electron imaging in the SEM revealed the presence of thin, regularly spaced hypermineralized incremental lines in the outer enamel portion. These lines exhibited the same spacing as the laminations and were, thus, likewise regarded as daily incremental markings. Between two successive daily incremental markings, subdaily growth marks were discernible in light microscopic and in BSE-SEM images. These subdaily growth marks closely resembled the (daily) prism-cross striations of human enamel. Supra-daily growth marks were not identified in the minipig enamel. The results of this study parallels previous findings in sheep enamel. It is cautioned that CFT of ungulate teeth may be considerably overestimated if the periodicity established for growth marks in human enamel is uncritically transferred to the analysis of morphologically similar growth marks in ungulate enamel. Anat Rec, 297:1935–1949, 2014. © 2014 Wiley Periodicals, Inc.

Mature dental enamel is the most heavily mineralized and therefore the most durable tissue of the mammalian body. Enamel is formed in an incremental way, and because it does not remodel or repair, microstructural growth marks in enamel represent a faithful record of the conditions of growth (both normal and pathological) experienced by an individual during the period of tooth crown formation (Boyde, 1989; Dean, 2000; Hillson, 2005; Schwartz et al., 2006; FitzGerald and Rose, 2008; Smith, 2008; Antoine et al., 2009). Such information is of major relevance in studies that use teeth as archives for life-history reconstruction in evolutionary, demographic, (paleo-) pathological, (paleo-) ecological, or forensic contexts (Goodman and Rose, 1990; Shellis, 1998; Dean, 2000, 2006; Dirks et al., 2002; Schwartz et al., 2006, 2002; Guatelli-Steinberg et al., 2005; Witzel et al., 2006, 2008; Smith, 2008; Bromage et al., 2009, 2012).

Differentiation of ameloblasts (enamel forming cells) starts over the tip(s) of the dentin horn(s) and progresses in cervical direction along the (future) enamel-dentin junction (EDJ). The sequence of amelogenesis comprises the secretory stage and the maturation stage (Boyde, 1989; Nanci, 2013). Newly secreted enamel matrix is soft and gel-like. It contains enamel specific proteins (mostly amelogenins) that regulate the growth of the thin enamel crystallites (consisting of a bioapatite approximating hydroxyapatite) that almost instantaneously form in the secreted enamel matrix (Nanci, 2013). During the secretory stage, the enamel obtains a mineral content of about 30% by weight. In the subsequent maturation stage, progressive removal of organic matrix and enamel fluid occurs together with an expansion of the apatite crystallites in width and thickness. At the end of this process, the enamel has achieved its final mineral content of about 96 % by weight (Smith, 1998; Nanci, 2013).

At the onset of amelogenesis, the ameloblast possesses only a short cellular extension (Tomes' process) at its dentin-facing pole. Release of enamel matrix at this flat secretory surface leads to the formation of an initial enamel layer onto the mantle dentin. All crystallites within this initial layer show the same orientation, and the enamel is aprismatic. Intensification of matrix production and secretion is then associated with the development of the distal portion of the Tomes' process (Boyde, 1989; Nanci, 2013). When this process is fully established, the ameloblast possesses two distinct sites of matrix release. Secretion at the first site, which encircles the base (proximal portion) of the Tomes' process, forms the interprismatic enamel that delimits a pit into which the distal portion of the Tomes' process protrudes. Secretion along one face of the distal portion of the Tomes' process produces the matrix of the enamel prism, a rod-like structure that fills in the previously formed pits bordered by interprismatic enamel. Due to the topography of the secretory sites, deposition of interprismatic enamel occurs slightly peripheral to that of contemporaneously formed enamel prisms. Near the end of enamel matrix secretion, the distal portion of the Tomes' process frequently regresses, so that again only a single flat secretory surface is present in the ameloblast. In consequence, in places a thin outer aprismatic enamel layer is formed whose crystallites are oriented in the same way as those of the interprismatic enamel (Boyde, 1989; Nanci, 2013). Depending on the shape of the Tomes' processes of fully active secretory ameloblasts and the resulting topography of the developing enamel surface, three principal prism patterns have been described in mammalian enamel (Boyde, 1965, 1967, 1989).

Ameloblasts show rhythmic fluctuations in the rate of enamel matrix secretion that are reflected by the presence of regular incremental markings in forming and mature enamel (Boyde, 1989; Dean, 2000; Hillson, 2005; FitzGerald and Rose, 2008; Simmer et al., 2010). In primate enamel, the microstructural growth marks can be allocated to two principal categories: short-period markings and long-period markings. Short-period markings have been associated with regular daily (circadian) fluctuations in the rate of enamel matrix formation (Boyde, 1989; Dean, 2000; Hillson, 2005; FitzGerald and Rose, 2008). In ground sections viewed in transmitted light, these markings appear as alternating bright and dark bands oriented perpendicular to the course of the enamel prisms. Varicosities and constrictions along the prism course that are visible in the scanning electron microscope (SEM) have been paralleled with these cross striations (Boyde, 1989; Shellis, 1998; Dean, 2000; Hillson, 2005; FitzGerald and Rose, 2008; Nanci, 2013). However, some authors (Risnes, 1999; Li and Risnes, 2004) have questioned a strict correspondence between the prism cross-striations seen in the light microscope and the prism varicosities and constrictions seen in the SEM. One bright/dark unit of prism cross-striation and one varicosity/constriction unit along the prism course are considered to represent the enamel formed over a 24 hr growth period (Boyde, 1989; Shellis, 1998; Dean, 2000; Hillson, 2005; FitzGerald and Rose, 2008; Nanci, 2013).

In backscattered electron (BSE) SEM images, the constrictions and varicosities of the enamel prisms have been shown to be associated with regular variations in mineral density, with the varicosities showing a relatively higher mineral content (Boyde and Jones, 1983). The periodic variation in mineral content is thought to reflect regular physiologic changes in the acid-base-balance of the organism, leading to a concomitant variation in the carbonate content and the crystallinity of the apatite crystallites formed (Okada, 1943; Boyde, 1989; Shellis, 1998).

