Dentinal Changes in Attrition and Abrasion – A Scanning Electron Microscopic Study

INTRODUCTION

The pulpo-dentinal complex is capable of mounting a defensive response against external injuries like caries, attrition, abrasion, erosion and cavity preparation¹. The light microscopic changes secondary to attrition and abrasion (due to exposure of dentinal tubules), include dead tracts, sclerosis and reparative dentin formation.

Dead tracts are the areas of dentin containing nonfunctional dentinal tubules². In Sclerotic dentin, the dentinal tubules gradually become occluded with calcific material. The refractive indices of the areas in which the tubules are occluded are equalized and such areas look transparent / white under transmitted light and appear dark in the reflected light^3-5. Reparative dentin, known by a variety of names is deposited at sites of the pulpal aspects of dentin, corresponding to areas of external irritations.²

There are only few ultrastructural studies on the changes of dentin secondary to attrition and abrasion.

The greater depth of the field of Scanning electron microscope compared with conventional light microscopy allows assessment of the three-dimensional morphology of the specimen⁶. As Scanning electron microscopic image complements the information available from the light microscope, the present study is to find out the ultrastructural changes occurring in dentin due to attrition and abrasion.

MATERIALS AND METHODS

Thirty extracted teeth (14 attrited, 10 abraded anteriors and 6 normal premolars, extracted for orthodontic purpose) were collected. Only the attrited and abraded teeth which were clinically free of caries and restoration were selected. Eight teeth were discarded due to a variety of reasons. Sample collection and specimen preparation were difficult tasks which made the number of cases non uniform among three study groups.

Twenty extracted permanent teeth (10 attrited, 6 abraded anteriors and 4 normal premolars) constituted the study material. After fixation in 10% formalin for 24-48 hours, the teeth were debrided by using a B P Blade attached to a handle.

The root portion of the attrited teeth was removed by using dental lathe with intermittent cooling. A groove was made on the crown with an inverted cone diamond point, and the teeth were mounted in dental stone. Chisel and mallet were used to fracture these teeth along their longitudinal axes. The fractured fragments were placed again in 10% formalin.

For abraded teeth, a portion of crown and root was removed, using dental lathe again with intermittent cooling. A groove was made over the coronal portion with an inverted cone diamond point and the teeth were fractured along their longitudinal axes after mounting in dental stone. The fractured fragments were placed again in 10% formalin.

One half of the each tooth was made into an approximately 100µ thick section using Silicon Carbide stone followed by fine grain black Emery paper (No.400) and was mounted with DPX on a glass slide and cover slip placed over it (Figure 1). This section of the tooth was visualized under a Stereomicroscope (Olympus SZX 12, Japan) under transmitted light to observe dentin. Images of Stereomicroscope were captured on the computer using a 3 Chip CCD Camera (Proview, Media Cybernetics, USA) and frame grabber card.

The specimens were not allowed to dry at any stage, during the preparation of ground section. Even the SEM halves of the specimens were not allowed to dry till they were kept in the vacuum for gold sputtering.

The images of the fractured surface of other half (SEM half) of the tooth specimen were captured by Stereomicroscope under reflected light prior to scanning under Scanning Electron Microscope. Based on the microscopic findings observed in ground sections under transmitted light (Figure 2), corresponding areas were selected for study under the Scanning Electron Microscope (Figure 3).

The SEM halves of the specimens were coated with gold by cathodic sputtering with JEOL FINE COAT ION SPUTTER JFC-1100 for two minutes and fed into the JSM 5600LV (JEOL) Scanning Electron Microscope, after mounting over the metallic stub. Each specimen was scanned from two sides, first the lesional surface and secondly the fractured surface at an accelerating voltage of 20kV. The chosen magnifications were x850, x2200, x5500. Magnification having higher than x5500 was only used to visualize some interesting structures, when noticed. Maximum fields in the areas of interest on the SEM halves were observed. As the specimens were fractured and not cut, the fracture surfaces were highly uneven under high magnification. The areas selected for photography were those where maximum length of the dentinal tubules was visible. Only the images of the representative areas were photographed.

The Scanning Electron Microscopy was carried out from the Department of Solid State and Structural Chemistry, Indian Institute of Science, Bangalore initially and later at Regional Research Laboratory, Trivendrum, India.

The normal samples were scanned first to form a base for the interpretation of samples with lesions. Then the attrited and abraded samples were subjected to a preliminary scan to note the pathologic features.

Based on preliminary trial scan and review of literature, the following features were looked for in both the cases of attrited and abraded teeth.

1. Lesional surface features of exposed dentin, in areas of dead tracts and sclerosis

2. Diameter and density of dentinal tubules in affected area

3. Distinctness of peritubular and intertubular dentin

4. Integrity of dentinal tubular surface and margins

5. Presence of crystals within the tubules and its nature

6. Features of reparative dentin

The data obtained were entered in a master chart for further interpretation and analysis.

