Study of developmental enamel defects of permanent

Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)
Study of developmental enamel defects of permanent teeth by atomic
force microscopy
E. Kaplova 1, K. Tomankova2, H. Kolarova 2, P. Krejci1.
1
Clinique of Dental Medicine, Faculty of Medicine and Dentistry, Palacky University, Palackeho 12, Olomouc, Czech
Republic
2
Department of Medical Biophysics, Faculty of Medicine and Dentistry, Institute of Translation Medicine, Palacky
University, Hnevotinska 3, Olomouc, Czech Republic
J. O. Turner was the first who described the developmental defect of permanent tooth caused by periapical inflammation
of temporary predecessor. This disorder is most common in the upper front teeth and premolars. These disorders are
collectively called developmental defects of enamel (DDE). They are visible deviations from the normal translucent
enamel resulting from enamel organ dysfunction. According to the macroscopic appearance we distinguish three types:
enamel hypoplasia, diffuse enamel opacity, demarcated enamel opacity. The combination of types of enamel defects found
in one tooth is also quite common. Results from studies at sheep and studies of macroscopic appearance, hardness values
and SEM examination of human enamel defects of permanent teeth support this division and demonstrate that the
pathogenesis of each type is different. DDE was imaged by atomic force microscopy Bruker Bioscope Catalyst using Peak
force Quantitative NanoMechanical measurement for imaging and mechanical characterization. Differences between
enamel of health part of tooth and DDE were studied.
Keywords Developmental defects of enamel; DDE index; Permanent teeth
1. Introduction
In 1906 J.O. Turner described a development disorder of permanent tooth caused by inflammation of periapical
temporary predecessor [16]. This disorder is most common by the upper anterior teeth and premolars because of the
close anatomical relationship between the roots of deciduous teeth and dental germs [17]. It manifests as discolorations,
opacities and enamel hypoplastic lesions on permanent teeth. The most common causes of inflammation of deciduous
teeth is Early Childhood Caries (ECC) and its complications. The relationship between caries in the deciduous dentition
and finding opacities and hypoplasia demonstrated Lo et al. in the study of Chinese children in nonfluoridated area [9].
As a result of high prevalence of Early Childhood Caries in the pediatric population we can also expect a higher
incidence of Developmental Defects of Enamel (DDE) at permanent teeth. It entails an aesthetic hendicap and
associated complications DDE such as failure of eruption of permanent teeth, calcifications in pulp cavity or more
frequent development of periodontitis of DDE suffering teeth [9, 1]. A DDE index is used to divide enamel defects into
one of three main types [15]:
(1)Hypoplasia, characterized by a reduced thickness of enamel. The absence of enamel may be partial or complete.
The extent vary, resulting in pit(s), groove(s), or larger areas of missing enamel.
(2)Demarcated opacities, have an abnormality in the translucency but no in the thickness of enamel. These lesions
have a clear boundary separating abnormal and normal enamel. Variations occur in their color, which ranges
from white to cream, yellow, and in degree of translucency.
(3)Diffuse opacities are alterations in translucency and have no change in enamel thickness. They do not have a
clearly defined margin. Fluoride-induced lesions are found in this type.
Dental enamel is the hardest substance of human body. The ultrastructure has been widely investigated, showing that
the inorganic components are distributed in the form of rods or prisms, which are composed of hydroxideapatite crystals
[6, 12].
The aim of this research is to analyze the differences between the surface of healthy enamel and DDE area with using
an atomic force microscope.
2. Material and Methods
2.1 Material and Instruments
In our experiments we used a human tooth with DDE-demarcated opacity, as a biological material. It was the permanent
incisor of 7 yars old girl, whitch was lost due to avulsion. In medical history of the patient was ECC with extraction of
deciduous incisors for periodontitis. On the labial area were two bordered creamy white opaque irregular opacities.
