We propose a new theory for enamel cupping lesions formation. At early stages, naturally formed cupping lesions showed increased porosity at two structural prismatic traits: the central cone extending into the enamel-dentine junction and the type-I Hunther-Schreger bands (HSB), suggesting them to be the main drivers for cupping lesion formation and development. In addition, these lesions were circumscribed by type-II HSBs, which present lower surface porosity and higher resistance to wear. This theory was verified in in vitro observations, where both the central cone and the type-I HSB of cuspal enamel showed higher susceptibility to wear, potentially elucidating the mechanisms involved on cupping lesion formation.
Cup-shaped (or cupping) lesions develop at the cusp tips of posterior teeth, present a concave morphology, and are often associated to erosive tooth wear (ETW). Recent in vitro simulation has provided evidence that the combination of tooth-to-tooth abrasion (dental attrition) and acid exposure (dental erosion) are the main factors explaining the initiation of cupping lesion on the enamel cusp tip, with later involvement of dentine [Ruben et al., 2019]. Theoretically, the combined mechanical loading and acid exposure would provide enlargement of interprismatic spaces at the enamel surface, facilitating acid diffusion into subsurface enamel (to a depth of ∼100–200 μm) and loosening prisms, which in turn, would be dislodged by lateral forces resulting in wear with a decreasing gradient from the cusp tip to the cusp periphery. The bottom of the cup-shaped lesion would be located at the original cusp tip, progressing toward the underlying dentine horn [Ruben et al., 2019].
Investigation of the histopathology of natural enamel cupping lesions on the cusp tips of posterior teeth can provide valuable information on their development mechanisms; however, this has received limited attention [Khan et al., 2001]. In a laboratory exercise observing extracted posterior human teeth, we have identified the presence of a pit-type enamel feature with low size/depth aspect ratio. This feature may be present in one or more cusp tips of the same tooth. We hypothesize that this enamel pit-type feature may represent early stages of the cupping lesions and/or be the precursor driving their development by promoting a specific type of tooth wear. The clinical appearance and histopathologic features of enamel anatomical features have not been previously reported in the literature. Here, we characterize them and provide a model explaining how they could lead to the formation of cupping lesions on the cusp tips of posterior teeth.
Materials and Methods
A total of 100 extracted human teeth (unidentified, 85 third molars and 15 premolars) presenting natural enamel cupping lesions were selected from a tooth-bank at the Oral Health Research Institute, Indiana University School of Dentistry. This study protocol was reviewed and approved by the Indiana University IRB, approval number NSO 911-07.
Stereomicroscopy and Optical Profilometry
All teeth were photographed under stereomicroscopy (SMZ 1500, NIKON, Japan) using ×15 magnification. From the 100 teeth, only a subsample of 20 third molars presenting at least one cup-shaped lesion including a pit in the center (pit-like lesion) had their occlusal surfaces analyzed under optical profilometry [Proscan, 2000; Scantron, England] with a vertical resolution of 0.3 mm.
Microradiography and Polarizing Microscopy
Selected teeth (n = 10) with typical features were sectioned in a hard tissue microtome (Silverstone microtome) equipped with diamond disc under water spray stirring, producing thin sections (90–110 mm). Sections were submitted to qualitative digital microradiography (OS X-ray system, Thermo-Kevex PXS5-928WB) and polarizing microscopy (SMZ 1500, Nikon, Japan, equipped with crossed polarizers and Red I retardance filter) under immersion in water and quinoline [De Medeiros et al., 2012].
Laboratory Simulation of Enamel Cupping Formation
Three teeth with early pit-like cupping lesion were selected. After removal and discard of the root with a diamond disc under water stirring, the remaining sectioned surface was polished flat and mounted on polishing disks, keeping the cusp of interest at the highest position. The tip of the cusp was submitted to a grinding process in a polishing machine (RotoForce-4, Struers, USA) using 4000-grit silicon carbide grinding paper and a force of 10 N, under water irrigation. After grinding off the existing cupping lesion using water irrigation, flowed by sonication for 3 min in 2% aqueous detergent solution (Micro-90 concentrated cleaning solution; Cole-Palmer, Burlington, VT, USA) [Wang et al., 2013], the flat exposed cuspal enamel surface was submitted to surface microhardness assessment (Leco 247 AT; equipped with Vickers indenter using 200 g for 11 s). Subsequently, simulations of two and three-body abrasive wear were performed on the test area by further polishing the enamel surface as described above, with irrigation of either water or abrasive slurry (30% w/w of pumice fine powder in 0.5% carboxymetyl cellulose), respectively.