The daily nature of prism cross-striations in primate enamel has been demonstrated using labeling of forming enamel and by histological analysis of enamel from individuals with a known age at death whose tooth crowns were still forming when they died. In the labeled enamel, the number of dark prism cross-striations present between two consecutive labels corresponded to the number in days between successive label administrations, while the number of dark cross-striations in the postnatally formed enamel of individuals with known age at death closely matched their age in days (Schour and Poncher, 1937; Okada and Mimura, 1940; Bromage, 1991; Smith, 2006; Antoine et al., 2009).

Recently, the expression of circadian clock genes has been reported in mouse molars and it has been suggested that these genes are involved in the regulation of ameloblast and odontoblast activities, such as enamel and dentin matrix secretion and mineralization (Zheng et al., 2011). In line with this assumption, it was demonstrated that the expression of several clock genes (Bmal1, Clock; Per 1, and Per 2) and clock dependent genes (e.g., Amelx) shows a circadian oscillation in ameloblasts or ameloblast-like cell lines (Lacruz et al., 2012; Zheng et al., 2013).

The regular long-period markings of primate enamel are known as striae of Retzius or Retzius lines. They correspond to successive positions of the enamel forming front. In lateral (imbricational) enamel, the striae of Retzius terminate at the outer enamel surface (OES) in shallow furrows (= perikyma grooves) that run horizontally around the enamel crown. In cuspal (appositional) enamel, the striae of Retzius are dome-shaped over the dentin horn and do not reach the OES (Risnes, 1984, 1998; Boyde 1989; Shellis, 1998; Simmer et al., 2010; Nanci, 2013). The time interval between the formation of consecutive striae of Retzius is called repeat interval and can be determined by counting the number of prism cross-striations between them. In nonhuman primates, the repeat interval ranges between 1 and 11 days, with an overall positive correlation between the length of the repeat interval and the body mass of the species (Smith, 2008; Bromage et al., 2012). In human enamel, the repeat interval typically ranges between 6 and 12 days (Reid and Dean, 2006, Reid and Ferrel, 2006). It has been hypothesized that stria of Retzius formation is caused by the interaction of two not fully synchronized rhythms that overlap at regular time intervals (Newman and Poole, 1974). Risnes (1998) supposed that striae of Retzius formation may reflect a periodic accentuation of the factors responsible for the development of prism cross-striations.

Another type of incremental markings recorded in mammalian enamel are so called laminations or laminated striations (Okada and Mimura, 1940, Ripa et al., 1966; Kodaka et al., 1991, 1989, 1995; Risnes, 1998). Laminations seem to be the most prominent growth marks in ungulate enamel. In the light microscope, they appear as incremental lines that follow a course corresponding to that of striae of Retzius in primate enamel (Okada and Mimura, 1940; Iinuma et al., 2004; Tafforeau et al., 2007; Jordana and Köhler, 2011; Kierdorf et al., 2013, 2012).

Experiments in deer (Iinuma et al., 2004) and sheep (Kierdorf et al., 2013) using vital labeling of forming enamel have proven that laminations constitute daily incremental markings. While there is consensus regarding their periodicity, the nature of laminations is debated. Smith (2006) argues that they may result from the circadian rhythm manifesting across the entire secretory front, that is, involve both the prismatic and interprismatic enamel growth regions. In contrast, Tafforeau et al. (2007) consider laminations to constitute three-dimensional alignments, and thus structural equivalents, of prism cross-striations. Kierdorf et al. (2013) demonstrated that in the inner portion of sheep enamel, laminations exhibit the same orientation as the sheets of interprismatic enamel, suggesting that the formation of laminations in this enamel zone is related to the proximal portion of the Tomes' process.

The only previous study analyzing incremental markings in porcine enamel reported the presence of laminations (called “Parallelstreifen”) with a one-day periodicity in animals injected intravenously with sodium fluoride (Okada and Mimura, 1940). Like in deer and sheep enamel (Iinuma et al., 2004; Kierdorf et al., 2013, 2012), long-period (supra-daily) incremental markings were not discovered in the pig enamel studied by Okada and Mimura (1940). In contrast, these authors found that in the enamel of Macaca cyclopis subjected to the same fluoride injection schedule as the pigs, in addition to the “Parallellstreifen” with a one-day periodicity also incremental lines with a five-day periodicity were visible. The latter were regarded as equivalents of striae of Retzius in human enamel. These findings suggest that the enamel of ungulates may differ from primate enamel with respect to the presence of long-period (supra-daily) incremental markings.

The increasing use of minipigs as an animal model in dental and orofacial research (Wang et al., 2007) requires a better understanding of basic developmental processes of dental hard tissues in pigs than is presently available. The same applies to the use of enamel hypoplasia in pig molars for the reconstruction of stress episodes experienced by animals from recent and archaeological pig populations (Dobney and Ervynck, 2000; Dobney et al., 2004). In addition, our general understanding of the significance of enamel incremental markings in different mammalian taxa is limited since research in this area has so far mainly focused on primate species. The aims of this article were therefore: (1) to analyze the structural characteristics and the periodicity of incremental markings in the enamel of pig molars, (2) to derive basic crown growth parameters of pig molars based on the characterization of the enamel incremental markings, and (3) to contribute to the knowledge of possible differences between ungulate and primate enamel regarding the structure and periodicity of enamel growth marks.

Materials and Methods

The study was performed on mandibular third molars of nine Hanford miniature pigs (Charles River Breeding, Wilmington, MA) that had been selected from a larger group of animals used in an experiment studying the effects of sodium fluoride and alendronate on biomechanical and histomorphometric properties of pig bones (Lafage et al., 1995). The original experiment had been approved by the Institutional Animal Care and Use Committee of the Merck Research Laboratories, West Point, PA (Lafage et al., 1995). A previous study on mandibular third molars from these animals demonstrated that the enamel of the fluoride group exhibited hypoplasia, marked hypomineralization, especially of the outer portion, and an accentuation of the incremental markings (Kierdorf et al., 2004).

