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Ultrastructural Pathology
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Analysis of Collagen Distribution in Human Crown
Dentin by Confocal Laser Scanning Microscopy
Alberta Lucchese a; Giovanni Pietro Pilolli a; Massimo Petruzzi a; Vito Crincoli a;
Michele Scivetti a; Gianfranco Favia a
a
Department of Odontostomatology and Surgery, Faculty of Medicine, University of
Bari, Bari, Italy

Online Publication Date: 01 June 2008

To cite this Article: Lucchese, Alberta, Pilolli, Giovanni Pietro, Petruzzi, Massimo,
Crincoli, Vito, Scivetti, Michele and Favia, Gianfranco (2008) 'Analysis of Collagen
Distribution in Human Crown Dentin by Confocal Laser Scanning Microscopy',
Ultrastructural Pathology, 32:3, 107 — 111
To link to this article: DOI: 10.1080/01913120801897216
URL: http://dx.doi.org/10.1080/01913120801897216

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 Ultrastructural Pathology, 32:107–111, 2008
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ISSN: 0191-3123 print /1521-0758 online
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DOI: 10.1080/01913120801897216
Analysis of Collagen Distribution in Human
Crown Dentin by Confocal Laser Scanning
Microscopy
Alberta Lucchese, DMD, PhD,
Giovanni Pietro Pilolli, DMD,
Massimo Petruzzi, DMD, PhD,
Vito Crincoli, DMD,
Michele Scivetti, DMD, and
Gianfranco Favia, MD, DMD
Department of
Odontostomatology and
Surgery, Faculty of Medicine,
University of Bari, Bari, Italy
Received 20 November 2007;
Accepted 11 December 2007.
Address correspondence to Alberta
Lucchese, DMD, PhD, University of
Bari, Department of
Odontostomatology and Surgery,
P.zza G. Cesare 11, 70124 Bari, Italy.
E-mail: alucchese@hotmail.com
ABSTRACT The authors used confocal laser scanning microscope to
analyze human crown dentin. Specimens from 10 teeth were divided in two
groups, one of which was decalcified and stained with hematoxylin and
eosin. In the second group an undecalcified section was analyzed. Both
groups were scanned by confocal microscope to generate optically
sectioned images. All of the analyzed samples presented an intense auto-
fluorescent that was ascribed to collagens. The degree of autofluorescence
intensity was variable and might be due to collagen expression. The results
indicate that a confocal microscope may be of help in analyzing and
defining the nature and extent of collagen fibrils in human dentin.
KEYWORDS autofluorescence, collagen, confocal laser scanning microscope,
dentin, microhardness
Human teeth are composed mainly of dentin, a mineralized tissue that
constitutes the bulk of the tooth and it is covered by a thin hard layer of
enamel. While mature enamel is composed almost entirely of mineral
(495% by volume), dentin is formed by carbonated nanocrystalline apatite
mineral phase, organic matrix, and water (respectively 50, 30, and 20% per
volume) [1, 2]. Dentine is composed of oriented tubules that run from the
dentin–enamel junction to the pulp in the crown and from the cementum–
dentin junction to the pulp canal in the root. The tubules are surrounded by
a mineralized peritubular zone (peritubular dentine), embedded in an
intertubular organic matrix.
The most recently formed dentin is called predentin, a 10- to 40-mm-thick
layer of unmineralized matrix, which separates the odontoblast layer from
the mineralized dentine. The dentin that outlines the pulp chamber may be
referred to as circumpulpal dentin, while the outermost layer of the dentin,
just under the enamel, is a narrow zone called mantle dentin. It is a product
of the first mineralization reaction by newly differentiated odontoblasts, and
has slightly different composition than circumpulpal dentin [3, 4].
107
 The dentin organic matrix primarily consists of perpendicular to the tooth axis. The sections were
fibrous collagens and other dentine-specific non- adhered to positively charged slides.
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collagenous glycoproteins in smaller amounts [5].
The matrix provides a framework for mineralization.
Collagens comprise 90% of the dentin matrix, and
are principally type I [6, 7].
Dentin morphology and composition has been
studied for many years. It was first characterized
using optical microscopy, and later using electron
microscopy. In recent years atomic force microscopy
has been utilized to measure both its microstructural
and mechanical properties [8, 9]. Harmonic genera-
tion microscopy (HGM) was applied to investigate
three dimensions of the dentin microstructure [10].
Microcomputerized tomography (mCT) using an
X-ray source also produces 3D images of the tooth
hard tissues, but the spatial resolution does not
allow detection of objects smaller than 10 mm [11].
In such a context, we undertook a confocal laser
scanning microscope (CLSM) analysis of the organic
matrix in human dentin to further our understanding
of the nature of this peculiar tissue and the
correlation between its structure and function.
Histological examination was carried out using a
Nikon Eclipse E600 microscope (Nikon Corporation,
Tokyo, Japan), equipped with argon–ion and
helium–neon lasers, emitting at 488- and 543-nm
wavelengths, which allows optical and confocal
laser scanning microscopic analyses. The Nikon EZ
C1 software (Nikon Corporation, version 2.10, Coord
Automatisering) was used for image processing.
RESULTS
CLSM analysis shows a different fluorescence of
different tissues. Specifically, the technique consists
of illuminating the sample with a monochromatic
punctiform laser source. The resulting emission
energy is detected by a spatially filtered optical
system, the pinhole, which eliminates light signals
arising from out-of-focus planes.
When analyzed by CLSM, the samples of both
groups showed a strong natural fluorescence or
autofluorescence pattern that was not homogenous,

