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Occlusal fissures in equine cheek teeth: nullCT and histological findings

E. Pollaris, C. Staszyk, K. Proost, M.N. Boone, I. Josipovic, B.

Pardon, L. Vlaminck

PII: S1090-0233(19)30154-6
DOI: https://doi.org/10.1016/j.tvjl.2019.105421
Reference: YTVJL 105421

To appear in: The Veterinary Journal

Accepted Date: 10 December 2019

Please cite this article as: Pollaris E, Staszyk C, Proost K, Boone MN, Josipovic I, Pardon B,
Vlaminck L, Occlusal fissures in equine cheek teeth: micro;CT and histological findings, The
Veterinary Journal (2019), doi: https://doi.org/10.1016/j.tvjl.2019.105421

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Occlusal fissures in equine cheek teeth: µCT and histological findings

E. Pollaris a, C. Staszyk b, K. Proost a, M.N. Boone c, I. Josipovic c, B. Pardon d, L. Vlaminck

a, *
lieven.vlaminck@ugent.be
a
Department of Surgery and Anaesthesiology of Large Animals, Faculty of Veterinary
Medicine, Ghent University, Salisburylaan 133, 8920 Merelbeke, Belgium
b
Institute of Veterinary-Anatomy, -Histology and -Embryology, Faculty of Veterinary
Medicine, Justus-Liebig-University Giessen, Ludwigstraße 23, 35390, Germany
c
Department of Physics and Astronomy – Radiation Physics, Faculty of Science, RP-UGCT,
Ghent University, Proeftuinstraat 86, 9000 Ghent, Belgium
d
Department of Large Animal Internal medicine, Faculty of Veterinary Medicine, Ghent
University, Salisburylaan 133, 8920 Merelbeke, Belgium
*
Corresponding author. Tel.: +32 9 264 76 18., (L. Vlaminck).

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Highlights

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 Equine occlusal cheek teeth fissures were commonly observed.



the pulp.
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µCT imaging showed the potential depth of these fissures and close relationship with

Histological examinations demonstrated that fissures can provide pathways for

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bacteria, potentially resulting in local pulpal inflammation.
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Abstract
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Fissures on the occlusal surface of equine cheek teeth are commonly encountered

during oral examination. Generally, their presence is considered abnormal but their
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aetiopathogenesis and clinical impact on pulp disease is still undetermined. The aims of this
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research were to study the extent of occlusal cheek teeth fissures using high-resolution X-ray

computed tomography (µCT) imaging and their histological characteristics. Twenty-seven

teeth (of 15 horses) were scanned using µCT imaging to analyse fissure extent. Histological

examination of a subset of teeth (n = 7) was performed.

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 Forty-three type 1 fissures (35 type 1a, eight type 1b) were identified. The mean length

of the fissure of type 1a and type 1b on the occlusal surface was 3.47 +/- 1.60 mm and 13.64

+/- 7.40 mm, respectively. Their mean depth was 13.22 +/- 10.76 mm and 7.42 +/- 6.42 mm,

respectively. Potential risk factors associated with fissure depth were identified using a

multivariable mixed model The location of the fissure and Triadan number were significantly

associated with fissure depth. Fissures could be identified on histological sections with the

presence of organic material inside the fissure, microorganisms in the continuation of the

fissure extending into the dentinal tubules and the presence of reactionary dentine. This study

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suggests that fissures can provide a pathway for microorganisms to enter the dentinal tubules,

potentially resulting in local pulpal inflammation. It appears that in healthy teeth, vital

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odontoblasts react by producing reactionary (tertiary) dentin, which lacks patent tubules and

thus provides a proper seal. -p

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Keywords: Equine Dentistry; Fracture; Imaging; Molars; Pulpitis
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Introduction

Occlusal fissures are common findings during equine dental examination (Dacre et al.,
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2007, 2008a, 2008b; Simhofer et al., 2008; van den Enden and Dixon, 2008; Casey et al.,

2015; Ramzan and Palmer, 2010; Menzies et al., 2011; Galloway and Easley, 2013). Ramzan
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and Palmer (2010) stated that fissures represent a ‘crack’ in the hard dental tissues due to their
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varying length and involvement of different hard dental tissues. It has been hypothesised that

fissures can play a role in the development of dental pathological changes (Dacre et al.,

2008a, 2008b; Simhofer et al., 2008; van den Enden and Dixon, 2008; Casey et al., 2015;

Ramzan and Palmer, 2010), but their clinical impact remains unknown.

