Non-carious cervical lesions imaged by focus variation microscopy

Abstract

Objective

The aim of this study is to characterize the morphological and histological features of NCCLs in a group of extracted teeth using a focus variation optical microscopic technique that is capable of detecting minute variation in surface topography.

Methods

Twenty three extracted teeth containing NCCLs were collected. Histological features of the surface and longitudinal cross sections were examined using a focus variation microscope (FVM).

Results

The sample included 8 teeth with wedge-shaped lesions; the remaining 15 contained saucer-shaped lesions. Wedge-shaped lesions showed obliterated dentinal tubules, parallel furrows and micro-fractures in the surface; longitudinal cross sections revealed subsurface micro-fractures extending toward the pulp chamber. The surfaces of typical saucer-shaped lesions were smooth and relatively featureless.

Conclusions

FVM imaging shows microstructures that are consistent with simultaneous erosion and toothbrush abrasion. Saucer shaped lesions have a smooth featureless surface as well as craters and dimples that could be attributed to uneven acid attack. In wedge-shaped lesions, the presence of scratches and furrows could be attributed to mechanical forces such as tooth brush abrasion. The furrows and in-surface micro-factures of wedge shaped lesions suggest a possible role of tensile stresses but require further investigation.

Clinical significance

NCCLs present in two morphologies, either saucer-shaped or wedge-shaped. Erosion and toothbrush abrasion are important in both types of lesions. Tensile stresses due to occlusal loading may be important in some wedge-shaped lesions. Knowing the etiology of cervical lesions is the key for their prevention.

Introduction

Non-carious cervical lesions (NCCLs) are defects at the necks of teeth that are not caused by caries. Cervical lesions are considered an increasingly important factor when considering long term health of the dentition because they put dental structure at risk. In addition to having unacceptable aesthetics, the exposed dentin could become hypersensitive and lesion progression and eventually pulp exposure becomes more likely. Stresses within the tooth are increased by the tooth’s reduced cross sectional area near the lesion and by the stress magnification at notched lesions, thus, the risk of tooth fracture increases significantly .

These lesions present a restorative challenge to the dentist since they are close to the gingiva and may suffer from great enamel loss, this enamel is necessary for achieving satisfactory bond strength. In addition to the fact that the restoration placed would be prey to the stresses and strains that caused the lesion in the first place; the bonds between dentin and restorative material may subsequently break leading to the loss of restorative material . Since NCCLs pose a risk to the longevity of the dentition and are difficult to reliably treat, understanding their etiology is very important. The optimal way forward may be to use knowledge of their etiology to prevent these lesions in the first place.

The etiology of these lesions is considered as a controversial topic. Historically, different etiologies have been identified. In 1908, G.V Black suggested several possible causes, including: faults in the formation of teeth, friction from an abrasive tooth powder, action of an unknown acid, secretion from a diseased salivary gland, physiological resorption like that seen in deciduous teeth, acid associated with gouty diarethis and action of alkaline fluids on calcium salts. Experimentally neither the friction from abrasive tooth powder or the action of acids produced lesions with sharp clean cut margins as those found in the oral cavity, nor the action of alkalis could dissolve enamel and dentin until calcium salts had been removed. In addition, NCCLs occur in teeth that lack contact with diseased mucous membranes or salivary glands, and several well marked cases of gout lacked any evidence of NCCLs. Therefore, Black concluded that he had no theory to offer that could explain the formation of these lesions . The majority of the recent literature speculates that the etiology of NCCLs is multifactorial, with combinations of different processes, including: abrasion, erosion, and possibly abfraction .

The condition which was referred to as “abfraction” was first proposed by Lee and Eakle in 1984 . They presented a tensile stress hypothesis, stating that in case of traumatic occlusion; excessive flexural stresses will lead to severe bending forces resulting in compression on one side of the tooth and tension on the opposite side. It is well known that enamel has a weak edge strength, enamel is prone to fracture at sites loaded in tension, producing sharp-angled wedge-shaped lesions at such sites. The authors proposed that the position of these lesions depends on direction of lateral forces and its size depends on the magnitude of the force . The term “abfraction” was suggested by Grippo in 1991, Grippo assumed that abfraction is the main cause of NCCLs . He defined those lesions as; the pathologic loss of tooth substance caused by bio-mechanical loading forces, he further explained that cuspal flexure caused by heavy occlusal loading leads to concentration of tensile forces at the neck of the tooth with subsequent micro-crack formation by disrupting bonds between hydroxyapatite crystals in enamel and dentine . These cracks make the tooth more vulnerable to erosion and/or abrasion.