MATERIAL AND METHODS

This study included 10 intact teeth (5 third molars
and 5 premolars extracted for orthodontics pro-
blems) of patients referred to the Department of
Odontostomatology and Surgery at the University of
Bari, following informed consent and approval from
the Internal Ethical Committee. All of the patients
were in good health with a mean age  standard
deviation of 21.2  3.5 years. The 10 teeth were
divided into two groups. The first group (A) was
immersed in an appropriate fixing and decalcifying
solution (Mielodec, Bio Optica, Milan, Italy) for
90 min, rinsed in 70% ethyl alcohol for 30 min, and
then conventionally processed for histopathologic
paraffin embedding, thin sectioning at 5 mm per-
pendicular to the tooth axis, and staining with
hematoxylin–eosin. The second group (B) was
progressively dehydrated in ethanol, cleared with FIGURE 1 Confocal laser scanning microscopy of undecalci-
fied section exhibited variable degrees of autofluorescence
xylene, and embedded in methylmetacrylate. After
intensity, where higher scale values are related to higher fluor-
polymerization, the blocks were trimmed with an escence intensity. Different layers of dentin showed different
Isomet diamond saw (Buehler, Lake Bluff, IL, USA) fluorescence extent: (1) predentin and secondary dentin, an
excellent resolution; (2) circumpulpal dentin, well resolved, and
and subsequently thin-cut at 100 mm with a Polycut (3) mantle dentin a diffuse but weak fluorescence. Original
E microtome (Reichert-Jung, Heidelberg, Germany) magnification  40.

A. Lucchese et al. 108

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FIGURE 2 Histological evaluation of mantle dentin and dental enamel junction. (A) Undecalcified section of DEJ. (B) Confocal laser
scanning microscopy of the previous sample. DEJ appears as thin fluorescent bundles departing from the mantle dentin in the inner
portion of enamel. (C) Decalcified section showed the remnants of DEJ, which was completely destroyed by the decalcification process.
(D) Confocal laser scanning of the previous image: the decalcification process negatively affects the fluorescence of DEJ. Hematoxylin &
eosin, original magnification  200.

either quantitatively or qualitatively. In fact, Figure 1
exhibited variable degrees of autofluorescence
intensity, where higher scale values are related to
higher fluorescence intensity. Different layers of
dentin showed different fluorescence extent: (1)
predentin and secondary dentin, an excellent reso-
lution; (2) circumpulpal dentin, well resolved, and
(3) mantle dentin, a diffuse but weak fluorescence.
The autofluorescence variability was also qualitative.
As can be seen from Figure 2B–D, autofluorescence
had a characteristic pattern by which it progressed from
the bulk dentin toward the enamel–dentin junction
(DEJ), where thin fluorescent bundles completely
departed from the dentin surface were present.
The comparative analysis of Figure 2B (group B,
undecalc) and 2D (group A, decalcified) led to the
identification of these brightly autofluorescence as
organic matrix. In addition, the well-known intrinsic
autofluorescence of collagen [12, 13], which is the
main constituent of pulp tissue and dentin, further