Measurements were carried out on AFM Bioscope Catalyst (Bruker USA) and with transmission microscope IX81
© 2012 FORMATEX
555
Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)
(Olympus, Japan), using silicon tip on nitride lever ScanAsyst Air tip (Bruker, USA). Tooth was put on the adhesive
tape.
Figure 1 The investigated tooth with two demarcated opacities. In our research we examined the bigger one in the middle.
2.2 Sample preparation
Freshly extracted permanent incisor was cleaned and stored in 10% formaldehyde solution in. It was cut into 3 mm
thick disc of hand grinding wheel. This disc was the whole labial part of tooth. It was not modified, only dried.
2.3 AFM imaging
The tooth was imaged with a scan rate of 0.1 Hz. We used ScanAsyst Air tip with a resonant frequency of 50 - 90 kHz
and a force constant of 0.4 N.m-1. AFM surface images were acquired in a Peak Force QNM imaging mode. All images
were processed by Nanoscope Analysis (Bruker, USA). After recording scan, the tip was placed on the identified place
of tooth (DDE, enamel) and force curves were measured in each point (pixel) of scan. The modulus of elaticity
(stiffness) was obtained by fitting the retract curve using the Derjaguin, Muller, Toropov (DMT) model [5].
3. Results and Discussion
Enamel ist the hardest substance of the body because of its high mineral content (92- 96%). However, it has relatively
low resistance to fracture, which is attenuated by the particular arrangement of the inorganic components distributed in
the form of rods or prisms [7]. The rods stand upright on the surface of the dentine and run throuhg the whole thickness
of the enamel layer [6]. In our research we investigated with using the AFM microscope the surface of healthy enamel
and enamel with DDE-demarcated opacity. We compared the modulus of elasticity and the altitude profile of healthy
and modified enamel. The first pair of images (Figure 2) shows a comparison of the surface of healthy enamel to the
surface of DDE in the magnification 100 × 100 µm. There is clearly shown the difference in structure of hard dental
tissue. The surface of DDE is rougher, wrinkled and irregular. The same situation but at higher magnification shows
following series of images (Figure 3 and 4).
Figure 2 The 3D topography images of enamel (left) and DDE (right) part of tooth in 100 × 100 µm scan. The scan was obtained by
Peak Force QNM imaging mode. Parameters of the pictures are: resolution 256 pixels scan rate 0.1 Hz. Height of enamel structure is
between 0 (dark fields) – 4.4 µm (light fields), height of DDE is 0 – 5.3 µm.
© 2012 FORMATEX
556
Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)
Graph 1 The line profile of enamel and DDE samples in line number 220.
Significant height differences of surface structures normal enamel and DDE can be seen on the graphs 1 – 3. In some
places inequality amounts to 2 µm of high. Surface structure corresponds with the morphology of mammalian enamel.
Mammals have in the most superficial region of enamel a very thin layer of apatite crystals oriented perpendicular to the
surface, so called aprismatic enamel [2, 4, 8].
Figure 3 The 3D topography images of enamel (left) and DDE (right) part of tooth in 10 × 10 µm scan. The scan was obtained by
Peak Force QNM imaging mode. Parameters of the pictures are: resolution 256 pixels, scan rate 0.1 Hz. Height of enamel structure is
between 0 (dark fields) – 300 nm (light fields), height of DDE is 0 – 1.7 µm.
Graph 2 The line profile of enamel and DDE samples in line number 215.
© 2012 FORMATEX
557
Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)
The organization of crystals is complicated, they are packed in very close contact and create clusters. The projections
of clusters on the surface in image Figure 4 look like aggregated globular particles of varying sizes. In our observations
of the shapes of particles in aprismatic enamel we find no geometrical symmetry. The smaller particles in images in
work of Farina et al. was in range 75nm and may represent cross sections of individual apatite crystals. Most of
crystalittes are closely attached to each other so particles in images present more than one enamel crystal [6]. Better
imaging of the structure of enamel prisms is obtained by cutting the DDE area parallel to the axial axis. We can study
the course of prisms from the dentinoenamel junction to the enamel surface. This we plan to do as a next part of our
research.