RESULTS

Light microscopic findings:

None of the normal teeth showed areas suggestive of dead tracts, dentinal sclerosis or reparative dentin formation. However, one premolar showed a small area resembling dead tract, which was interpreted as an artifact (Table 1)

All the attrited and abraded teeth showed the formation of dead tracts. Few abraded teeth showed reparative dentin formation and smaller areas resembling dentinal sclerosis. All the attrited teeth showed the presence of reparative dentin but no areas of dentinal sclerosis.

Areas of reparative dentin and dentinal sclerosis were identified based on their location and their appearances under transmitted and reflected lights.

Areas identified as dead tracts, dentinal sclerosis and reparative dentin were subjected to observation under the SEM, in addition to lesional surfaces.

Scanning electron microscopic findings:

The points observed while scanning the fractured surfaces were :

1. Diameter and density of dentinal tubules in affected area

2. Distinctness of peritubular and intertubular dentin

3. Integrity of dentinal tubular surface and margins

4. Presence of crystals within the tubules and its nature

5. Occlusion of tubules

In addition to the above, the lesional surfaces were scanned.

Different grades were given for assessing the distinctness of peritubular and intertubular dentin. This method was followed to reduce subjectivity. The grading was done by two observers jointly. 

The normal teeth were scanned first to form a base for the interpretation of samples with lesions. The nature of the normal dentin was observed in different areas and the findings noted down.

To ensure uniformity, for each specimen in every group, two fields, one near the pulpal end and one near the dentino-enamel junction area were selected under x850 and/or x2200 magnification, among the photographs taken and the changes were interpreted. The findings were tabulated in a master chart for further analysis and interpretation.

Analysis of the findings revealed that, the diameters and density of the tubules in normal, attrited and abraded teeth were more near the pulpal end compared to the dentino-enamel junction area (Tables 5a & 5b).

In normal teeth, many of the dentinal tubules showed distinct peritubular and intertubular dentin; the prominence was more towards the incisal area or near the dentino-enamel junction (Table 2). The peritubular dentin was distinctly denser and finely crystalline in comparison to coarse crystalline nature of intertubular dentin. The margins/walls of the tubules were smooth and there was no evidence of any crystals within the tubules (Table 2). Openings of the lateral branches of the odontoblastic processes were visible within the tubular wall (Figure 4).

The significant finding in attrited and abraded dentin was the presence of diseased or affected tubules with roughening of walls and margins (Tables 3, 4 & 6). The changes of the tubules in attrited and abraded teeth were common to both. The changes were either the presence of small globular calcifications on the walls of tubules, causing roughening of the walls and the fracture margins of the tubules, or rhomboid crystals (Figure 4) attached to walls either randomly or in clusters. The changes within individual tubules were interestingly similar. However, completely unaffected tubules (Figure 4) without any degenerative changes as in the case of normal dentin were found occasionally adjacent to the affected tubules. There were no distinctive changes in the peritubular or intertubular dentin in either attrited or abraded teeth compared to normal.

Variation in the dentinal tubule diameters in a single field was observed within attrited, abraded and normal teeth, but was more marked in attrited and abraded dentin (Tables 5a & 5b).

The Odontoblastic processes were universally absent in all the teeth studied except in one or two dentinal tubules.

The tubules adjacent to lesional surface were found to be packed with debris to a considerable depth. There was space between the plug and the walls of the tubules (Figure 5).

Plugging of the openings of the dentinal tubules of the lesional surface of both attrited and abraded teeth was very obvious, as the openings were not very distinct. The locations of openings of tubules were seen as round or ovoid depressions (Figure 6). The diameter of the openings was found to be 3µ. This was larger than what might be expected (Figure 7, Tables 5a & 5b).

The smaller areas resembling dentinal sclerosis, seen in few abraded teeth under light microscopy could not be identified by SEM. There was no evidence of complete occlusion of tubules in these areas. The reparative dentin area seen in all attrited but few abraded teeth under light microscopy showed no tubular morphology under scanning electron microscopy (Figure 8). The area of reparative dentin appeared as an irregular mass of tissue without any specific organization into peritubular and intertubular dentin.

DISCUSSION

The light microscopic changes of the dentin produced by attrition and abrasion have been well described in the text books. The appearance of dead tracts, dentinal sclerosis and reparative dentin has been secondary to attrition and abrasion, is well known. Even though a number of studies have been reported using Transmission Electron Microscopy, Scanning electron microscopy, Microradiography and other techniques^7-22, a clear consensus on what causes these effects is not obtained. Hence the present study was undertaken to observe the changes in the dentin and within the dentinal tubules in attrited and abraded teeth. Also the nature of reparative dentin in relation to normal circumpulpal dentin was observed.

Scanning electron microscopy (SEM) has been used since it gives extremely high resolution pictures of high magnification of surfaces and depth of field. SEM was chosen specifically to study the changes in the dentinal tubules, peritubular and intertubular dentin and tubular contents in areas of dead tract formation and dentinal sclerosis.

Fracturing of tooth was preferred over sectioning, as sectioning with rotary instruments would cause obscuring of tubules with debris, thus masking the changes within the tubules. Cleaning with ultrasonic cleaners may result in loss of tubular contents if any.