For this study, molars of four animals from the control group that had received neither NaF nor alendronate and of five animals from the fluoride group were analyzed. Both groups were fed a low-fluoride standard foodstuff. The pigs of the fluoride group received a daily oral dose of 2 mg of sodium fluoride (∼0.91 mg fluoride) per kilogram of body weight added to their foodstuff for 12 months starting on November 6th of the year of birth. Mean age in days of the selected animals at the start of the experiment was 266 (range: 255–280) in the control and 270 (range: 255–280) in the fluoride group. Five to fifteen days after the end of the experiment, the animals were killed by injection of sodium pentobarbital and their mandibles were collected and stored at −18°C until further processing.

Mandibular third molars (M3s) were chosen for study on the assumption that most of the secretory stage and the complete maturation stage of their enamel formation were covered by the study period. Only few reliable data on crown formation time (CFT) are available for the M3 of different pig breeds. In larger breeds, like the Danish landrace, crown formation of the M3 covers a time span of ∼12 months, starting prior to 7 months of age and being completed at about 19 months (Robinson et al., 1987). In minipigs, data on crown formation of the M3 are available for Göttingen minipigs (Davies, 1990) and Clawn strain miniature pigs (Ide et al., 2013). These data indicate that crown formation of mandibular third molars in minipigs starts at an age of ∼8–9 months and is completed at around 17 months of age. In the minipigs from this study, M3 crown formation was completed in all animals at the end of the experiment and considerable root formation had already taken place. The teeth showed either no or only slight cuspal wear.

For microscopic analysis, the right M3s were extracted, cleaned from adhering soft tissues and dried. The extracted teeth were embedded in epoxy resin (Biodur E12, Biodur products, Heidelberg, Germany) and subsequently sectioned axiobuccolingually through the highest points of the central tooth cusps (Fig. 1a). In the case that larger hypoplastic defects were present in the designated section plane (teeth from the fluoride group), the section plane was slightly shifted in mesial or distal direction to avoid that it included the defect area. In one tooth, a second section was cut tangentially to the OES to determine the cross-sectional prism outline in surface enamel.

Figure 1.

Overview of tooth section and schematic illustration of areas for measurement of crown growth parameters in lingual enamel. (a) Buccolingually sectioned M3 from the fluoride group viewed in plain transmitted light. Asterisk marks the EDJ, arrow marks the apical enamel border on the lingual tooth side. D: dentin; E: enamel. (b) Schematic illustration of lingual enamel with indication of the three regions where measurements were performed along the crown flank. Linear enamel thickness was determined perpendicular to the EDJ in the three regions. Outcrop of the first imbricational incremental line at the OES is indicated by an arrowhead; arrow points to the apical enamel border.

For imaging in the SEM, the cut surfaces of the blocks were first smoothed with silicon carbide sandpaper (grits: 600–2,400). This was followed by polishing on a motorized rotor polisher (Labopol-5, Struers, Copenhagen, Denmark) with a diamond suspension of 3 µm particle diameter (DiaPro Dac 3, Struers) and a final polishing step using a colloidal silica suspension (OP-S, Struers). BSE-SEM imaging of the uncoated polished surfaces was performed with an FEI Quanta 600 FEG ESEM that uses high resolution Schottky field emission and was operated in a low vacuum mode at an accelerating voltage of 20 kV. To enhance the visibility of the enamel structure, some of the polished block surfaces were etched with 34% (v/v) phosphoric acid for 3 sec after obtaining the initial BSE-SEM images. The etched surfaces were thoroughly rinsed with water, air dried, and again examined in the SEM. For secondary electron (SE) imaging, selected specimen were sputter-coated with gold and viewed in a Hitachi S 520 SEM operated at 10 kV.

For light microscopy, tooth blocks were mounted with their polished sides down on glass slides using the epoxy resin as glue. Slices of about 0.5 to 1 mm thickness were sectioned from the blocks and then ground and polished to a final thickness of about 50 µm as described previously (Kierdorf et al., 2013). Sections were viewed and photographed in transmitted light using an Axioskop 2 Plus microscope (Zeiss, Jena, Germany) equipped with a digital camera. For determination of crown growth parameters the acquired images were stitched together using Adobe Photoshop software (Adobe, San Jose) and subsequently analyzed using the tools of the Digimizer program (MedCalc Software, Ostend, Belgium).

Once the periodicity of the regular enamel incremental markings had been determined, several enamel growth parameters were recorded. First, the number of daily growth marks visible in the section plane of each tooth was recorded in lingual and buccal enamel. Second, daily enamel apposition or secretion rate (DSR) was determined in the lingual and buccal crown flanks at three locations (upper, middle, and lower crown region; Fig. 1b). DSRs were recorded as the distances (in µm) between the inner borders of consecutive daily growths marks along a reconstructed prism course at either the prism or the interprism growth front, depending on the visibility of the marks.

In each of the three regions of the tooth flank, the enamel layer was divided into three zones (inner, central, and outer third), and the values reported are arithmetic means of apposition rates recorded in the respective zones. Third, the duration of appositional growth, that is, the number of days during which a single ameloblast had secreted matrix, was reconstructed by counting the number of daily growth marks along the path of a prism on its way from the EDJ to the OES. Fourth, the enamel extension rate (EER), that is, the rate at which new secretory ameloblasts had differentiated from precursor cells along the presumptive EDJ from the tip of the dentin horn towards the future apical enamel border, was determined in lingual enamel. For this, in each section, the length of the EDJ was divided into deciles and the distances between the starting points of successive daily growth marks at the EDJ were recorded. EERs are presented as arithmetic means of the distances between consecutive daily growth marks that started at the EDJ in the respective decile. If the stretch of enamel between two consecutive daily growth marks spread over the border between two adjacent deciles, the respective value was assigned to the cervically located decile. The number of daily growth marks starting at the EDJ was also determined for each decile.

Results

In ground sections viewed in transmitted light with phase contrast, a prominent, regular incremental pattern was visible in the enamel of animals from the control group and the fluoride group (Fig. 2a,b). When viewed at low magnification, the incremental pattern presented as regularly alternating bright and dark lines/bands that formed a steep angle with the EDJ and gave the enamel a laminated appearance (Fig. 2a,b). Due to their steep inclination, in the majority of the studied sections >50% of the incremental lines (laminations) were located in cuspal (appositional) enamel, that is, did not crop out at the OES (Fig. 1b). Outcrop of the incremental lines at the OES was not regularly associated with the presence of a distinct groove on the enamel surface (Figs. 2a,b, 4a,b, 5b).

