Correlating the Densities of Hunter-schreger Bands With Function and Surfaces of Teeth: A Micrometric Analysis

2.1 Sample calculation

This study was approved by the Institutional Ethics Committee (IEC ref. no. 23094). The extracted human teeth were collected from the Department of Oral and Maxillofacial Surgery; all the samples were anonymized and used following institutional guidelines. Tooth samples that had intact crowns; no evidence of attrition, abrasion, or erosion; and were visually free from hypoplasia and fractures were included in the study. On the basis of the study by Lynch et al. (2010),¹ Table 1 shows the HSB densities on various tooth types. The highest standard deviation was noted in premolars, at 2.2 units. Sample size was determined from published variability in HSB density¹ where the largest observed standard deviation was approximately σ = 2.2. For a two-sample comparison, using a two-sided α = 0.05 and power = 0.80, the required sample size per group to detect a between-group difference of d = 4.0 units is approximately n ≈ 5 per group (calculation based on the standard two-sample formula). Given the study design (incisor/canine/premolar/molar × maxilla/mandible with two section orientations), we allocated 80 teeth in total (10 teeth per tooth class per arch, further subdivided into two sectioning orientations of n = 5 each). Because some subgroup cells are small, subgroup analyses and interaction effects should be interpreted cautiously; we therefore provide effect sizes and 95% confidence intervals alongside p-values.

The formula used is $N=\frac{2{(Z_{1-\frac{\mathrm{\alpha }}{2\ast k}}+Z_{1-\mathrm{\beta }})}^2{\mathrm{\sigma }}^2}{d^2}$ where σ is the standard deviation.

Eighty teeth were selected and segregated into incisors, canines, premolars, and molars. A total of 10 samples were included in each category for both jaws, maxillary and mandible. Furthermore, each category was subdivided into 2 groups of five each. One group was used for mesiodistal sectioning, and the other was used for labiolingual sectioning. The occlusal aspect was also studied in each of the above groups. The distribution of teeth is shown in Figure 1.

Figure 1. Tooth-class allocation and sectioning methods used in the study: Maxillary and mandibular arches.

2.2 Preparation of samples

A total of eighty wax molds, each measuring 2 × 2 inches, were fabricated. A thin mix of dental stone was poured into each mold, and once the initial set had occurred, the tooth was positioned such that half of the crown and root were embedded while the remaining portion projected outward. The samples were divided into two groups for mesiodistal and labiolingual sectioning, as both planes are essential for evaluating specific anatomical and microstructural aspects of tooth morphology, particularly the orientation and distribution of Hunter–Schreger bands (HSBs).

Each specimen was first sectioned along the mesio-distal plane, which enabled simultaneous visualization of the mesial, distal, and occlusal surfaces. This orientation facilitated the assessment of band patterns in relation to proximal surfaces and their continuity toward the occlusal aspect.

Subsequently, the same specimens were sectioned in the buccolingual plane to allow visualization of the buccal, lingual, and occlusal surfaces. This approach ensured comprehensive assessment of enamel banding patterns in both orthogonal planes, thereby enabling evaluation of structural variations across the entire crown.

The rationale for employing both mesio-distal and buccolingual sectioning was based on the variation in functional loading and stress distribution across different tooth surfaces. In maxillary teeth, the palatal cusps function as the primary load-bearing cusps, whereas in mandibular teeth, the buccal cusps serve this role. Additionally, mesial and distal surfaces are subjected to differing stress patterns during mastication. These functional differences were considered critical for interpreting the orientation and distribution of Hunter–Schreger bands in relation to biomechanical forces ( Figure 2).

Figure 2. Comparative representation of mesiodistal and buccolingual sectioning of a tooth with corresponding photomicrographs of Hunter–Schreger bands.

(A) Mesiodistal section showing crown width and longitudinal orientation of Hunter–Schreger bands (Scale bar = 100 μm). (B) Buccolingual section illustrating crown thickness and comparatively parallel pattern of Hunter–Schreger bands (Scale bar = 100 μm).

