Download collagen in odontogenic tumours

Medical Technology SA
Volume 26 No. 1 | June 2012
Peer reviewed ORIGINAL ARTICLE
COLLAGEN IN ODONTOGENIC TUMOURS: A HISTOCHEMICAL- AND
IMMUNOHISTOCHEMICAL STUDY OF 19 CASES
J Hangelbroek (NHD Med Tech), EJ Raubenheimer (MChD, PhD, DSc), R Vorster (NHD Med Tech), SP Ngwenya (MDent)
Unit 2: Pathology, School of Oral Health Sciences, Medunsa Campus, University of Limpopo, South Africa
Correspondence to: EJ Raubenheimer | tel: +27 12 521 4838 | fax: +27 12 521 4274 | email: [email protected]
Abstract
This study investigated the collagen content of odontogenic tumours. Wax blocks of 19 odontogenic tumours (ameloblastoma n=3,
adenomatoid odontogenic tumour n=3, ameloblastic fibroma n=3, ameloblastic fibro-odontoma and ameloblastic fibro-dentinoma
n=4, odontogenic myxoma n=3 and odontogenic fibroma n=3) were sectioned and stained by the H&E-, Picrosirius-, Reticulin-,
Masson Trichrome methods and the immunoperoxidase technique for collagen type IV. The distribution of collagen types I-IV was
recorded for each tumour. The basement membrane zones of ameloblastomas showed perpendicular oriented collagen type I fibres
which anastomosed with the stromal collagen. The collagen content correlated with the shapes of the epithelial components in
ameloblastomas, adenomatoid odontogenic tumours, ameloblastic fibromas and odontogenic fibromas. Formation of collagen was
found to be the first indication of stromal induction. In odontogenic myxomas, coarse type I collagen fibres were intersected at
obtuse angles by delicate type III fibres. Except for around blood vessels, no collagen type IV was found in odontogenic tumours.
The distribution of collagen is unique for each odontogenic tumour type.
Keywords
Collagen, odontogenic tumours, histochemistry, immunohistochemistry
INTRODUCTION
Odontogenic tumours arise from the epithelium- and/or ectomesenchyme from which teeth and their supporting structures originate. The epithelial component gives rise to dental
enamel and the ectomesenchyme to the remainder of the tooth,
periodontal ligament and alveolar bone. During embryogenesis
complex interactions between these tissues result in the orderly
formation of a tooth and its supporting structures. Pathological proliferations with varying levels of induction between the
remnants of these primordial tissues may lead to the development of complex tumours which differ in microscopic appearance and biological behaviour. The World Health Organization (WHO) classification, which has recently been refined,
categorizes odontogenic tumours into those of odontogenic
epithelial origin, odontogenic ectomesenchymal origin and a
third group comprised of a mixture of odontogenic epithelium
and odontogenic ectomesenchyme [1]. In several odontogenic
tumours the deposition of basement membrane material, dental
hard tissue (enamel, dentin and cementum), bone and fibrous
connective tissue occur and an accurate diagnosis depends
on identification of these tissue types. The organic component
of most of the mineralized odontogenic tissues is collagen. In
pathological proliferations, microscopic distinction between
cementum, bone and dentin may be difficult and distinction
often depends on the associations- and spatial arrangement of
these mineralized deposits.
Collagen is the most important structural component of human
connective tissue. At least 10 distinct collagen types have been
described [2]. Collagen types I, II and III consists of a helix of
3 coiled polypeptide chains and represent the most important
structural collagens. Collagen type I is typically found in the
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dermis of the skin and type II in bone and cartilage where their
most important function is to provide strength to the tissue. Collagen type III is found in reticulin fibres around blood vessels
and the viscera where their flexibility allows movement, expansion and contraction [2]. Collagen types I-III are deposited by fibroblasts and other cells of mesenchymal origin. Collagen type
IV is synthesized by cells of epithelial- or endothelial origin,
often as a component of basement membranes. Their reticular
distribution facilitates a supportive function [3].
Studies on collagen distribution and typing in odontogenic
tumours are infrequent in the literature. The purpose of this
study was to record the distribution of collagen in odontogenic
tumours of epithelial-, ectomesenchymal- and mixed epithelial and ectomesenchymal origin (keratocystic odontogenic
tumours were excluded from this study).