Figure 2.

Ground sections of lingual enamel of M3s from the fluoride (a) and the control group (b) viewed in transmitted light with phase contrast. Overview of lingual enamel in the lower crown region showing a prominent regular incremental pattern. Incremental markings appear as alternating bright and dark lines. Outcrop of the incremental lines at the OES is not regularly associated with the presence of a groove. White lines indicate the reconstructed paths of two prisms. Asterisks mark EDJ. D: dentin; E: enamel. Occlusal to top. Bars: 250 µm.

The reconstructed course of individual enamel prisms was less steep than the course followed by the incremental lines (Fig. 2a,b). In the inner and central enamel zones, the prism course formed an angle of ∼45° with the EDJ, while in the outer enamel zone the prisms gradually changed to a more or less horizontal course and terminated perpendicular to the OES (Figs. 2a,b, 4a–c, 5b). Accordingly, in the outer enamel the incremental lines were oriented perpendicular to the prism course (Figs. 2a,b, 4a–c, 5b).

At higher magnifications, it was apparent that in both, the control and the fluorotic specimens, the incremental pattern comprised regularly spaced incremental lines that delimited broader portions of enamel (Figs. 3-5). In ground sections of control teeth viewed in plain transmitted light, these incremental lines appeared brownish while in transmitted light with phase contrast they appeared bright (Figs. 3, 4a,b). At higher magnification, it was further discernible that the enamel prisms exhibited a number of alternating dark and bright cross striations between successive incremental lines. Mostly five pairs of dark and bright prism cross striations that were oriented perpendicular to the prism long axis could be distinguished between successive incremental lines (Figs. 3a,b, 5b,c).

Figure 3.

Ground section of central lingual enamel of an M3 from the control group viewed in plain transmitted light (a) and in transmitted light with phase contrast (b). Evenly spaced growth marks (laminations, black arrowheads) separate broader enamel portions. Laminations are caused by the staggered alignment of accentuated prism cross striations. In places, five pairs of alternating bright and dark prism-cross-striations (small arrows) are discernible between consecutive laminations. Large arrows indicate overall prism direction. Occlusal to top, OES to the right. Bars: 50 µm.

Figure 4.

Ground section of outer lingual enamel of an M3 from the control group viewed in plain transmitted light (a) and in transmitted light with phase contrast (b), and SE-SEM image (c) of etched surface enamel from a control M3. Prism course (indicated by arrows) is horizontal in outer enamel. In the transitional zone between central and outer enamel, which is visible in Fig. 4a,b, both types of regular growth marks (laminations) that are either associated with the enamel prisms (black arrowheads) or with the interprismatic enamel (white arrowheads) are discernible. In the outer enamel, the laminations are associated with the interprismatic enamel. Occlusal to top. Bars: 100 µm in a and b and 15 µm in c.

Figure 5.

Higher magnifications of ground section through the inner (a), outer (b), and central (c) lingual enamel of an M3 from the fluoride group viewed in transmitted light with phase contrast. Occlusal to top, OES to the right in all figures. (a) Regular growth marks (laminations, white arrowheads) reflecting consecutive positions of the growth front of the interprismatic enamel form an angle of about 40° with the prism course (indicated by white arrow). Asterisk marks EDJ. Bar: 50 µm. (b) Horizontally oriented prisms (white arrow) in outer enamel terminate perpendicular to the OES. Laminations that denote former positions of the prism growth front (black arrowheads) and the growth front of the interprismatic enamel (white arrowheads) are discernible. Note presence of dark and bright cross striations between the two outer marked laminations. Bar: 50 µm. (c) Incremental pattern in central enamel. Black arrowheads mark laminations that denote former positions of the prism growth front; white arrowheads mark laminations indicating former positions of the growth front of interprismatic enamel. Small arrows indicate subdaily growth marks; large arrows indicate prism direction. The topography of the former growth fronts of prisms and interprismatic enamel is visible in the encircled area. Bar: 50 µm.

In central enamel of teeth from the control group, the optical impression of the incremental lines was caused by the staggered alignment of accentuated prism cross striations along successive positions of the prism growth front (Fig. 3a,b). In this enamel zone, the incremental lines were relatively broad and formed an acute angle of about 50° with the enamel prisms (Fig. 3a,b). In the transitional area between the central and the outer enamel portion, a change of the incremental pattern occurred (Fig. 4a,b). Due to the gradual shift of the prism course to a horizontal orientation, the staggered alignment of prism cross-striations along the former prism forming front was more and more lost and instead continuous, that is, uninterrupted, thin incremental lines that were oriented perpendicular to the prism course became increasingly visible (Fig. 4a,b). SEM images showed that these thin incremental lines were associated with the interprismatic enamel (Fig. 4c).

Compared with the control group, the visibility of the incremental markings was markedly enhanced in the enamel from the fluoride group (Fig. 5a–c). When viewed in transmitted light with phase contrast, the fluorosed enamel exhibited relatively broad, regularly spaced dark (bluish) bands that in the central enamel formed an acute angle of about 50° with the course of the enamel prisms (Fig. 5c). In places, the inclination of the former prism growth front was discernible within the prism course at the peripheral border of the dark incremental bands (Fig. 5c). This morphology is regarded to reflect the inclination of the secretory surface at the distal, prism-forming portion of the Tomes' process. Like in the enamel of the control teeth, the relatively broad incremental bands are therefore regarded to correspond to successive positions of the prism growth front.

Thin, uninterrupted incremental lines that appeared bright in transmitted light with phase contrast were also present in the fluorosed enamel. These lines were most prominent in the inner and outer enamel portions, but were in places also visible in the central enamel (Fig. 5a–c). In the latter, these thin lines delimited prism portions of roughly triangular outline that bordered on the internally adjacent broad incremental band (Fig. 5c). These triangular prism portions are regarded to reflect the shape of the pits of the enamel forming front that had previously been occupied by the projecting, distal portions of the Tomes' processes. At the occlusal margin of a single prisms, the thin incremental line was typically positioned a few microns peripheral to the dark incremental band that marked the position of the prism growth front. In contrast, at the cervical margin of the prisms, the two growth marks were positioned adjacent to each other.