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 A classification of fissure types was proposed in a previous study based on their

orientation and extent, with type 1 fissures involving the secondary dentine overlying pulp

horns. They are further subdivided in type 1a (T1a) that have a perpendicular orientation to

the surrounding enamel fold and type 1b (T1b) demonstrating a more mesial-distal path. Type

2 (T2) fissures never involve the secondary dentine (Pollaris et al., 2018). Previous research is

limited to the observation of fissures on the occlusal surface and information on the depth of

fissures is lacking.

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The objectives of this study were to determine the extent of occlusal cheek teeth

fissures using high-resolution X-ray computed tomography (µCT) imaging, to identify factors

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associated with the depth of fissures and to describe histological features of fissures.

Materials and methods

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Cheek teeth extracted without damaging the occlusal surface were collected from

clinical cases at Ghent University (2015 – 2017). In addition, healthy cheek teeth were
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extracted less than 12 h post mortem from a horse euthanased for reasons other than dental

disease. All teeth were examined macroscopically for the presence of fissures and the
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classification system previously described was used for categorisation (Pollaris et al., 2018).

The teeth were stored in 4% formaldehyde until further processing.

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High-resolution µCT imaging was performed on teeth that represented all fissure types

and fissure characteristics were evaluated. Each tooth was scanned individually in the custom-

built scanner system HECTOR (tube voltage: 150kV; tube current: 222µA; Masschaele et al.,

2013). Using a geometrical magnification of 4.92, a reconstructed voxel size of 40.3µm was

achieved. Per sample, 2001 projection images of 1000ms integration time were acquired over

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a full 360° rotation. The projection data was reconstructed to a 3D volume using the in-house

developed software package (Vlassenbroeck et al., 2007). A commercial 3D rendering

software package (VGStudioMAX and myVGL, Volume Graphics GmbH, Germany) was

used to measure different parameters including horizontal fissure length at the level of the

occlusal surface, fissure depth from the occlusal surface and the horizontal length of the

fissure at its most apical position. The minimal distance between the fissure and the pulp was

measured. Data were recorded on an excel spreadsheet and descriptive statistics were

generated to establish mean values.

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Histological examination was performed on a subset of teeth selected to roughly

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represent all fissure types. Specific zones for histological examination were selected from
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each tooth individually based on results from µCT imaging. Samples were decalcified using

an EDTA-based decalcifier, followed by routine processing and embedding in paraffin wax.

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Sections were cut 7 µm thick and a haematoxylin and eosin staining was performed. The
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decalcified sections were microscopically assessed for the presence of fissures.

Statistical analysis
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To determine factors associated with fissure depth, a multivariable mixed model was

constructed with ‘fissure depth’ (continuous) as the outcome variable and ‘fissure type’
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(1a,1b), ‘jaw’ (lower jaw, upper jaw), ‘tooth age’ (young, 0 – 14 years old; middle-aged, 15 –
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20 years old; geriatric, >20 years old), ‘tooth’ (Triadan position 07, 08, 09 or 10), ‘location’

(buccal, lingual) and ‘dental disease’ (periodontitis, apical infection, no dental disease) as

potential factors. Log transformation was required to reach a normal distribution for the

outcome variable. The results of the descriptive statistics are presented as the mean and

standard deviation (SD). Horse was added as a random effect to account for clustering of teeth

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in each of the models. The F-test was used to calculate test statistics. Satterthwaite’s degrees

of freedom method was used to obtain the P-values in each of the models.

In the first step, the univariable association of each factor with the outcome variable

was tested. Predictors with a P-value < 0.2 were withheld for the multivariable model. The

multivariable model was constructed using a backward elimination procedure gradually

excluding non-significant factors. Interactions between significant main effects were tested.