In ground sections, areas of dead tract formation in relation to attrition and abrasion appear, dark under transmitted light and bright under reflected light. What causes the appearance of the dead tracts in the exposed dentinal tubules has been controversial. The most common explanation has been that the sealed off dentinal tubules contain air and this results in the appearance of dead tracts^3-5. The other explanation is the 2 accumulation of crystals within the dead tracts (Toda et al² ). In the present study the diseased and unaffected tubules were quite obvious in the dead tract area. The most common findings of the diseased tubules had been the roughening of the walls of the tubules and the presence of crystals within the diseased tubules. Even though the presence of air within the tubules may play a role, the presence of crystals and roughening of the walls of the tubules can also play a central role in the appearance of dead tracts in the case of exposed dentinal tubules. The diseased tubules in this study, in the area of dead tracts, presented both roughening of the tubular wall and a variety of crystals. Both these would hinder the passage of light by scattering and refraction of the rays.

The roughening of the walls was probably due to globular deposition of calcified material. This finding was consistent with the transmission electron microscopic findings of Tronstad and Langeland²³ who found deposition of fine needle like crystals on the walls of the tubules in a centripetal direction. In addition to these, there were distinct angular cuboidal crystals within the tubules that were distinct from debris caused by fracturing process. The crystals could be identified by their definite morphology and pattern of arrangement, which was lacking in debri. It was obvious that these crystals have originated from within the tubules and not been introduced from outside. The two possibilities that one can think of are: 1. the mineralization induced by the damaged odontoblastic process and 2. the acidic media (probably either due to clinically non evident dental erosion or action of microorganisms in dental plaque) penetrating the tubules might have caused demineralization of the tubular wall and when neutralized, the supersaturated solution might have induced mineralization on the walls of the tubules and crystal formation around central nidi.

It was interesting to note that odontoblastic processes were universally absent in all tooth samples studied except in one or two dentinal tubules. This would probably be due to the failure of fixatives to reach the deeper part of the tubules resulting in subsequent liquefaction degeneration and disappearance. Remnants of odontoblastic process were observed in one or two dentinal tubules.

Adjacent to lesional surface the tubules were found to be packed with debris to a considerable depth. There was space between the plug and the walls of the tubules most likely produced by the contraction of the material due to drying while in the vacuum chamber of the gold sputtering machine or the vacuum chamber of the SEM. The shrinkage denotes that they are either purely organic or organic material with very little mineral content. For all likelihood the material may have been introduced from the external surface and probably consist of salivary proteins, food debris and microorganisms pushed in by mechanical action. Part of it could have been formed by microorganisms as in the case of plaque and subsequently mineralized as in the case of calculus. The presence of these materials closely packed within the tubules made the distinction between the lumen, peritubular dentin and intertubular dentin difficult, in areas adjacent to the lesional surface, especially in case of abrasion.

The scanning electron microscopy of the lesional surfaces of both attrited and abraded teeth showed plugging of the openings of the dentinal tubules, since the openings were not very distinct. The diameter of the openings of the dentinal tubules (which appeared as round or ovoid depressions) was larger than what might be expected, possibly due to certain amount of decalcification of the surface, resulting in shrinkage of adjacent dentin due to dehydration eventually resulting in enlargement of the openings.

In the present study, smaller areas resembling dentinal sclerosis were seen in few abraded teeth under transmitted light. These areas could not be identified by SEM. Probably because very few areas of sclerosis were present in association with abrasion in the specimens studied and also because it was difficult to identify these small areas under the SEM, the areas of dentinal sclerosis were not detected. There was no evidence of complete occlusion of tubules in these areas.

Reparative dentin is deposited at sites of the pulpal aspects of primary or secondary dentin, corresponding to areas of external irritations⁷ . Either damaged odontoblasts or replacement cells recruited from pulp are responsible for the formation of reparative dentin. Its rate of deposition varies directly with the degree of injury. It is characterized by having fewer and twisted tubules. Reparative dentin is less mineralized than normal dentin. Sometimes, its formative cells may become inclusions in it (osteodentin)^3,4. Because of the absence of tubules, reparative dentin appears bright in transmitted light and dark in reflected light⁴ .

In the present study, under light microscopy, variable amounts of reparative dentin formation was found on the pulpal aspect of all attrited teeth but only in few of abraded teeth. As the sections had not passed through the pulp while fracturing, the presence of reparative dentin could not be appreciated in the remaining abraded teeth. Under scanning electron microscopy the area of reparative dentin, appeared as an irregular mass of tissue without any specific organization into peritubular and intertubular dentin.

CONCLUSION

The present study revealed that all cases of attrition and abrasion with dentin exposure showed the formation of dead tracts and variable amounts of reparative dentin, under light microscopy. Under SEM, the predominant finding in the areas of dead tracts of both attrited and abraded dentin was the presence of diseased or affected tubules.

Areas resembling dentinal sclerosis seen in few abraded teeth under transmitted light could not be identified by SEM. There were no areas of complete occlusion of tubules as was anticipated in dentinal sclerosis.

The reparative dentin formation observed in all attrited but few abraded (as the fracture plane did not pass through the pulp in some abraded teeth) teeth under transmitted light, appeared as an irregular mass of tissue without any specific organization into peritubular and intertubular dentin.