MATERIALS AND METHODS
Wax blocks of 19 cases representative of the epithelial-, ectomesenchymal- and mixed epithelial ectomesenchymal categories of odontogenic tumours were retrieved from the archives
of our laboratory (Table 1). Although odontogenic keratocysts
are classified as keratocystic odontogenic tumours in the recent
WHO classification [1], we did not include examples in this
study. Five- 3 micron sections were prepared and stained with
haematoxilin and eosin, Picrosirius-4, Gomori’s reticulin- and
the Masson Trichrome techniques and the Pasqual modification
of the immunoperoxidase technique for Collagen Type IV (Dako
monoclonal anti-human collagen IV M0785). All sections were
viewed by conventional light microscopy and the Picrosirius
stained sections by employing high intensity polarized light.
The distribution of the collagen types was recorded. Areas of
inflammation were not analyzed. Collagen type I is reported
Medical Technology SA
Volume 26 No. 1 | June 2012
to appear yellow and red with strong birefringence, collagen
type II red with weak birefringence and collagen type III, green
with weak birefringence in sections stained by the Picrosirius
technique and examined by high intensity polarized light [4].
Table 1: Tumour type and numbers examined.
TUMOUR TYPE
NO. OF
CASES
Odontogenic Tumours of Epithelial Origin
Ameloblastoma
3
Adenomatoid odontogenic tumour
3
Mixed Odontogenic Tumours
Ameloblastic fibroma
3
Ameloblastic fibro odontoma
2
Ameloblastic fibro dentinoma
2
Odontogenic tumours of Ectomesenchymal Origin
Odontogenic myxoma
3
Odontogenic fibroma
3
Reticulin stains are reported to be indiscriminately positive for
collagen types I, II and III [5].
RESULTS
In all odontogenic tumours the capsule consisted of Type I
collagen. No collagen Type IV was found as a product of any
tumour in the study, except for positive staining of the basement
membranes of blood vessels within and around the tumours.
As expected, collagen deposits in the ectomesenchymal- and
mixed categories of odontogenic tumours were more extensive
than in those of epithelial origin. In ameloblastomas, argyrophilic collagen type I fibres radiated from the basal area of
the ameloblasts perpendicular through the epithelial basement
membrane zone into the surrounding coarse stromal collagen.
Delicate type III fibres were present between the type I fibres
(Figures 1A and 1B). Areas of plexiform change in ameloblastomas showed dense collagen type I fibres between and linear
to the plexiform strands of ameloblastic epithelium. The shape
of the plexiform strands generally correlated with the direction
and volume of the collagen bundles in the connective tissue
stroma.
At the periphery of the solid epithelial nodules in adenomatoid
odontogenic tumours, foci showing accumulations of delicate
wavy collagen fibres correlated with basement membrane- like
deposits surrounding the anastomosing chords of epithelium
seen in reticulin stained sections (Figure 2). Calcifications had a
tendency of occurring in areas where these delicate fibres were
found. The central spaces of the rosette- and duct like structures
in the solid epithelial nodules contained varying densities of
entangled collagen type I fibres.
Figure 1A: Collagen distribution in the basement membrane zone of
ameloblastomas. Collagen type I fibres pass perpendicular through the
basement membrane zone (arrows). Note the delicate type III fibres between
the type I fibres (Picrosirius stain viewed with high intensity polarized light,
magnification X400).
In the ameloblastic fibromas, irregularly distributed volumes
of collagen were present around the epithelial follicles. In the
zones characterized by dense woven collagen, thick fibres
radiated into the ectomesenchymal stroma and anastomosed
with a more delicate fibre network around individual plump
mesenchymal cells. Further away from the epithelial follicle, fibres associated with less plump mesenchymal cells, followed a
course parallel to the layer of ameloblasts of the follicle (Figure
3A). The density of the fibres around the epithelial follicles was
generally found to impact on the direction of enlargement- and
shape of the epithelial follicle (Figure 3B).
Figure 1B: Delicate argyrophylic fibres passing from the basal region of the
ameloblastic epithelium (A) through the basement membrane zone (arrows)
into the connective tissue (C) (Reticulin stain, magnification X400).
Figure 2: Adenomatoid odontogenic tumour. Flattened epithelial chords
surrounded by abundant delicate fibres in the peripheral zones of the tumour
(arrows) (Reticulin stain, magnification X40).
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Volume 26 No. 1 | June 2012
Figure 3A: Ameloblastic fibroma. Note the epithelial follicle (E), dense array
of delicate fibres in the basement membrane zone (arrow), radiating coarse
fibers merging with the fibre network around plump ectomesenchymal cells
and parallel arrays of fibres in the peripheral connective tissue (CT) (Reticulin
stain, X200).