Figure 4 The 3D topography images of enamel (left) and DDE (right) part of tooth in 1 × 1 µm scan. The scan was
obtained by Peak Force QNM imaging mode. Parameters of the pictures are: resolution 256 pixels, scan rate 0.1 Hz.
Height of enamel structure is between 0 (dark fields) – 55 nm (light fields), height of DDE is 0 – 260 nm.
Graph 3The line profile of enamel and DDE samples in line number 135.
The DMT modulus (elasticity) is imaged in Figure 4. The reduced elastic modulus E* was obtained by fitting the
retract curve using the Derjaguin, Muller, Toropov (DMT) model [5]:
F=
4 *
E Rd 3 + Fadh ,(1)
3
where F is the force, R is the tip radius, d is the separation, Fadh is the adhesion force, and E* is the reduced elastic
modulus. By assuming infinite elastic modulus for the tip (Etip) and knowing the Poisson’s ratio of the sample νs, the
Young’s modulus for the sample (Es) can be calculated using following equations [10]:
2

 1 − ν s2 1 − ν tip
.
+
E* = 
 E

E
tip 
 s
(2)
DMT modulus determined the elasticity of enamel and ist modificaton. From the perspective of flexibility, the
enamel is compact structure without significant changes in elasticity of the structure (see Figure 4). Conversely, DDE
© 2012 FORMATEX
558
Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)
region of the tooth shows the changes in elasticity (stiffness) in the whole surfaces, namely in lower part of structures.
The brithest places of studied area determined the higher stiffness (lower elasticity) than dark places. Relative units of
elasticity Arb (Arbitary) suggest the DDE elasticity modulus to 85.5 Arb unlike enamel to 14.9 Arb. After calibration
by standard samples would be reached to absolut values of Young´s module in GPa. The greater magnifications of
sample, the higher stiffness was measured, as can be seen in Table 1. Another organic or anorganic materials reached
very low value of elasticity in comparison with enamel. For example thin glass surface can reached values up to 1.9
Arb. On the other hand, cell nucleus can acquire stiffness about 15 mArb.
Figure 5 The DMT modulus images of enamel (left) and DDE (right) part of tooth in 10 × 10 µm scan. The scan was
obtained by Peak Force QNM imaging mode. Parameters of the pictures are: resolution 256 pixels, scan rate 0.1 Hz. Dark
fields marks high elasticity (low stiffness), light fields marks low elasticity (high stiffness).
The enamel is an extracellular product of ectodermal cells ameloblasts with 3 distinguishing features- apatite
crystallites, organic matrix with collagen, few remnants of organic matrix after complete of mineralization.
Systemic or local disorders of ameloblasts during their active involvement can result in developmental defects of
enamel [14]. Although there are only a few types of enamel defects, Small and Murray [13] and Pindborg [11]
suggested more than 50 etiological factors of DDE in addition to fluoride, which can be divided into systemic and local
factors. Most studies have focused on enamel defects associated with systemic factors [9]. Our research deals with
disorders of dental hard tissues formed in connection with local irritation. In this case it was an inflamation resulting
from ECC development what mutilate the germin of permanent incisor. Nearly all the visible defects of the enamel of
human teeth can be classified according to macroscopic appearance into one of three types (DDE index) - hypoplasia,
diffuse and demarcated opacity [14, 3]. The combination of multiple types of enamel defects found in one tooth is also
quite common. Results from studies of macroscopic appearance, hardness values and SEM examination of human
enamel defects of permanent teeth support this division and demonstrate the different pathogenesis of each type [14].
Hardness values in Table 1 show that in our research is the DDE region much harder than healthy enamel. The work
of Suckling [15] conversely evaluates the defect Demarcated opacity as much softer. This discrepancy may be caused
by a completely different technique of hardness measure - Suckling determined hardness of DDE on the cut from the
surface to the dentino-enamel junction, our research focused on determining the hardness values from the surface layer
of enamel.