Although there is no formal morphological classification for NCCLs, two distinct cross- sectional shapes have been identified: 1) wedge-shaped and 2) saucer-shaped lesions . Although it is tempting to associate particular etiologies to the shape of the lesion, some reviewers have cautioned against doing this

Additional study of the histological features of cervical lesions may clarify what appears to be the complex etiology of these lesions. Previous investigations of the histology of NCCLs have used scanning electron microscopy (SEM) , light microscopy of lesion cross sections , and stereo imagery . Most of these studies have examined these lesions at relatively low resolutions and only two have examined the subsurface microstructure of these lesions. Much work remains to be done before the histology of NCCLs is completely characterized.

The aim of this study is to characterize the morphological and histological features of NCCLs in a collection of extracted teeth. For the first time, focus variation microscopy (FVM) has been used to study NCCLs. In FVM, the three dimensional (3D) image of a rough surface is constructed from a stack of scanned images collected by a scanning optical microscope (SOM) as the specimen’s surface is moved vertically away from the microscope’s objective lens . FV microscopy has several advantages, including: resolution in the vertical direction is very high, easy specimen preparation, images can be used to quantitate surface topography and roughness, and images are in true color.

Materials and methods

Approximately 200 freshly extracted human teeth that had been extracted for periodontal reasons were examined for the presence of NCCLs. Samples with intact NCCLs were selected, regardless of tooth type, lesion shape or lesion size, only samples free of caries, fractures and defects were included. From this collection of teeth, twenty-three teeth fit the inclusion criteria and were utilized in this study. Each tooth was thoroughly washed, scraped to remove shreds of periodontal ligament and ultrasonically scaled to remove plaque and calculus. All teeth were sterilized in ethylene oxide gas chamber for 8 h then stored in deionized water with thymol. The teeth were inspected visually to identify the following; tooth type, morphology of the lesion (wedge-shaped or saucer-shaped), location of the lesion (buccal or lingual), and presence of wear facets (attrition).

Sample preparation & imaging

The root(s) of each tooth were embedded in self-cured acrylic resin (Jet Set-4™ Powder & liquid; Lang Dental Manufacturing Co., Inc.; Wheeling, IL; USA), leaving the lesion and surrounding cementum, dentin, and enamel exposed. A three dimensional image of the surface of the lesion was obtained using a FVM, (InfiniteFocus ® G3, Alicona ® Imaging GmbH, Graz, Austria). Each area of interest was scanned at two magnifications, 50× and 100×. The lateral resolution was 10 μm and the vertical resolution was 20 nm. Software (IFM version 1.4, Alicona ® Imaging GmbH, Graz, Austria) that came with the microscope was used to acquire and store the images ( Fig. 1 ).

Fig. 1
Infinite focus images are in focus over a long depth of field as shown for this tilted surface.

An earlier version of the commercial FVM used in this study has been described in detail . This is an “optical” microscope, one that uses an objective lens to focus reflected visible light to form a magnified image. Such lenses have a very narrow depth of field, meaning that the surface of the object is in-focus over a very narrow range of distances from the lens. Images are collected consecutively as the vertical distance between the lens and the surface is changed. A vertical stack of images is collected and analyzed using proprietary algorithms to determine which that parts of each image have the highest contrast and therefore are at the best focus . The software uses the three-dimensional coordinates of the points of best focus and the images collected at best focus are assembled to construct an in-focus three-dimensional image of the surface. The lateral resolution of FVM images, like that of other optical microscopes, is limited by the wavelength of visible light (∼10 μm). The advantage of FVM is that it has a high vertical resolution; it is capable of resolving changes in height as small as 10 nm.

Preparation of tooth cross sections

Each tooth was bisected using a water-lubricated diamond blade mounted in a low-speed cutter (SMART CUT™ 4005 Low-Speed Sectioning Saw, Valenicia, CA, USA). This cut was through the midline of the tooth and lesion, producing two longitudinal sections, each in the buccolingual plane. The sectioned teeth were then polished with water-lubricated 320, 600, and finally 1200 grit silicon carbide paper, using a precision lapping polishing machine (SMART CUT LP, Valencia, CA, USA) . Each section was ultrasonically cleaned to remove debris created by sectioning and polishing. The FVM images of each section were then made at several magnifications (5×, 10×, 20× and 50×). Other data was collected after visual inspection of the teeth. Notes were made about the qualitative appearance of the lesions.

Materials and methods

Approximately 200 freshly extracted human teeth that had been extracted for periodontal reasons were examined for the presence of NCCLs. Samples with intact NCCLs were selected, regardless of tooth type, lesion shape or lesion size, only samples free of caries, fractures and defects were included. From this collection of teeth, twenty-three teeth fit the inclusion criteria and were utilized in this study. Each tooth was thoroughly washed, scraped to remove shreds of periodontal ligament and ultrasonically scaled to remove plaque and calculus. All teeth were sterilized in ethylene oxide gas chamber for 8 h then stored in deionized water with thymol. The teeth were inspected visually to identify the following; tooth type, morphology of the lesion (wedge-shaped or saucer-shaped), location of the lesion (buccal or lingual), and presence of wear facets (attrition).