Figure 3B: Epithelial follicle surrounded eccentrically by dense radiating
collagen Type I fibres that restrict expansion in the associated part of the
follicle (black arrows). The white arrows indicate the direction of expansion of
the follicle towards a zone unrestricted by collagen (Picrosirius stain viewed
with high intensity polarized light, magnification X400).
Figure 4A: Ameloblastic fibro-odontoma. Homogenization of collagen adjacent
to the epithelial follicle (ameloblast indicated by black arrow) with ectomesenchymal cells incorporated in lacunae (bold white arrows). Note the linear
arrangement of plump ectomesenchymal cells on the surface of the deposit
(delicate white arrows) (Reticulin stain, X200).
Figure 4B: Epithelial basement membrane zone (between the white arrows),
mature dentin with a globular appearance (asterisk) and more recent
deposited dentin (black arrow). Note the odontoblastic tubules in the latter
(Masson trichrome stain, tissue decalcified, X200).
Figure 5: Odontogenic fibroma. Note the collagen bundles running parallel
with the orientation of the dormant epithelial rests (Masson Trichrome stain,
magnification X 200).
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The biopsy samples of the mixed epithelial and ectomesenchymal category with induction of dental hard tissue formation
(ameloblastic fibro-odontoma and ameloblastic fibro-dentinoma) were decalcified due to their mineralized content. The
earliest sign of induction of mineralized tissue was demonstrable by employing the reticulin staining method which showed
homogenization of fibres which enveloped plump ectomesenchymal cells in lacunae close to the epithelial follicle. In these
areas the mineralized tissue resembled bone. In other foci, a
linear arrangement of plump ectomesenchymal cells on the surface of the area where mineralized tissue formation occurred
was noted (Figure 4A). In areas of more advanced tubular dentin
formation, odontoblastic tubules were present. Tubular dentin
adjacent to the epithelial follicle stained red and the more recently deposited dentin stained blue by the Masson Trichrome
technique. The transition between the red and blue areas was
sharp (Figure 4B). The outlines of enamel rods (which remain
after demineralization) stain red with the Masson Trichrome
Medical Technology SA
Volume 26 No. 1 | June 2012
Figure 6A: Odontogenic myxoma. Thick type I collagen bundles exhibiting
birefringence intersecting at obtuse angles to the delicate type III collagen
which shows birefringence (arrows) (Picrosirius stain viewed with high
intensity polarized light, X100).
Figure 6B: Calcifications surrounded by radiating thick collagen bundles. The
core of the acellular calcification was torn from the block during sectioning.
(Masson Trichrome stain, magnification X200).
technique, black with Reticulin stains and were non birefringent when stained by the Picrosirius method and viewed with
high intensity polarized light.
bres blue. The argyrophilia of the delicate reticulin fibres makes
the Reticulin technique appropriate for their identification unlike the Masson Trichrome technique which stains the coarser
fibres blue and fails to demonstrate the delicate reticulin fibres.
Sections stained with the Picrosirius technique are viewed with
high intensity polarized light making differentiation between
the three most common collagen types (I, II and III) possible.
At least six tumours in each of the major categories of odontogenic tumours (epithelial, mixed and ectomesenchymal) were
included in the study.
The odontogenic tumours in the ectomesenchymal group
(odontogenic fibroma and odontogenic myxoma) differed in
the quality of the ectomesenchymal component. In odontogenic fibromas, mature collagen type I fibres (yellow with strong
birefringence) staining blue with the Masson Trichrome technique (Figure 5) and black and wavy with Reticulin stains, were
present. The fibres ran roughly parallel to the inactive epithelial
strands. Foci of mineralization were surrounded by concentric
thick type I collagen bundles. Collagen in the myxoid interstitial
tissue in odontogenic myxomas varied in quantity between the
cases examined. In general long, coarse and wavy collagen type
I fibres were oriented roughly parallel to each other and at obtuse angles to delicate and shorter collagen type III fibres (Figure
6A). Acellular calcifications in the stroma were surrounded by
thick radiating type I collagen fibres (Figure 6B). The dormant
odontogenic epithelial remnants failed to show a structured
basement membrane zone.