The relationships between the categorical predictors were explored by chi-squared tests. The

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assumptions of the multivariable mixed model were checked. Normality was confirmed using

a QQ-plot and the Shapiro Wilk Normality Test. The residuals were plotted versus the fitted

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values to assess linearity. Homoscedasticity and the absence of outliers were confirmed by the
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construction of residual plots. Model fit was evaluated using Akaike information criterion and

r2 values and by inspection of residuals. Statistical significance was set at P<0.05. Statistics
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were carried out using R V3.5.21.
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Results

Seventy-seven teeth from 54 horses were extracted for various reasons: periodontal
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disease (n=47), apical infection (n=22) and other (n=8). In 23/77 teeth a total of 41 fissures

were identified (27 T1a, seven T1b and seven T2). Twenty cheek teeth extracted from a horse
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euthanased for non-dental related problems showed fissures including 14 T1a, six T1b and 20
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T2 fissures.

µCT imaging

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See: The R Project for Statistical Computing. https://www.r-project.org (Accessed 10 December 2019).

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 Twenty-seven teeth from 15 horses were selected for µCT imaging (Table 1). Based

on macroscopic findings, 44 type 1 fissures were expected to be visualised. After µCT

imaging, 5 (T1b) macroscopically identified fissures were not identified as fissures. These

were interpreted as linear areas of secondary dentine. Four additional fissures, not

macroscopically visible, were identified on µCT imaging in contact with the occlusal surface

(3 T1a, 1 T1b).

A total of 43 type 1 fissures were observed (35 T1a, eight T1b) extending into primary

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dentin (Appendix: Supplementary material). The peripheral enamel was involved in 26 and

the cementum in eight T1a fissures. One T1b fissure involved the enamel ring and no T1b

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fissure involved the cementum. Fissures usually showed a linear configuration, but y-shaped
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fissures were observed. Fissures extended apically in the same plane as the occlusal surface,

although one spiralling fissure was observed. Superficial T2 fissures only involving the
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enamel ring were commonly visualised. Measurement results of T1 fissures are summarised
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in Table 2. Fifteen T1a fissures and one T1b fissure extended from the occlusal surface to a

level at or below the occlusal tip of the corresponding pulp (Fig. 1). In six (four T1a, two T1b)

fissures, the relationship to the pulp could not be determined (open pulp, pulp canal
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obliterated with dentin).

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The mean fissure depth in mandibular teeth was 15.43 mm (+/- 11.44 mm) and 7.23
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mm (+/- 4.50 mm) in maxillary teeth. The mean fissure depth on the buccal side of the teeth

was 15.17 mm (+/- 10.95 mm) and 6.00 mm (+/- 4.60 mm) on the lingual side. Of the

potential factors associated with fissure depth, only the location of the fissure (P < 0.001) and

tooth number (P = 0.002) were significantly associated with fissure depth in the final model

(Table 3). The expected fissure depth for a fissure on the buccal aspect of the tooth was 7.54

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mm (95% confidence intervals [CI] 3.22-19.26 mm) deeper as compared to the lingual side.

Fissures tended to be deeper in Triadan 09 (Fig. 2), with a significant difference compared to

Triadan 10 and Triadan 07, but given the low number of fissures in Triadian 07 observed in

this study population, caution must be taken with interpreting this result. The expected fissure

depth was 3.44 mm (95% CI 1.12-10.88 mm) deeper for an ‘09’ tooth compared to the ’10’

position (Table 3).

Histological findings

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Seven teeth were selected for histological examination. During sectioning of the teeth

it was observed that fissures on and close to the occlusal surface were dark stained. In the

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deeper sections, the fissures had a macroscopically whiter appearance (Fig. 3A-C).
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In the first specimen (tooth 3), a T1a fissure was identified both macroscopically and
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on µCT imaging at the level of pulp horn (PH)1 (depth 14.56 mm) and PH 2 (depth 18.95
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mm). Histological sections were made at both pulp horn locations 15mm subocclusally (SO).

At the level of PH1, a fissure was identified traversing through primary and secondary dentin

towards the pulp (in this section occluded with dentine). Microorganisms were identified
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around the tip of the fissure (in the secondary dentine) and further invading the secondary

dentine towards the pulp horn where reactionary dentin was deposited (Fig. 3). At the level of
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PH2 the fissure did not directly communicate with the vital pulp, however blue stained micro-
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organisms were identified invading the secondary dentine further towards the pulp and with

reactionary dentin present which could be followed to the periphery of the pulp (Fig. 4). The

pulp was vital although a lymphocytic infiltration was present.