In inner enamel, the continuous thin bright incremental lines were the dominating growth marks (Fig. 5a). Here, they formed an acute angle of about 40° with the prism course. In outer enamel, the dark bands and the thin bright incremental lines were oriented parallel to each other and at right angles to the prism course (Fig. 5b).

SEM images revealed that in the inner and central enamel zones the prisms followed an undulating course, causing the presence of distinct Hunter-Schreger bands in these areas (Fig. 6). Prisms were separated by prominent interrow sheets of interprismatic enamel, and the enamel exhibited a typical pattern 2 structure according to Boyde's (1989) classification. In contrast, in the outer enamel the prisms were arranged in parallel and followed a nearly straight course towards the OES (Figs. 4c, 7a,c,d). Transversely cut prisms in this area exhibited a round profile and were delimited by a complete, cylindrical prism boundary and enveloped by a layer of interprismatic enamel (Fig. 7b), which is typical for a pattern 1 prism arrangement (Boyde, 1989).

Figure 6.

SE- SEM image of etched inner enamel of a minipig M3 from the control group showing longitudinally sectioned prisms (parazone, PZ) and more or less transversely cut prisms (diazone, DZ). Arrow indicates prism direction. IP: Sheets of interprismatic enamel. Occlusal to top, OES to the right. Bar: 30 µm.

Figure 7.

BSE-SEM images of buccolingually (a,c,d) and tangentially sectioned (b) lingual enamel of M3s from the fluoride group. (a) Boundary between central (left) and outer (right) enamel zone. The central zone shows a typical Hunter-Schreger pattern with alternating bands of more or less longitudinally (parazones, PZ) or transversely cut (diazones, DZ) prisms. In the outer enamel zone, prisms are arranged in parallel and follow a nearly horizontal course to the OES. Thin incremental lines (white arrowheads) become increasingly more discernible in the outer enamel zone. Occlusal to top. Bar: 100 µm. (b) Transversely cut enamel prisms in outer enamel. Prisms exhibit a round cross-sectional profile and are delimited by a complete prism sheath. Occlusal to top. Etched section. Bar: 20 µm. (c) Higher magnification of enamel near the OES (toward the right). Fine, regularly spaced bright hypermineralized incremental lines are present in interprismatic enamel (white arrowheads) and in the adjacent enamel prisms (black arrowheads). Note that these bright lines are positioned a few micrometer further peripherally in interprismatic enamel (IP) compared with the adjacent enamel prisms (P). Where prisms are exposed tangentially over a larger distance, subdaily growth marks in form of varicosities and constrictions are visible along the prism long axis between two consecutive hypermineralized lines. Occlusal to top. Bar: 20 µm. (d) Higher magnification of enamel near the OES (toward the right). Note presence of numerous very fine (subdaily) incremental markings in the interprismatic enamel between adjacent hypermineralized lines (white arrowheads). The phenomenon is best visible between lines 2 and 3, and lines 4 and 5. Occlusal to top. Etched section. Bar: 20 µm.

Incremental markings were not readily visible in SEM images of the inner enamel zone. However, fine incremental lines were discernible in the more peripheral areas of the central enamel and especially in the outer enamel (Fig. 7a,c,d). Here, BSE-SEM images showed regularly spaced thin bright lines that were oriented perpendicular to the prism course in both enamel prisms and interprismatic enamel (Fig. 7c,d). The brightness of these lines indicated that they were more highly mineralized than the surrounding enamel. The hypermineralized lines were present in the enamel of the teeth from both, the control and the fluoride group. However, in the latter they were more prominent, as the outer enamel of the fluorotic molars was overall markedly hypomineralized.

The hypermineralized lines in the interprismatic enamel were always located a few micrometers further towards the OES than the corresponding lines in the adjacent enamel prisms (Fig. 7c). This topography reflects the different positions of the matrix releasing sites at the Tomes' processes of fully active secretory ameloblasts with formation of interprismatic enamel occurring at the proximal and formation of the enamel prism at the distal portion. Thus, simultaneously secreted and initially mineralized matrix at these sites is separated by a distance corresponding to the length of the distal portion of the Tomes' process. The fact that the hypermineralized lines in prismatic and interprismatic enamel were oriented parallel to each other also indicates that during formation of the outer enamel the respective matrix releasing sites of the ameloblasts exhibited a parallel orientation, as is typical for pattern 1 enamel.

As could be seen when the section plane was located over larger distances in the interprismatic enamel, the hypermineralized lines formed a continuous structure (Figs. 4c, 7d). In places, the BSE-SEM images further revealed the presence of numerous (c. 10–12) alternating very fine incremental markings in the interprismatic enamel between consecutive hypermineralized lines (Fig. 7d). The distance between consecutive hypermineralized lines in the outer enamel averaged about 20 µm and thus closely matched the distance recorded between successive incremental lines in microscopic sections of this enamel area. Where prisms were exposed tangentially over a larger distance in the section plane, presence of multiple alternating constrictions and varicosities was discernible along their long axis between consecutive hypermineralized lines (Fig. 7c). The varying brightness of these growth marks in BSE-SEM images suggests that the varicosities and constrictions differed in mineral content.

In the ground sections, the recorded numbers (arithmetic mean ± SD) of incremental lines (laminations), which denote successive position of the enamel forming front, within the section planes were 232 (±22) in buccal and 248 (±32) in lingual enamel of teeth from the control group. For the teeth from the fluoride group, the numbers were 239 (±17) in buccal and 240 (±13) in lingual enamel. Assuming a CFT for minipig M3 of 8–9 months (Davies, 1990; Ide et al., 2013), this finding suggests that laminations constitute daily growth marks. It is therefore concluded that the enamel located between the inner borders of two adjacent laminations (formed at either the prism growth front or the interprismatic growth front) reflects growths over a 24 h period. The same is assumed to be the case for the enamel portion located between two consecutive hypermineralized lines seen in the SEM. Consequently, the prism cross-striations observed in the light microscope between consecutive laminations and the varicosities and constrictions recorded between consecutive hypermineralized lines in the SEM are regarded to constitute subdaily growth marks.