Sample preparation & imaging

The root(s) of each tooth were embedded in self-cured acrylic resin (Jet Set-4™ Powder & liquid; Lang Dental Manufacturing Co., Inc.; Wheeling, IL; USA), leaving the lesion and surrounding cementum, dentin, and enamel exposed. A three dimensional image of the surface of the lesion was obtained using a FVM, (InfiniteFocus ® G3, Alicona ® Imaging GmbH, Graz, Austria). Each area of interest was scanned at two magnifications, 50× and 100×. The lateral resolution was 10 μm and the vertical resolution was 20 nm. Software (IFM version 1.4, Alicona ® Imaging GmbH, Graz, Austria) that came with the microscope was used to acquire and store the images ( Fig. 1 ).

Fig. 1
Infinite focus images are in focus over a long depth of field as shown for this tilted surface.

An earlier version of the commercial FVM used in this study has been described in detail . This is an “optical” microscope, one that uses an objective lens to focus reflected visible light to form a magnified image. Such lenses have a very narrow depth of field, meaning that the surface of the object is in-focus over a very narrow range of distances from the lens. Images are collected consecutively as the vertical distance between the lens and the surface is changed. A vertical stack of images is collected and analyzed using proprietary algorithms to determine which that parts of each image have the highest contrast and therefore are at the best focus . The software uses the three-dimensional coordinates of the points of best focus and the images collected at best focus are assembled to construct an in-focus three-dimensional image of the surface. The lateral resolution of FVM images, like that of other optical microscopes, is limited by the wavelength of visible light (∼10 μm). The advantage of FVM is that it has a high vertical resolution; it is capable of resolving changes in height as small as 10 nm.

Preparation of tooth cross sections

Each tooth was bisected using a water-lubricated diamond blade mounted in a low-speed cutter (SMART CUT™ 4005 Low-Speed Sectioning Saw, Valenicia, CA, USA). This cut was through the midline of the tooth and lesion, producing two longitudinal sections, each in the buccolingual plane. The sectioned teeth were then polished with water-lubricated 320, 600, and finally 1200 grit silicon carbide paper, using a precision lapping polishing machine (SMART CUT LP, Valencia, CA, USA) . Each section was ultrasonically cleaned to remove debris created by sectioning and polishing. The FVM images of each section were then made at several magnifications (5×, 10×, 20× and 50×). Other data was collected after visual inspection of the teeth. Notes were made about the qualitative appearance of the lesions.

Results

Table 1 shows the distribution of teeth by tooth type. Twenty-one teeth were maxillary and only 2 were mandibular. Fifteen teeth contained a saucer-shaped lesion and 8 had a wedge-shaped lesion. In twenty of the teeth the lesion presented on the buccal surface while only 3 teeth presented with a lingual lesion. Wear facets were present in all eight teeth containing wedge-shaped lesions, while wear facets were present in only three of the 15 teeth containing saucer-shaped lesions.

Table 1
Distribution of teeth by position.
Tooth type Maxillary
Central
Maxillary
lateral
Maxillary
Canines
Maxillary
Premolar
Maxillary
Molars
Mandibular central Mandibular lateral
Number 4 3 9 3 4 1 1

Surfaces of the lesions

FVM micrographs revealed the presence of cracks, scratches, craters, dimples & horizontal furrows. The surface topography of wedge-shaped lesions was different from the saucer-shaped lesions in the following ways:

All 8 wedge-shaped lesions exhibited scratches on the surface, the scratches were in multiple different directions, some scratches were more distinct than others ( Fig. 2 ). On the other hand, saucer-shaped lesions showed fewer and fainter scratches ( Fig. 3 ), twelve out of the 15 saucer-shaped lesions exhibited no scratches. Some of the wedge-shaped lesions contained furrows ( Fig. 4 ), this feature was absent in saucer-shaped lesions. In contrast, craters (see Figs. 5 and 6 ) and dimples ( Figs. 6 and 7 ) were observed only in saucer-shaped lesions and were not observed in any of the wedge shaped lesions. Partially occluded cracks could be seen in the surfaces of some wedge-shaped lesions ( Fig. 8 ).

Fig. 2
This wedge-shaped lesion contains numerous scratches. Bar at upper left is 20 μm.

Fig. 3
Saucer-shaped lesions are generally featureless, containing few scratches and no cracks. Bar at upper left is 20 μm.

Fig. 4
Horizontal furrows within a wedge-shaped lesion. Bar at upper left is 10 μm.

Fig. 5
Arrows point to craters within this saucer-shaped lesion. Bar at upper left is 10 μm.

Jun 17, 2018 | Posted by in General Dentistry | Comments Off on Non-carious cervical lesions imaged by focus variation microscopy
Premium Wordpress Themes by UFO Themes