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 The second specimen (tooth 7) showed a T1a fissure (depth 23.09 mm) at the level of

PH1. This fissure was identified up to 13 mm with the same characteristics as described for

the first specimen but disappeared 15mm SO. Furthermore, a T1b fissure was found 3mm SO

in a path between PH 1 and 4 with blue stained microorganisms present on both sides of the

fissure end, invading the dentine in a path towards the centre of the corresponding pulp horns.

This fissure was observed macroscopically but was not interpreted as a true fissure on µCT

imaging but rather as secondary dentin (Fig. 5).

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The third specimen (tooth 14) showed a T1b fissure (depth 7.28 mm) at the level of

PH1. There was a defect in the secondary dentine of PH1. The fissure was identified

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histologically 3mm SO in a path towards the patent pulp canal. The fissure did not directly
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communicate with the canal, but bacteria were observed from the fissure spreading into the

patent pulp canal. At a section 4mm SO the fissure was still observed, although at this section
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it was further distanced from the canal, a lesser amount of bacteria were present and irregular
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dentine was observed (closed canal). Another T1a fissure (PH4; depth 3.7 mm) traversed

primary and secondary dentine at 3mm SO in a path towards the pulp horn with bacteria

further invading the dentine towards the centre of the pulp horn. Two other fissures were
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found in this section in a mesial-distal direction in the secondary dentine overlying the pulp

horn. The three fissures did not appear to be directly connected but bacteria could be followed
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from one fissure to the other. One mm deeper, the T1a fissure was shorter and fewer bacteria
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were present.

The fourth specimen (tooth 15) macroscopically showed a T1b fissure connecting PH

1 and 2 but was not identified as a fissure on µCT imaging. Histologically 3mm SO, the

fissure was identified in the secondary dentine with bacteria present at both sides of the

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fissure. At 8mm SO, the fissure was only identified at the level of PH 2, whereas bacteria

were still present at the level of PH1. A second T1b fissure connecting PH 3 and 4 was

identified macroscopically, although not on µCT imaging. However, 8mm SO (PH 3) the

fissure was identified in the secondary dentine following a path towards PH 4. Bacteria were

present at each end of the fissure, invading the dentine until the centre of PH3.

The fifth specimen (tooth 19) had a T1a fissure at the level of PH3 (depth 3.25 mm)

and PH 4 (depth 9.68mm), and a T1b fissure (depth 16.5 mm) connecting PH 3 and 4 (Fig. 6).

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At a depth of 3 mm SO, all fissures were identified. Four mm SO, part of the T1b fissure was

identified at the level of PH3 including bacteria and reactionary dentine. At the level of PH4

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4mm SO, both T1a and T1b fissures were identified with bacteria and reactionary dentine

present at the end of the fissures. -p

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The sixth specimen (tooth 21) showed a T1a fissure at the level of PH 1 (depth 1.16
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mm). As expected, no fissure was identified 6mm SO. On the last specimen (tooth 23), a T1a

fissure was observed at the level of PH 1 (depth 20.63 mm) and PH 2 (15.34 mm). The PH 1

fissure was identified 2mm SO, reaching the centre of the pulp horn location. This fissure was
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visualised up to 18mm SO where it extended less far into the dentine (more distant from the

PH) and with less bacteria present. Similar findings were observed up to 18mm SO at PH 2
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level. At 11mm SO, bacteria were still present in a path from the fissure towards the vital pulp
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with at the end reactionary dentine present. At 18 mm SO, bacteria were no longer visible but

reactionary dentin was observed.

In total, 29 fissures were identified on microscopic examination (Appendix:

Supplementary material). Organic material (plant material, segments dentine) was present

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inside these fissures in 11 cases (37%) (Fig. 6B). Stained microorganisms were found in

28/29 fissures (97%) predominantly towards the pulp location and invading the dentinal

tubules (Fig. 3-6). In continuation of a fissure, reactionary dentine was visible at the distal

aspect of 20/29 (69%) fissure fractures (Fig. 3-6). The bulk of reactionary dentine was usually

present at the border between primary and secondary dentine, narrowing down in the direction

of the pulp horn.