DISCUSSION
Reports on the collagen fibre content of odontogenic tumours
are infrequent in the literature. In a recent study on the collagen
in the connective tissue walls of odontogenic cysts, employing only the Picrosirius staining technique, it was demonstrated
that differences exist in the birefringent nature of collagen in
the walls of inflamed- (dentigerous- and radicular cysts) versus
non inflamed cysts (dentigerous- and odontogenic keratocysts)
[6]
. For this reason, areas of inflammation were not examined
in our study. In order to avoid the shift in colour during polarization when viewing sections of different thicknesses [7],
meticulous care was taken to prepare all sections at a thickness
of 3 microns. Our study exploited several routine histochemical methods and immuno-histochemistry for collagen type IV to
highlight- and type collagen in routine embedded wax blocks of
several odontogenic tumours. The techniques included Reticulin
stains which exploit the argyrophilic characteristic of collagen
and in particular the delicate reticulin fibers and the colourful
Masson Trichrome technique which stains course collagen fi-
The capsules of all odontogenic tumours contained mainly type
I collagen. Type IV collagen was not found in any tumour except for positivity in the basement membranes of blood vessels.
The collagen fibres in the basement membranes of ameloblasts
in ameloblastomas were found to be spatially organized with
thick type I collagen passing perpendicularly through the basement membrane zone and merging with the collagen in the
capsule and fibrous septa between the epithelial follicles. The
Picrosirius staining technique showed delicate fibres with the
staining characteristics for collagen type III between the coarse
type I fibres. The organized arrangement of the collagen in the
basement membranes of ameloblastomas may be helpful in
distinguishing ameloblastomas from other odontogenic lesions
with inactive epithelium. The lack of incorporation of plump
ectomesenchymal cells adjacent to the basement membrane
zone distinguishes ameloblastomas from ameloblastic fibromas
in which induction of ectomesenchymal cells are present. The
volume and direction of the collagen fibres deposited between
the ameloblastic follicles had a bearing on the shape of the epithelial follicle, with plexiform growth patterns exhibiting denser
collagen between the epithelium. Although the study did not
include a case of desmoplastic ameloblastoma, we propose that
extreme collagenization may be the reason for the flattened and
atrophic morphology of the chords of odontogenic epithelium
characteristic of this variant of ameloblastoma. Further research
is indicated in order to determine whether the structure of the
basement membrane zone of the desmoplastic ameloblastoma
follows the description of the ameloblastomas reported in this
study.
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Volume 26 No. 1 | June 2012
In a paper on the collagen fibre component of adenomatoid
odontogenic tumours, El Labban (1992) [8] describe a layer of
fine filaments running perpendicular to the epithelial basal
lamina, similar to that seen during induction of dentin formation. Stromal calcifications in these tumours mainly involve fine
fibrils resembling amyloid, an observation supported by our
study. The absence of an organized collagen fibre arrangement
(as in ameloblastomas) in the basement membrane zones close
to the epithelium in adenomatoid odontogenic tumours was
noteworthy. The shape of the narrow anastomosing epithelial
strands (which are reported to be present mainly at the periphery of adenomatoid odontogenic tumors [9]) was found to be the
result of accumulation of collagen which appeared to restrict
spatial enlargement of the epithelium.
teodentin” is recommended. Although structurally similar, the
term “cementum” should be reserved for mineralized deposits
on the surface of a tooth root only. In our study odontoblastic
tubules were apparent only in areas where formation of hard
tissue was at a more advanced stage. Linear arrangement of
plump ectomesenchymal cells on the surface of homogenized
collagen, as demonstrated in our study, may be the first indication of tubular dentin formation as the arrangement of the cells
mimics those of odontoblasts in the pulp of a tooth. The distinct
difference in the colour- and sharp transition between recently
formed- and more mature tubular dentin as demonstrated by
the Masson Trichrome staining technique cannot be explained
satisfactorily, except that it may reflect a chemical difference
between the two dentin types.
The fibres in the basement membrane zone of ameloblastic
fibromas resembled those described in ameloblastomas. Adjacent to the basement membrane zone further differentiation of
the ectomesenchyme was present. Collagen fibres surrounded
individual plump ectomesenchymal cells, mimicking the first
step in induction of mineralized tissue deposition. Due to the
formation of collagen and subsequent expansion of the extracellular space, the cells in this zone were plump and it appeared less cellular than in the surrounding connective tissue.