The atomic force microscopy was used to study enamel surface with aim to compare the pattern of particle
distribution in healthy enamel and in DDE. AFM gives high contrast, high-resolution images and is an important tool as
a source of structural information.
Table 1 Elasticity modulus of teeth surface in arbitary unit (Arb)
Tooth surface magnification
DDE 1x
DDE 10x
DDE 100x
Enamel 1x
Enamel 10x
Enamel 100x
© 2012 FORMATEX
Elasticity modulus DMT (Arb)
188.4
85.8
49.5
22
14.9
10.6
559
Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)
Acknowledgements. This work was supported by CZ.1.05/2.1.00/01.0030 and LF 2012/019. Thanks to Trystom company Olomouc
for processing of the biopsy sample.
References
[1] Bhushan BA, Garg S, Sharma,D, Jain M. Esthetic and endosurgical management of Turner´s hypoplasia, a sequlae of trauma to
developing tooth germ. J.Ind.Soc.ped.prev.dent. 26, 2008;7:121-124.
[2] Boyde A. Comparative histology of mammalian teeth,in Dahlberg, A. A. (Ed.), Dental Morphology and Evolution, Chicago
[3] Clarkson,J. Review of terminology, Classifications and Idices of developmental defects of enamel. Abstract.1989.
[4] Costa, LRRS, Watanabe I, Fava M. A study of prismless enamel in non-erupted human deciduous teeth. Braz. J. Morphol. Sci.
13,1996; 219–223.
[5] Derjaguin BV, Muller VM, Toropov YP. Effect of contact deformations on the adhesion of particles. Journal of Colloid and
Interface Science. 1975;53:314-326.
[6] Farina M, Schemmel A, Weissmuller G, Cruz R, Kachar B,Bisch PM. Atomic force microscopy study of tooth surfaces. Journal
of Structural Biology. 1999;125:39–49.
[7] Fawcet D in Fawcet DW (Ed.) A Text Book of Histology, Saunders,Philadelphia,PA,1989;602- 618.
[8] Kodaka, T, Kuroiwa and Higashi, S. Structural and distribution patterns of surface ‘‘prismless’’ enamel in human
permanent teeth. Caries Res. 1991;25:7–20.
[9] Lo ECM, Zheng,CG, King,NM. Relationship between the Presence of Demarcated Opacities and Hypoplasia in Permanent Teeth
and Caries in their primary Predecessors. Caries Res. 2003;37:456-461
[10] Morsi SM, Pakzad A, Amin A, Yassar RS, Heiden PA. Chemical and nanomechanical analysis of rice husk modified by
ATRP-grafted oligomer. Journal of Colloid and Interface Science. 2011;360:377-385.
[11] Pindborg JJ. Aetiology od developmental enamel defects not related to fluorosis. Int Dent J. 1982;32:123-134.
[12] Poggio C, Lombardini M, Colombo M, Bianchi S. Impact of two toothpastes on repairing enamel erosion produced by a soft
drink: An AFM in vitro study. Jurnal of dentistry. 2010;38:868-874.
[13] Small BW and Murray JJ. Enamel Opacities Prevalence, Classifications and Etiological Considerations. Dent. 1978;6:33-42.
[14] Suckling GW. Developmental Defects of Enamel - Historical and Present-Day Perspectives of Their Pathogenesis. Adv. Dent.
Res. 1989; 3(2):87-94.
[15] Suckling GW, Nelson DGA and Patel MJ. Macroscopic and scanning electron microscopic appearance and hardness values
of developmental defects in human permanent tooth enamel. Adv. Dent. Res. 1989; 3(2):219-233.
[16] Turner JG. Effects of abscess arising from temporary teeth. Br. J. Dent. Sci. 1906;49:562-564.
[17] Turner JG. Two cases of hypoplasia of enamel. Br. J.Dent. Sci. 1912;55:227-228.
© 2012 FORMATEX
560