Discussion

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High resolution µCT imaging provided detailed information and enabled precise

measurements of the extent of type 1 fissures in equine cheek teeth. Microscopic fissure

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location corresponded with µCT images indicating that they are true fissure fractures that can
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act as an entry portal for bacteria to invade the tooth and potentially contaminate the dentino-

pulp complex. The occlusal surface of the enamel folds contained localised fractures
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(superficial T2 fissures) which were commonly observed both macroscopically and on µCT
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imaging but were not further investigated since their impact on dental pathology was

considered very limited. Previously described by Kilic et al. (1997), they could be compared

to craze lines (traumatic cracks in the enamel) reported in human teeth (Berman and Rotstein,
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2016).
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Studies in human dentistry showed that the correlation between histology and µCT
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imaging is high, in accordance with the results of the present study with 23/24 fissures

confirmed on histology following µCT imaging (Jung et al., 2005). Five macroscopically

identified fissures (T1b) were not confirmed as fissures based on µCT imaging but were

interpreted as linear areas of secondary dentine. However, three of these were histologically

reconfirmed to be fissures. We suggest that it is difficult to visually identify a fissure within

10
secondary dentin on µCT images due to the highly similar grey scale (Fig. 5). Furthermore,

four fissures (three T1a, one T1b) were identified on µCT imaging whereas not

macroscopically observed. These were likely very recent fissures due to the absence of brown

staining. Wellman (2018) also reported on fissures that are not macroscopically visible.

Macroscopic equine cheek teeth fractures are predominantly found in the Triadan 09

teeth and on the buccal aspect of cheek teeth (Dacre et al., 2007; Dixon et al., 2000, 2007;

Taylor and Dixon, 2007). Results of the present study showed fissures on the buccal side of

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the teeth and fissures present in Triadan 09 teeth to be deeper, making it tempting to speculate

that fissures are an initial stage before tooth fracture. It has been observed that enamel was

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particularly thick at the buccal aspect of the upper and at the lingual aspect of the lower cheek
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teeth and it was suggested that these areas withstand the greatest masticatory pressure while

grinding food (Kilic et al., 1997). Dentine was found to be thicker on the lingual/palatal
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aspect, compared to the buccal aspects of pulp horns (Shaw et al., 2008). Since there are
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indications that there is an interactive mechanical supportive role for dentine (and cementum)

with respect to enamel (Shaw et al., 2008), it can be speculated that sites with thicker enamel

and less dentine are more prone for mechanical trauma. This further supports the hypothesis
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that certain fissure types in particular locations are more prone to progress into gross fractures

(e.g. slab fractures).

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Organic material could be found in superficial sections indicating its migration into the

defect in the tooth along a fissure from within the oral cavity. In the continuation of the

fissure fracture towards the pulp horn location, microorganisms within the dentinal tubules

were identified associated with reactionary dentine. In most specimens, the area of the

reactionary dentine became smaller and could be followed up to the centre of the former pulp

11
horn. Based on µCT images, fissure communication with the pulp was recorded in 4/16 cases.

However, this was not confirmed by microscopic examination in any of these cases. This is

attributed by the difficulty to create a histological section at the exact identified level of

contact between the fissure and the pulp. Wellmann (2018) did show direct communication

between a fissure and the pulp tissue on histological examination.

The present study demonstrated the close proximity of fissures to pulp tissue (Fig. 4).

In three sections, the deeper part of the fissure propagated to a level adjacent to vital pulp.