Based on the assumed one-day periodicity of the laminations, DSRs were determined in the ground sections (Table 1). In lingual and buccal enamel of both groups, mean values for DSR increased from the inner to the central and further to the outer enamel zone. Lowest mean DSRs, recorded in inner buccal enamel, ranged between 11.1 and 12.8 µm in the fluoride group and between 12.7 and 14.9 µm in the control group. In the corresponding enamel region of the lingual side, values ranged between 12.4 and 14.2 µm (fluoride group) and between 13.0 and 14.5 µm (control group). In the controls, mean values around 20 µm/day occurred in the outer enamel zone where a maximum mean value of 24 µm/day and a single absolute maximum value of 28 µm/day were recorded in upper buccal enamel. In all but one of the 18 analyzed enamel zones, enamel apposition rates were lower in the fluoride group compared with the control group, thereby demonstrating a negative effect of the fluoride exposure on enamel matrix secretion. Compared with the control group, mean daily matrix production was overall reduced by 7.5% in lingual and by 14.7% in buccal enamel of the fluoride group. Comparison of mean DSRs revealed significant differences between the fluoride and the control group for the inner (P = 0.044), central (P = 0.001) and outer thirds (P = 0.014) of the enamel (two-tailed t-tests for independent samples). Differences between groups remained significant (P < 0.05) for the central and outer enamel thirds following Bonferroni adjustment of P-values for three tests.

Table 1. Mean DSRs (μm/day) with standard errors (SE) in the different enamel portions as well as linear enamel thicknesses (μm) determined in the three analyzed enamel regions of M3s from the fluoride and the control group
 GroupInner thirdCentral thirdOuter thirdEnamel thickness
MeanSEnMeanSEnMeanSEnMeanSEn
  1. n = Number of analyzed sections.

Buccal
Upper crown regionFluoride11.10.7314.90.7418.20.441720.338.74
Control14.90.9319.70.4324.02.431732.040.83
Middle crown regionFluoride12.81.0514.50.4519.20.551659.638.75
Control12.71.1316.90.7319.91.131695.632.73
Lower crown regionFluoride11.80.5515.20.2518.70.251540.479.15
Control13.61.1317.91.0321.30.531554.652.73
Lingual
Upper crown regionFluoride14.21.2415.70.5419.30.841741.422.24
Control14.51.9416.60.8419.90.931813.716.04
Middle crown regionFluoride12.40.7514.60.3519.70.451691.365.55
Control13.01.6417.00.3420.90.541714.1117.44
Lower crown regionFluoride12.40.5515.20.3517.80.451472.065.35
Control13.61.1417.60.9419.80.541579.6117.94

In lingual enamel, mean distances between successive laminations at the EDJ dropped from 120 µm in the first (cuspal-most) decile to 19 µm in the 10th decile for the control group and from 155 µm (first decile) to 20 µm (10th decile) in the fluoride group (Fig. 8). Mean number of laminations starting at the EDJ per decile increased from low numbers in the first decile (fluoride group: 7.3; control group: 9.4) to highest values in the 10th decile (fluoride group: 47.8; control group 48.5). In consequence, >50% (53.6% in the fluoride group and 55.7% in the control group) of the laminations started at the EDJ in the cervical 30% of the EDJ extension. Our data thus demonstrate a very high EER in upper cuspal enamel of the pig molars and a constant decrease of this rate in cervical direction. The reconstructed durations of ameloblasts secretory lifespans varied between 104 and 150 days in the control group and between 98 and 157 days in the fluoride group. Values for the duration of ameloblasts secretory activity in the different crown regions are given in Table 2.

Figure 8.

Mean daily enamel extension rates (x) and mean number of laminations (Δ) starting at the EDJ per decile of EDJ length in the lingual enamel of the M3s from the fluoride group (a, n = 5) and the control group (b, n = 4). Vertical bars indicate one standard error.

Table 2. Means and standard errors (SE) of ameloblast secretory activity (duration of appositional growth) in days in the different crown regions of M3s from the fluoride and the control group
 Upper crown regionMiddle crown regionLower crown region
MeanSEnMeanSEnMeanSEn
  1. n = Number of analyzed sections.

Buccal
Fluoride group145.35.24139.02.35128.45.15
Control group121.09.23141.36.33119.33.53
Lingual
Fluoride group137.88.14135.25.65120.37.55
Control group133.38.13134.34.14112.34.34

Discussion

This is the first study providing detailed information on incremental markings and growth parameters of porcine molar enamel. The appearance and course of the prominent regular incremental markings observed by light microscopy in ground sections of minipig enamel match those of laminations previously described in the enamel of other ungulate species (Iinuma et al., 2004; Tafforeau et al., 2007; Jordana and Köhler, 2011; Kierdorf et al., 2013, 2012). Based on the numbers of laminations present in the section planes and the reported CFT of 8 to 9 months for the M3 of minipigs (Davies, 1990; Ide et al., 2013), we conclude that the laminations in minipig enamel constitute daily incremental markings.

Our results corroborate the findings of Okada and Mimura (1940) who described laminations (“Parallelstreifen”) with a periodicity of one day in their study on labeled forming pig enamel. A daily periodicity of laminations was previously also demonstrated in experimentally labeled enamel of deer and sheep (Iinuma et al., 2004; Kierdorf et al., 2013). The fact that longer-period (supra-daily) incremental markings were not discernible in the microscopic sections of minipig enamel likewise parallels previous observations in the enamel of pig, deer, and sheep (Okada and Mimura, 1940, Iinuma et al., 2004; Kierdorf et al., 2013). In contrast, Tafforeau et al. (2007) described presence of Retzius lines with a repeat interval of seven (laminations) in teeth of fossil and extant rhinocerotids.

Considering the reported topography of the secretory front of pattern 1 and pattern 2 enamel (Boyde 1965, 1967, 1989; Kierdorf et al., 1991), the findings in the minipig molars indicate that periodic circadian fluctuations of enamel matrix secretion and initial mineralization had caused the occurrence of incremental markings along the forming fronts of both, enamel prisms and interprismatic enamel. The relatively broad incremental bands formed by the staggered alignment of prism cross striations in our interpretation reflect consecutive position of the prism growth front while the thin, continuous incremental lines that are especially prominent in inner and outer porcine enamel reflect successive position of the interprismatic growth front. It is further concluded that these growth marks are formed simultaneously at the secretory sites located at the distal and at the proximal portion of the Tomes' process of individual ameloblasts. This means that the distance between consecutive growth marks in either the enamel prisms or the interprismatic enamel can be used to determine the daily rate of enamel matrix secretion. For reconstructing the total number of consecutive daily positions of the enamel forming front during crown formation, either type of growth mark may thus be used.