Two sectioning orientations were thus followed: one aligning the mesiodistal plane with the study plane and the other aligning the labiolingual plane. After complete setting, the specimens were mounted on a lathe, and half of the exposed tooth surface was carefully reduced to achieve a sectioned half-profile, similar to the methodology described by Lynch et al.,¹ who emphasized controlled reduction to preserve structural detail. To improve visualization, the samples were subsequently polished using ground sectioning followed by pumice paste. Finally, the cleaned and prepared specimens were subjected to imaging.

2.3 Imaging

Photomicrographs were captured using the microscope camera (Olympus CX20i) at a 4× objective and exported as uncompressed TIFF files. Images were calibrated with a stage micrometer and analysed in ImageJ version 1.59 (published by the National Institutes of Health, NIH, USA) as depicted in Figure 3. The number of HSB pairs was counted within defined ROIs (1 dark + 1 adjacent light band = 1 pair).

Figure 3. Representative reflected-light photomicrographs showing HSBs on different tooth surfaces.

Panels: (A) maxillary incisor labial; (B) maxillary incisor lingual; (C) maxillary canine labial; (D) maxillary canine lingual; (E) mandibular premolar buccal; (F) mandibular molar buccal; (G) mandibular molar lingual; (H) mandibular molar occlusal; (I) mandibular molar lingual; (J) the mandibular molar occlusal. Images captured with 4× objective (Olympus CX20i); scale bar = 100 μm. Original TIFFs archived at Zenodo DOI: 10.5281/zenodo.17919950. Original unprocessed TIFF photomicrographs are available in the Zenodo deposit (see Data Availability) — file names correspond to the samples reported in Table 1.

The density of the HSB was calculated as follows:

The density of the HSB was then tabulated in Microsoft Excel and coded for the surface or the type of tooth.

2.4 Reliability

A subset of images (n = 10) was re-analysed by the primary observer and independently analysed by a second observer. Intraclass correlation coefficients (ICC) were calculated using a two-way mixed-effects model for absolute agreement (single measures).

2.5 Statistical analysis

Statistical analyses were conducted using SPSS version 20.0 (IBM Corp., Armonk, NY). Normality of continuous variables was assessed with the Shapiro–Wilk test. Between-arch comparisons used independent-samples t-tests (two-sided) with Cohen’s d reported as the effect size (Hedges’ g reported when n is small). Within-tooth surface differences were assessed with repeated-measures ANOVA (surface as the within-subject factor; arch and tooth class as between-subject factors). Mauchly’s test was used to assess sphericity; where violated, Greenhouse–Geisser or Huynh–Feldt corrections are reported. Partial eta-squared (ηp²) is reported as the effect size for ANOVA. Post hoc pairwise comparisons used Bonferroni correction. All tests used α = 0.05. Exact p-values, degrees of freedom, F statistics, effect sizes and 95% CIs are reported in text and tables.

3.1 Independent t tests to compare the densities of HSB

The most pronounced differences in HSB density were observed on the incisal/occlusal surfaces, with mandibular incisors showing greater densities than their maxillary counterparts did (1.17 ± 0.14 vs. 0.51 ± 0.12, p < 0.001) (see Table 1). This finding suggests a stronger adaptive response to masticatory forces in mandibular incisors. Similarly, a substantial difference was noted in the incisal/occlusal surface of the canines, where maxillary canines presented higher HSB density than mandibular canines did (0.91 ± 0.11 vs. 0.70 ± 0.16, p = 0.042), possibly reflecting differences in functional demands. Premolars consistently demonstrated greater HSB densities in mandibular teeth, especially on the buccal and lingual surfaces, with a notable difference on the buccal side (1.50 ± 0.16 vs. 1.06 ± 0.10, p = 0.001) ( Table 1). In contrast, molars presented minimal variation, with no statistically significant differences across most surfaces, indicating a more uniform HSB distribution in this tooth class.