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Microorganisms and reactionary dentine were present as described above which illustrates the

response of vital odontoblasts to occlude dentinal tubules with mineralised material to arrest

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bacterial colonisation (Yu and Abbott, 2007). However, it shows that microorganisms
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colonise the tooth by migrating along fissures and dentinal tubules deeper towards the pulp

horn, which identifies fissures as possible routes for development of pulpitis. This supports a
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hypothesis that fissures observed oroscopically may not be innocuous in all cases and could
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act as a portal of entry of microorganisms to inoculate dental pulp. This is the most common

route of entry of microorganisms in human dental pulp with the consequent development of

pulpitis (Love and Jenkinson, 2002). Previous equine studies have shown that the SO
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secondary dentine can be as little as 2 mm in thickness (White and Dixon, 2010), indicating

that even fissures with a limited depth could compromise the pulp. The same study showed
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that the thickness of SO secondary dentine varies greatly between individual pulp horns, teeth
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and individual horses through which we assume that a tooth position or pulp location is not

less or more susceptible for bacterial invasion via a fissure. It is believed that a healthy pulp is

capable of responding to a certain level of bacterial invasion by producing reactionary dentin

that lacks patent tubules thus sealing further access to pulp tissue (Dacre et al. 2007;

12
Lundstrom and Wattle, 2016). Why the pulp defence system is overrun in certain teeth is still

very poorly understood.

Conclusions

The results of the present study suggest that fissures are real fracture lines most likely

induced by masticatory forces. The fissure fractures caused entry portals into the dentinal

tubules and may provide a direct or indirect (through the dentinal tubules) pathway for

microorganisms to invade the pulp system thus representing another etiological route for

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pulpitis.

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Conflict of interest statement
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None of the authors has any financial or personal relationships that could

inappropriately influence or bias the content of the paper.

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Acknowledgements

Preliminary results were presented as an Abstract at the European Veterinary Dental

Forum, Utrecht, 16-18 May 2019. This research was supported by Ghent University, Faculty
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of Veterinary Medicine and the Justus-Liebig-Universität Giessen, Institute of Veterinary

Anatomy, Histology and Embryology.

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Wellmann, K.C., 2018. Assessment of the potentiol role of fissure fractures in the aetiology of
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Equine cheek teeth apical infection. Proceedings of the European Veterinary Dental
Forum, Innsbruck, Austria, 31 May – 2 June 2018 pp. 145.
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White, C., Dixon., P.M., 2010. A study of the thickness of cheek teeth subocclusal secondary
dentine in horses of different ages. Equine Veterinary Journal 42, 119-23.

Yu, C., Abbott., P.V., 2007. An overview of the dental pulp: Its functions and responses to
injury. Australian Dental Journal 52, S4-16.

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Table 1.

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An overview of teeth and fissures identified by µCT imaging.

Diseased teeth Sound teeth

Tooth Triadan Tooth age (years) Pathology Fissure Type Tooth Triadan Tooth age (years) Fissure Type

pr
1a 1b 1a 1b

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1 309 14 Periodontitis 1 0 16 109 17 1 1
2 107 20 Periodontitis 1 0 17 110 16 0 1

Pr
3 409 2 Periodontitis 2 0 18 208 14 0 1

4 409 25 Periodontitis 1 0 19 209 17 2 1

5 309 20 Periodontitis 2 0 20 210 16 0 2

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6 409 16 Periodontitis 1 0 21 307 15 1 0

7 409 28 Periodontitis 2 0 22 308 14 2 0

8 307 15 PA/A PH/D 1 0 23 309 17 2 0

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9 408 12 PA/A PH/D 2 1 24 310 16 2 0

10 209 14 PA/A PH/D 1 0 25 408 14 2 0

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11 209 21 PA/A PH/D 1 0 26 409 17 1 0

12 308 25 PA/A PH/D 2 0 27 410 16 1 0

13 208 11 PA/A PH/D 1 0 Total 14 6

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 14 210 23 PA/A 3 1

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15 109 5 PA/A 0 0

Total 21 2

PA/A, Periapical abscess; PH/D, Pulp horn defect

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Table 2.

f
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Micro CT measurements of Type 1 fissures in 43 equine cheek teeth.

Fissure type Mean horizontal length at Mean horizontal length at Mean minimal distance Mean depth (mm)

pr
the occlusal surface (mm) the most apical aspect (mm) to the pulp (mm)

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Type 1a 3.47 +/- 1.60 2.09 +/- 0.88 4.64 +/- 4.41 mm 13.22 +/- 10.76
(range, 1.19 – 10.63) (range, 0.81 – 4.45) (range, 0 – 14.59) (range, 1.16 – 49.06)

Pr
Type 1b 13.64 +/- 7.40 3.18 +/- 1.66 7.46 +/- 3.58 mm 7.42 +/- 6.42
(range, 2.87 – 23.20) (range, 1.00 – 5.74) (range, 0.65 – 11.03) (range, 1.87 – 18.79)

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Table 3.