In the inner enamel of the minipig molars, the light microscopic phenomenon of laminations is caused by growth marks present along former positions of the secretory front of the interprismatic enamel. A corresponding situation has previously been demonstrated in the inner enamel of sheep molars (Kierdorf et al., 2013) where it was attributed to the fact that interprismatic enamel accounts for >50% of the total volume fraction in the inner portion of sheep enamel (Grine et al., 1987). Since pig and sheep share a typical pattern 2 structure in their inner and central enamel portions, this explanation presumably also holds for pig enamel. Increasing secretory activity then causes a progressive shift in the volume occupancy between prismatic and interprismatic enamel. In consequence, in the central enamel portion, prismatic enamel occupies a considerably larger fraction (up to 80%) of the total enamel volume (Grine et al., 1987). Thus, in this enamel portion, the phenomenon of laminations seems to be mainly attributable to the three dimensional alignment of prism cross-striations as was previously pointed out by Tafforeau et al. (2007).

The recorded parallel orientation of the regular growth marks in enamel prism and interprismatic enamel in the outer enamel of minipigs can be related to the parallel orientation of the respective growth sites that is typical for pattern 1 enamel (Boyde, 1989). Moreover, in pattern 1 enamel, the prisms are completely enveloped by interprismatic enamel (Fig. 7b). This situation may contribute to the observation that in the outer minipig enamel the laminations were predominantly attributed to the interprismatic enamel. In contrast, during formation of pattern 2 enamel, the prism growth front at the distal portion of the Tomes' process is typically inclined in a cervical direction against the long axis of the ameloblast and thus against the secretory front of the interprismatic enamel (Boyde, 1989, Kierdorf et al., 1991). Due to this inclination, the growth fronts of prismatic and interprismatic enamel are separated in the cuspal and lateral walls of the Tomes' process pits while they meet at the cervical rim of the pits, a topography that could be reconstructed in the central enamel of the minipig molars (Fig. 5c).

We observed a considerable difference in the visibility of incremental markings between SEM images and light microscopic images. Thus, in SEM images of the inner enamel, incremental markings were not discernible, while light microscopic images of ground sections showed prominent incremental markings in this enamel zone. That SEM images of enamel reveal fewer incremental markings than light microscopic images of ground section has been ascribed to the fact that in transmitted light microscopy a summation of structural information from the whole section thickness is recorded while in the SEM only information from a thin superficial enamel layer is available (Li and Risnes, 2004). Our findings indicate that the visibility of the incremental markings is caused predominantly by compositional differences (mineral density) between them and the surrounding enamel.

The prominent hypermineralized lines visible in BSE-SEM images of the outer enamel of teeth from the control and fluoride groups point to a more pronounced difference in mineralization between the incremental lines and the surrounding enamel in the outer enamel compared to more deeper enamel zones. The hypermineralized lines exhibited a spacing of about 20 µm, which corresponds to the distance recorded between consecutive laminations in microscopic ground sections of this enamel zone. It was therefore concluded that the enamel stretch located between consecutive hypermineralized lines likewise reflects a growth increment formed within a 24 hr period. We assume that these hypermineralized lines correspond to the daily growth marks (laminations) that in the light microscopic images were attributed to the growth front of the interprismatic enamel. However, the exact structural relationship between the incremental markings recorded by transmitted light microscopy and by SEM imaging remains to be established.

The recorded enamel apposition rates in the third molars of the minipigs were very high, reaching means of about 20 µm in outer enamel. High DSRs have previously also been determined in the enamel of other ungulate species. In sheep molars, Kierdorf et al. (2013) found mean DSRs between 15.0 and 17.0 µm in buccal and between 11.6 and 13.4 µm in lingual enamel. In the minipig teeth, as in the sheep molars, DSR increased from the inner towards the outer enamel. For the inner enamel of mandibular first molars of sika deer, Iinuma et al. (2004) determined a mean DSR of 11.2 µm. DSRs reported for hominoid enamel are generally lower, ranging between a lower limit of 2–3 µm and an upper limit of 6–7 µm (Smith, 2006), with human molar enamel exhibiting mean DSRs between 2.3 µm in inner cervical and 5.1 µm in outer cuspal enamel (Hillson, 1996).

Mean DSRs in minipig M3s were only moderately higher than those recorded in corresponding crown regions of sheep M1s (Kierdorf et al., 2013). However, enamel thickness differs markedly between the molars of the two species. Whereas in sheep M1s, enamel thickness ranged between c. 450 and 650 µm (depending on the crown region), values for the minipig M3 ranged between c. 1,500 and 1,800 µm. Thus, the enamel layer of the minipig molars was approximately three times thicker than that of the sheep molars.

The two species also differ with respect to the duration of the secretory activity of single ameloblasts. Ameloblast secretory lifespan in the sheep M1 ranges between 35 and 53 days (Kierdorf et al., 2013), whereas the values determined for the minipig M3 ranged between 98 and 157 days. On the basis of these findings we suggest that an increase in the enamel thickness of ungulate teeth is basically achieved by an extension of the secretory lifespan of the ameloblasts, not by an increase in DSR. It may be speculated that a mean DSR in the range of 20–25 µm is at the upper limit of the physiological capability of secretory ameloblasts.

Enamel thickness in human molars is similar to that of the minipig M3 (Grine, 2005; Mahoney, 2008). Mean DSR is, however, much lower in human compared to pig molars. In consequence, the secretory lifespan of ameloblast is considerably longer in human than in pig molars, reaching average values of 473 days in human molar cuspal enamel (Dean, 1998). This is also reflected in much longer CFTs in human compared to pig molars. For the minipig M3, a CFT of 8 to 9 months has been reported (Davies, 1990; Ide et al., 2013), whereas CFTs of human molars range between 33 and 40 months (mean 37.4 months) for the M1 (Mahoney, 2008) and between 48 and 60 months for the M2 (El Nesr and Avery, 1994).