3.2 Measurement reliability

Intra-rater reliability was good (ICC = 0.87, 95% CI 0.74–0.94), indicating consistent measurement of HSB density across repeated trials. Inter-rater reliability was good (ICC = 0.84, 95% CI 0.69–0.93), demonstrating strong agreement between the two observers.

3.3 Repeated-measures ANOVA comparing HSB density

Repeated-measures ANOVA was used to assess the impact of the dental surface (Buccal, Lingual, Mesial, Distal, and Incisal/Occlusal) on Hunter-Schreger Band (HSB) density across different tooth classes (Incisor, Canine, Premolar, Molar) in both the maxilla and mandible. The analysis revealed several key findings: ( Table 2).

The main effect of arch (Surface × Arch) was not statistically significant (F(4,128) = 1.744, p = 0.144, partial ηp² = 0.052), indicating no consistent global arch difference when averaged across tooth types and surfaces. However, specific tooth×surface comparisons ( Table 1) revealed localized differences (for example, mandibular incisors and mandibular premolar buccal/lingual surfaces), indicating localized rather than global arch effects.

3.4 Effect of surface morphology/texture

The analysis revealed noteworthy differences in the HSB density across the five surfaces (F = 9.617, p < 0.001; Table 2). This suggests that the HSB density is not uniformly distributed across all the tooth surfaces. Specifically, the incisal/occlusal surfaces generally presented lower densities than did the other surfaces. For example, the incisal/occlusal surface of mandibular incisors was denser than that of maxillary incisors (mean ± SD: 1.17 ± 0.14 vs. 0.51 ± 0.12, p < 0.001; Table 2). Similarly, mandibular premolars presented greater HSB density on the buccal and lingual surfaces than did maxillary premolars (buccal: 1.50 ± 0.16 vs. 1.06 ± 0.10, p = 0.001; lingual: 1.42 ± 0.22 vs. 1.02 ± 0.15, p = 0.010; Table 2).

3.5 Effect of arch form

No noteworthy differences were observed between the maxilla and mandible regarding the surfaces of the teeth (F = 1.744, p = 0.144; Table 2). This indicates that, overall, the distribution of the HSB density is relatively consistent between the two arches when it is averaged across all tooth classes and surfaces. For example, while mandibular premolars and molars tended to show greater densities on the buccal and lingual surfaces, these differences were not statistically significant across all tooth classes (e.g., buccal:maxilla 1.06 ± 0.10 vs. mandible 1.50 ± 0.16, p = 0.001).

3.6 Interaction between tooth class and surface

The interaction effect between tooth class and surface type was nearly significant (F = 1.776, p = 0.059). This suggests that variations in HSB density across different surfaces may differ depending on the tooth class. For example, premolars showed more pronounced differences in HSB density between the buccal and lingual surfaces than incisors did (buccal: 1.06 ± 0.10 for premolars vs. 0.91 ± 0.32 for incisors; lingual: 1.42 ± 0.22 for premolars vs. 1.05 ± 0.29 for incisors; Table 2), indicating that premolars exhibit more variation in response to functional forces.

3.7 Three-way interaction among the tooth class, arch, and surface

A significant three-way interaction was found among tooth class, arch, and surface (F = 3.413, p < 0.001; Table 2). This major interaction highlights that the effect of the tooth class and surface on the HSB density is influenced by the arch in which the teeth are located. For example:

Mandibular molars had similar HSB densities on the buccal and lingual surfaces to those of maxillary molars (buccal: 1.29 ± 0.11 vs. 1.42 ± 0.19; lingual: 1.44 ± 0.14 vs. 1.57 ± 0.13), but differences were observed in other surfaces, such as the incisor/Occlusal (mandibular molars: 1.37 ± 0.18 vs. maxillary molars: 1.34 ± 0.11, p = 0.757; Table 2).