Final multivariable model of factors associated with fissure depth in 43 equine cheek teeth.

Variable Category n % Estimate SE 95% CI P

Location Lingual 14 32.6% -7.54 1.57 -19.26 – (-3.223) 0.0001
Buccal 29 67.4% Reference
Tooth Triadan 07 3 7.0% -46.11 2.560 -376.24 – (-5.773) 0.0003
Triadan 08 11 25.6% -3.08 1.807 -9.38 – (-1.009) 0.066
Triadan 09 20 46.5% Reference
Triadan 10 9 20.9% -3.44 1.815 -10.88 – (-1.121) 0.047

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SE, Standard error; CI, Confidence intervals

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Figure legends

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Fig. 1. A) µCT images of tooth 5 (309) at the occlusal surface. Two T1a fissures are visible

(pulp horn [PH]1 red arrow; PH2 green arrow). B) Transverse image 30 mm subocclusally

(SO). At the position of PH1 the canal is occluded with secondary dentine. At the level of

PH2, the fissure is present at the level of the corresponding pulp. C) Longitudinal section at

the level of the fissure at PH2. The fissure can be visualised (green arrowheads) until the

most apical aspect close to the pulp 49 mm SO (green arrow).

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Fig. 2. Boxplot illustrating the depth of the fissure according to tooth number.
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Fig. 3. T1a fissure at the level of PH 1 of tooth 23 (309) at the level of the occlusal surface

(A), 4 mm SO (B) and 15 mm SO (C). The fissure is stained dark in image A and B, whereas
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it has a whiter colour in image C (between red arrowheads). D) µCT image 4mm SO
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visualising the fissure (red arrowheads). E) Histological section (magnification 4x). The

fissure can be identified (between green arrowheads) with in continuation of the fissure blue
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stained microorganisms (blue arrowheads) invading the secondary dentine following a path

towards the centre of the pulp horn location. E) Histological section of an area corresponding
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to the red box in C (magnification 10x). The microorganisms (blue arrowheads) end where

reactionary dentine is present (orange arrowheads). p, primary dentin; s, secondary dentin.

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Fig. 4. Histological sections of tooth 3 (409), 15mm SO. A) Magnification 4x. The T1a

fissure is visualised in the proximity of vital pulp. The fissure extends in primary (p) and
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secondary (s) dentine. Microorganisms surround the fissure and follow a direction towards

the pulp. Reactionary dentin is present where the stained microorganisms or no longer
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visible. B) Section corresponding to the red box in A, magnification 10x. At the ‘end’ of the
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fissure, microorganisms are visible and colonise dentinal tubules in continuation of the

fissure towards the pulp. C) Section corresponding to the green box in A, Magnification 10x.

Tertiary dentine (orange arrow) is visible and can be followed to the vital pulp.

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Fig. 5. A) Photograph of the occlusal surface of tooth 7 (409). A T1a fissure is present at the
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level of PH1 (red arrowhead) and a T1b fissure is identified connecting PH1 and PH4 (green

arrowhead). B) µCT image 3mm SO with a T1a fissure (red arrowhead). Due to the smooth
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connection and similar grey scale, the macroscopically identified T1b fissure connecting PH1

and PH4 was identified as secondary dentin. C) Histological section 3mm SO corresponding
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to the area in the red box in B (magnification 4x). A T1b fissure is visible with

microorganisms surrounding the fissure and invading the dentinal tubules in the direction of
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PH1.

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Fig. 6. A) µCT image of tooth 19 (209) 4mm SO. Two T1b fissures are visualized (red and
-p
green arrowhead). B) Histological section 4mm SO, magnification 4x. A T1b fissure is

visible (top in the direction of PH 3, bottom toward PH4) containing plant material.
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Microorganisms are surrounding the fissure and on the top side sclerotic dentine is identified.
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