Collagen in the surrounding connective tissue was oriented in
parallel bundles and the ectomesenchymal cells were inactive- appearing and compressed in greater density. Early signs
of mineralized tissue induction, as described in ameloblastic
fibro-odontomas, were not present. The role of collagen in determining the shape of the epithelial follicles in ameloblastic
fibromas was noteworthy. Growth and enlargement of the epithelial follicles were generally found to be restricted in areas of
dense collagen deposits, leading to enlargement of the epithelial follicle along ectomesenchymal planes of lesser resistance.
The situation is analogous to the inflation of a balloon (epithelial follicle) in a closed hand, where the balloon will enlarge
through the spaces between the fingers. This study suggests that
although differences in the mitotic rate of the epithelium may
play a role as a determinant of the shape of the follicle, the
varying density of surrounding collagen undoubtedly determine
the planes of enlargement of the follicle. This study is the first
to propose that follicular, plexiform or multi-lobular shapes of
the odontogenic epithelial follicles in odontogenic tumours are
influenced by the density and spatial arrangement of collagen
in the ectomesenchymal tissue around the follicles.
Odontogenic fibromas are described microscopically as proliferations of varying cellularity with dispersed delicate collagen
fibers in a fibromyxoid background and scattered groups of
inactive appearing strands of odontogenic epithelium. In our
study the epithelium lacked the delicate arrangement of fibres
described for ameloblastomas and this feature may be helpful
in distinguishing these two tumour types. Metaplastic dysplastic
dentin, cementum or osteoid may be present [1]. The thick collagen fibres were suggested to distinguish central odontogenic
fibromas from the connective tissue of dilated tooth follicles [11].
In our study odontogenic fibromas showed thick and wavy type
I collagen bundles which were oriented roughly parallel to the
inactive odontogenic epithelial strands. The epithelial basement
membrane zones lacked the organized spatial arrangement of
collagen observed in ameloblastomas. Foci of mineralization
were surrounded by concentric collagen bundles in an unstructured way. These mineralized deposits, unlike those in odontogenic myxomas, could therefore be regarded as dystrophic
in nature.
With the aid of ultrastructural investigations, Josephsen, Larsson
and Fejerskov (1980) [10] demonstrated a rim of finely filamentous meshwork separating the epithelium from the connective
tissue in ameloblastic fibro-odontomas. This meshwork was
demonstrated in this study to consist of two layers. The centrifugal fibres close to the ameloblasts showed the spatial arrangement described in the basement membrane zone of ameloblastomas and ameloblastic fibromas. The second layer showed the
same morphology as in ameloblastic fibromas (plump ectomesenchymal cells surrounded by collagen), with foci of hard tissue
deposits. The earliest sign of dental hard tissue depositioning
was seen adjacent to the epithelial basement membrane zone
as an area of homogenization of fibers around plump ectomesenchymal cells. In situations where ectomesenchymal cells are
located within lacunae in a mineralized matrix and formation
of odontoblastic tubules cannot be demonstrated, the term “os-
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Odontogenic myxomas are characterized microscopically by
randomly arranged stellate, spindle shaped and round cells with
centrally placed nuclei with long pale cytoplasmic extensions
dispersed in abundant mucoid or myxoid stroma [1]. Only a few
collagen fibres are present and unlike odontogenic fibromas,
odontogenic epithelial remnants are scarce. In a study by Simes
(1975) [12] the stroma of odontogenic myxomas showed collagen bundles and smaller fibres similar to myxomas elsewhere
in the body. Our study showed a varying quantity of collagen
in odontogenic myxomas with thick type I collagen fibres that
run roughly parallel. Tumours with an increase in collagen are
generally referred to as fibromyxomas (or myxofibromas) [1].
There are no clear criteria reported in the literature for applying the latter terminologies and for this reason we recommend
the use thereof to be suspended. More delicate collagen type
III fibres intersect at obtuse angles to the main fibre type. This
feature may be helpful in distinguishing peripheral odontogenic
myxomas from oedematous irritation fibromas, a diagnostic pitfall which probably contributes to the low number of peripheral
odontogenic myxomas recorded in the literature [13, 14]. Unlike
the dystrophic calcifications identified in odontogenic fibromas,
the structured arrangement of the collagen around the acellular
calcifications in the stroma of odontogenic myxomas resembles
Sharpey’s fibres radiating from acellular dental cementum. As
cementum is defined by its location on the root surface of a
tooth, the term “cementoid” is suggested for these deposits.
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Volume 26 No. 1 | June 2012
Acknowledgement
The study was performed with the aid of a grant provided by the
Department of Education and Training in 2010.
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