109 Analysis of Dentin by Confocal Microscopy

 supports our conclusion that the strongly fluorescent
components are collagen.
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DISCUSSION
Confocal microscopy has found wide applicability
in the microscopy of different specimens and living
tissue because of its ability to obtain images from the
sample without interference of out-of-plane fluores-
cence. This type of microscope uses optical section-
ing by the laser light to visualize a tissue. One optical
image is obtained for each focal plane, giving a two-
dimensional image [14, 15].
Here we used CLSM as a support to already
available techniques for analyzing collagen dis-
tribution in human dentin. In the present analysis,
we have been able to observe high, clear auto-
fluorescence zones. Intrinsic autofluorescence of
dentin is well known as the correlation between the
autofluorescent signal and the microhardness of
dentine. We have inferred that such an intense
fluorescence is due to the collagen component of
the organic matrix, in agreement with (1) a number
of studies reporting high autofluorescence intensity
of collagen [12, 13], (2) the fluorescence pattern
resembling collagen distribution reported in the lit-
erature [16, 17], and (3) the finding that mineral loss
does not influence the autofluorescent behavior of
dentine [18]. Indeed, the comparative analysis of
confocal microscope images of groups A and B
(Figure 2B, D) led to identify strong autofluorescent
areas in both of them.
In this context, the different fluorescence extent
might be attribute to the different spatial orientation
and to the different percentage of organic matrix in
the dentin layers. In fact, the predentin and secondary
dentin, showing an excellent autofluorescence, are
less- or unmineralized and contain numerous col-
lagen fibrils. Moreover, some previous immunohis-
tochemical studies were able to demonstrate the
presence of type I and III collagens in the inner layer
of the dentin [16, 17]. The density of collagen in
dentine gradually decreases from the circumpulpal
dentine (well resolved by CLSM), which exhibits
thinner collagen fibrils toward the DEJ, which exhi-
bits a strong fluorescence. Collagen fibrils are orien-
ted perpendicularly to the tubules in the bulk dentin,
forming lumps of ordered collagen fibrils along the
A. Lucchese et al.
tubules. Collagen fibrils oriented along the tubules
long axis were found close to the DEJ (Figure 2C, D).
Indeed, the DEJ is less mineralized than both
enamel and dentin. On the other hand, the DEJ is
richer in collagen than either. It is related to the first
mineralized layer of dentin or mantle dentin [17]. In
undecalcified specimens, thin bundles of collagens
coming from the mantle dentin and extending in the
inner layer of enamel are evident (Figure 2A, B). The
decalcification process destroyed DEJ making harder
its resolution/detection. In decalcified images, thin
fluorescent bundles completely departed from the
dentin surface were evident, representing the DEJ
remnants after the decalcification (Figure 2C, D).
In addition, these data appear illustrative of the
correlation between the autofluorescent signal and
the microhardness of dentine, suggested by some
authors [19–21]. In this regard, the association
between dentin microhardness and mineral con-
centration is well known [1, 22]: Wang et al. [23]
demonstrated that the mantle dentin subjacent to the
enamel and the inner layer of dentin that surrounds
the pulp are softer than the underlying primary
dentin. Therefore, if higher natural fluorescence
corresponds to higher collagen composition and less
mineralization, the present study appears to indicate
that high degree of autofluorescence intensity is
related to low microhardness value.
To conclude, CLSM appears to be an optimum
auxiliary tool for investigating the presence and
distribution of collagens by fluorescence, so inviting
further experiments aimed to define our knowledge
about dentin, such as primary dentin development
versus secondary dentin, dentin bacterial infection,
and crack formation.
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Analysis of Dentin by Confocal Microscopy