Scanning Electron Microscopy
Four-thirds molars with naturally formed cup-shaped lesions and one artificial cup-shaped lesion were analyzed under scanning electron microscopy (SEM) (JEOL7800f, Jeol, USA; using 5 kV and lower electron detector with low depth of filed; magnification up to 11,000), after sonication for 3 min in 2% aqueous detergent solution (Micro-90 concentrated cleaning solution, Cole-Palmer) [Wang et al., 2013] and sputter-coating with gold in high vacuum.
Under stereomicroscopy, a low surface area size/depth aspect ratio depression at the enamel surface cup-shaped lesion with resemblance of enamel pits was observed in 54 out of the 100 occlusal surfaces with cup-shaped lesion (Fig. 1a, b, 2a1–d4). Histologically, the pit bottom is not close to any accentuated incremental lines and presented a deep cone-shape higher porosity area (translucent zone under quinoline immersion) extending from the enamel surface (where the cone base is located) into the enamel-dentine junction (EDJ) at the dentine horn, resembling the cone-shaped 3D arrangement of prisms in cuspal enamel [Osborn, 1968] (Fig. 1b). As one approaches the center of the cone, a decreasing microhardness can be found (Fig. 3). One or more cup-shaped lesions per occlusal surface presented a pit-like structure, and in one case, two pits were found at the same cup-shaped lesion under stereomicroscopy (Fig. 2a1, and online suppl. material; for all online suppl. material, see www.karger.com/doi/10.1159/000528844). Pits presented variation in color (dark brown, white spot, and identical to normal tooth color), depth (up to 700 mm), diameter at the opening, and flatness of peripherical walls (see online suppl. material). Pit diameter can be lower than the low-magnification stereomicroscopic examination resolution (Fig. 2d1, d2), so that differentiating cup-shaped lesion with or without pit requires higher resolution (either by profilometry or SEM). Frequently, cup-shaped lesions, regardless the presence of pit, presented areas of volume loss forming grooves or incomplete concentric arcs, the latter resembling the arrangement of Hunter-Schreger bands (HSBs) in cuspal enamel from the occlusal view (Fig. 2b2, a3, d3, d4). At the pit bottom, commonly filled with debris (Fig. 2a2, a3, c2, d3), the walls varied presenting either a smooth or a rough surface, the latter been found in combination with increased diameter at the prism bottom (Fig. 2c2).
The curved pits walls presented exposed prisms heads (with variable angulations) showing high surface porosity and arranged in circles/arcs concentric to the pit depression, forming a vortex (Fig. 2). A ring of lower surface porosity, polished-like texture, with no exposed prisms heads, can be found at the periphery of the pit wall (Fig. 2a4). Secondary pits can be found at the lateral pit walls and/or at the periphery of the cup-shaped depression, surrounded by secondary concentric circles of prisms (secondary vortices; Fig. 2d4).
The results of the simulation of an enamel cupping lesion are shown in Figure 3a–l. The artificial lesion was formed after grinding for 14 s with the use of abrasive slurry (three-body abrasion, Fig. 3e), with the depression located just subjacent to the bottom of the original pit-like cupping lesion, where a decreased surface microhardness had been detected. The surface became completely flat after subsequent grinding with water only (two-body abrasion), for 14 s, as visually demonstrated in Figure 3f. Then, a second grinding session with abrasive resulted in de novo artificial cupping lesion formation at the same location detected previously (Fig. 3g, h). The central depression was confirmed by optical profilometry and microcomputed tomography (see online suppl. material). Under SEM, the periphery of the artificial cupping lesion presented two patterns of surface texture, resembling the arrangement of HSBs in cuspal enamel, with two types of arcs concentric to the central depression. One arc type consisted of a depression (higher volume loss) with a, rough, etched-like, surface with exposed prisms heads surrounded by enlarged sheaths (Fig. 3l). The other type consists of relatively smooth, polished-like, surface, with prims running more parallel to the enamel surface, and presenting no volume loss (Fig. 3l).
This study reports, for the first time, histopathological features of naturally formed enamel cupping lesion with important repercussions for the understanding of their development on cups tips. Our results show evidence of wear associated with specific histological features of cuspal enamel, providing consistent characterization of ETW.