The long CFTs of human teeth are also influenced by their low EERs compared with ungulate teeth. Dean (1998) reported mean EERs between 13.6 µm/day in the most cuspally located and 3.0 µm/day in the most cervical enamel of human second permanent molars. Similarly, Guatelli-Steinberg et al. (2012) reported mean EER values around 8 µm/day, with individual maxima reaching about 15 µm/day in the most cuspally located enamel (first decile) of human permanent teeth. Values then markedly decreased in cervical direction, reaching mean values around 3 µm/day in the most cervical crown portion (10th decile) of human enamel.

By contrast, EER reached maximum values between 120 and 155 µm/day in the cuspal-most enamel (first decile) of the (brachydont) minipig M3 and decreased to lowest values between 19 and 20 µm/day in the most cervical (10th decile) enamel. Even higher EERs have been recorded in hyposodont sheep molars, with mean maximum values between 180 and 217 µm/day and individual maxima of 260 and 275 µm/day occurring in the most cuspally located enamel (Jordana and Köhler, 2011; Kierdorf et al., 2013). In their study on molar plate formation in elephantids, Dirks et al. (2012) reasoned that among elephantid species EER and not DSR is the primary mechanism for creating teeth with different crown heights. Based on these findings in the minipig molars and the previous observations in sheep molars (Jordana and Köhler, 2011; Kierdorf et al. 2013), the statement by Dirks et al. (2012) may be extendable to ungulates in general.

We observed that enamel thickness and DSR remained quite constant over large parts of the crown flank in the studied minipig M3s. In combination with the recorded marked variation in EER this implies that the formation of the cervical third of the crown of these teeth covers at least the same time span as that of the upper two thirds. Together with the recorded distribution of cuspal and lateral enamel along the crown flank, this fact is considered to contribute to the reported higher prevalence of enamel hypoplasia in the cervical compared with the upper and middle crown portions of pig M3s (Dobney and Ervynck, 2000; Dobney et al., 2004; Witzel et al., 2006).

Our study further revealed that in addition to daily incremental markings, also markings with a shorter (subdaily) periodicity are present in minipig enamel. In places, five pairs of alternating bright and dark prism cross-striations were visible between consecutive laminations in the ground sections. Subdaily incremental markings were also discernible in the BSE-SEM images. The subdaily growth marks in minipig enamel are morphologically similar to the prism cross-striations and the alternating prism varicosities and constrictions described as daily growth marks in human enamel (Boyde, 1989; Hillson, 2005; Nanci, 2013). The findings in Fig. 7d may point to the presence of an even shorter periodicity in matrix secretion/initial mineralization of porcine enamel.

Presence of five subdaily growth increments between consecutive (daily) laminations has previously also been described in sheep enamel (Kierdorf et al., 2013). The findings in sheep and pig enamel suggest that in these species, in addition to a daily (24 hr) growth cycle reflected by the laminations, a further oscillation of ameloblast activity with a periodicity of ∼5 hr occurs. The physiologic basis for the formation of subdaily growth increments in the enamel of pigs and sheep is presently unknown. We hypothesize that in ungulate teeth the need for the completion of the enamel layer within a relatively short time period requires a very high synthetic and secretory ameloblast activity that causes both, the occurrence of more frequent pulses of maximum secretory activity and a less pronounced difference between minimum and maximum ameloblast secretory activity than are present in the markedly slower forming human enamel.

The structural characteristics of the enamel growth marks and the rapidity of enamel formation recorded in ungulates may cause an erroneous interpretation of their periodicity when interpreted against the background established for the periodicity of incremental markings in human enamel. Morphologically, the laminations in ungulate enamel closely resemble the striae of Retzius of human enamel. However, the enamel located between consecutive laminations in ungulate enamel represents growth over a 24 hr period, whereas the enamel present between consecutive striae of Retzius in human enamel represents growth over a period of several days. Likewise, the (subdaily) prism cross-striations in ungulate enamel resemble those seen in human enamel, but reflect a considerably shorter period of enamel formation.

An uncritical transfer of the timing of incremental markings present in human enamel to incremental markings with a similar appearance in the enamel of ungulates will result in a misinterpretation of DSRs and CFTs in the latter. For example, in their study of cheek teeth from horses, Hoppe et al. (2004) depicted microscopic sections in which laminations where designated as striae of Retzius and prism cross striations present between consecutive laminations as daily growth marks. In consequence, these authors calculated a DSR of only c. 5 µm for horse enamel, a value that is very low compared to the DSRs in other ungulate species. Mistaking sub-daily for daily enamel growth marks will also result in an overestimation of CFTs. This is probably the cause for previously reported CFTs of 1035 days in first molars of Gazella granti or of 725 days in second molars of a small ruminant species like Kirk's Dik-dik (Madoqua kirkii) (Macho and Williamson, 2002). Interestingly, Macho and Williamson (2002) reported a cross-striation repeat interval of 4 or 5 days between the structures that they, presumably erroneously, identified as striae of Retzius instead of daily laminations. This may indicate the presence of 4–5 subdaily growth increments between consecutive laminations in the species studied by them, which would parallel our observation in sheep and minipig enamel.

In conclusion, our study provided evidence for the presence of regular daily and subdaily incremental markings in minipig enamel. Based on the analysis of these growth marks, very high DSRs could be reconstructed for the minipig molars. Regular supra-daily enamel markings were not recorded in the minipig enamel, which matches previous observations in the enamel of pigs (Okada and Mimura, 1940), deer (Iinuma et al., 2004), and sheep (Kierdorf et al., 2013, 2012). This can either mean that such supra-daily markings are not distinguishable from daily markings with the methods used in these studies or that, in contrast to human enamel, regular supra-daily incremental enamel markings are not present in these species.

ACKNOWLEDGEMENTS

The authors thank D. Klosa for his skillful help with BSE-SEM-imaging. The pig mandibles were kindly donated by Merck Sharp Dohme Research Laboratories, West Point, PA. The authors gratefully acknowledge the comments of two anonymous reviewers that helped to improve the article.

Ancillary