The specific histological features of cuspal enamel associated with wear include two prismatic traits: the variations in prismatic orientation in HSBs and the central cone [Osborn, 1968]. In HSBs, prisms oriented perpendicularly to the enamel surface (hereafter called type-I HSB) are more susceptible to acid dissolution and more resistant to wear when they are part of sound enamel, while acid etching renders those prims with relatively higher porosity and more prone to wear. HBS with prisms oriented parallel to the enamel surface (type-II HSB) behave in the opposite way [Osborn, 1973; Shimada and Tagami, 2003]. In cuspal enamel, in a horizontal plane perpendicular to the cusp main axis, the visual arrangement of HSBs consists of incomplete interlocking arcs concentric to one or more vortices close to the cusp tip [Osborn, 1968] (Fig. 3), playing a role in surface wear morphology. The central cone in cuspal enamel consists in a region of prims with increasing diameter from the EDJ toward the enamel surface, including subregions of cones with decreasing diameter toward the center of cuspal enamel [Osborn, 1968]. Our results, from both naturally and artificially formed cupping lesions, show features of acid-etched HSBs, with areas with exposed prism heads (type-I HSB) presenting etched-like surface texture (increased porosity) and increased volume loss compared to areas with prims were more parallelly oriented relative to the enamel surface (type-II HSB) that presented a polished-like surface texture and lower volume loss (Fig. 1, 2). The incomplete arcs (composed of HSBs types I and II) concentric to a vortex seen under SEM (Fig. 1, 2) are consistent with the arrangement of HSBs in cuspal enamel (Fig. 3) [Osborn, 1968]. The occurrence of more than one vortex per cup-shaped lesion confirms the original report of up to three vortices per cuspal enamel [Osborn, 1968]. Away from the vortex center, outlining the cup-shaped lesion periphery, arcs with a polished-like surface texture (type-II HSBs) shown here (Fig. 3i, j) have been reported in artificial enamel cup-shaped lesion induces by the combination of acid exposure, saliva, and mechanical load [Ruben et al., 2019].
The formation of artificially formed cupping could be explained by the three-body abrasive wear process, where the use of the third body (abrasive slurry) selectively enhances the wear of the softer areas [Sabrah et al., 2018]. Here, the softer area was located just subjacent to the bottom of a pit-like cup-shaped lesion and presented lower surface microhardness (Fig. 3). The artificial cupping induced here presented a sharper contrast between the types of HSBs surrounding the central depression (at the center of a vortex of HSBs), with type-I HSBs presenting higher volume loss compared to type-II HSBs. Such peripherical features are similar to those presented here in naturally formed, as well as in artificially formed enamel cupping lesions under the long-term exposure to acid, saliva, and mechanical load [Ruben et al., 2019]. The main difference between the two models is that in the present a single factor (abrasive wear only) was applied on an area subjacent to an existing cupping lesion, whereas the multifactor model by Ruben et al.  used sound cuspal enamel with no attempts to target specific cuspal enamel traits (HSBs vortices and central cone). Future research is necessary to answer the question of whether the origin of the more susceptible areas in cuspal enamel is environmental or developmental. Current knowledge does not provide clear answers.
The current observations and the arrangement of enamel prisms and HSB in cuspal enamel [Osborn, 1968] support our proposed theoretical model for the formation of enamel cupping lesion (Fig. 4a–c). The model predicts that the formation of enamel cup-shaped lesions results from the combination of dental erosion, dental abrasion (two or three-body), and the histological structure of the enamel cusp. The latter includes the enamel layer thickness, the distribution of prisms in cones (with the base on the enamel surface and the apex on the EDJ) in the center of the enamel cusp, and the variations in prismatic orientations in HSB. The model predicts that the combination of acid exposure, mechanical load with an abrasive (three-body wear), type-I HSB, and the vortex in the center of the HSB pattern in cuspal enamel would yield the highest probability of cupping formation.
The central prismatic cones in cuspal enamel [Osborn, 1968] would provide higher susceptibility to wear. The outward diffusion of ions in enamel (beneficial against acid inward diffusion) [Atkinson, 1947] would be hampered by the highest surface area/EDJ area aspect ratio in cuspal prismatic cones. As wear progresses, a central depression is formed surrounded by curved walls, continuously exposing additional HSB at new angles, thus exposing new groups of prisms heads to the oral environment (and to the acid). The wall of the pit/cup depression would be outlined by a HSB with prisms oriented more parallel to the enamel surface. The model provides a better understanding of where and how cupping lesions develop, opening new avenues for the study of more effective clinical detection, and further development of preventive and therapeutic measures.
Statement of Ethics
This study protocol was reviewed and approved by the Indiana University IRB, approval number NSO 911-07. This research complied with the guidelines for human studies and was conducted ethically in accordance with the World Medical Association Declaration of Helsinki.
Conflict of Interest Statement
Authors declare no conflict of interest.
Frederico Barbosa de Sousa received a visiting professor scholarship by the CAPES-PRINT program, offered jointly by the Brazilian Ministry of Education and the Federal University of Paraiba (Brazil).
Frederico Barbosa de Sousa and Anderson T. Hara conceived the idea, analyzed data, proposed the model, wrote, and revised the manuscript. Frederico Barbosa de Sousa collected data.
Data Availability Statement
Data generated or analyzed during this study consist mainly of images, the most representative of which are included in this article. Further inquiries can be directed to the corresponding author.