Enamel mineralization in the absence of maturation stage ameloblasts.

Abstract

The role of maturation stage ameloblasts is not clear yet. The aim of this study was to verify to which extent enamel mineralizes in the absence of these cells. Maturation stage ameloblasts and adjacent dental follicle cells from rat lower incisors were surgically removed and the limits of this removal were marked by notches made in the enamel. Histological analysis confirmed that the ameloblasts had been removed within the limits of the notches. The teeth erupted and when the notches appeared in the mouth, the enamel in the experimental teeth was hard but whitish compared to the yellowish colour of the contralateral incisors used as control. SEM images revealed similar enamel rod arrangement in both groups. Decreased mineral content was observed in some specimens by polarized light microscopy, and microhardness values were much lower in the experimental teeth. FTIR analysis showed that higher amounts of protein were found in most experimental teeth, compared with the control teeth. Enamel proteins could not be resolved on 15% SDS-PAGE gels, suggesting that most of them were below 5kDa. These results suggest that the enamel matured in the absence of ameloblasts has increased protein content and a much lower mineral content, suggesting that maturation stage ameloblasts are essential for proper enamel mineralization.

Full text

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Enamel mineralization in the absence of maturation
stage ameloblasts
Isabel Maria Portoa, Jose ´ Merzela, Frederico Barbosa de Sousab, Luciano Bachmannc,
Jaime Aparecido Curyd, Se ´rgio Roberto Peres Linea, Raquel Fernanda Gerlache,*
aDepartment of Morphology, Piracicaba Dental School, University of Campinas, Piracicaba, SP, Brazil
bDepartment of Morphology, Health Science Center, Federal University of Paraı ´ba, UFPB, Joa ˜o Pessoa, PB, Brazil
cDepartment of Physics and Mathematics, University of Sa ˜o Paulo, FFCLRP/USP, Ribeira ˜o Preto, SP, Brazil
dDepartment of Biochemistry, Piracicaba Dental School, University of Campinas, Piracicaba, SP, Brazil
eDepartment of Morphology, Stomatology and Physiology, Dental School of Ribeira ˜o Preto, University of Sa ˜o Paulo, FORP/USP,
Ribeira ˜o Preto, SP, Brazil
1. Introduction
Amelogenesis can be divided into at least three phases:
secretion, when most of the dental enamel proteins are
secreted; transition, when the ameloblasts lose most of their
secretory organelles, shorten and lose their Tomes processes,
and a capillary invasion occurs into the enamel organ;1and
maturation, when the enamel loses water and organic
material, which is replaced by crystal growth,1becoming
the most highly mineralized tissue in the body.2
Maturation stage ameloblasts cover the enamel matrix in
the maturation stage; however, the role played by these cells
in the maturation process is not fully understood.3Amongst
the roles proposed for these cells, we will highlight two
archives of oral biology 54 (2009) 313–321
a r t i c l e i n f o
Article history:
Accepted 17 January 2009
Keywords:
Mineralization
Enamel
Maturation stage ameloblasts
a b s t r a c t
The role of maturation stage ameloblasts is not clear yet. The aim of this study was to verify
to which extent enamel mineralizes in the absence of these cells. Maturation stage amelo-
blasts and adjacent dental follicle cells from rat lower incisors were surgically removed and
thelimitsof this removal weremarkedby notches made inthe enamel. Histological analysis
confirmed that the ameloblasts had been removed within the limits of the notches. The
teetheruptedand when thenotches appearedinthe mouth, theenamel intheexperimental
teeth was hard but whitish compared to the yellowish colour of the contralateral incisors
used as control. SEM images revealed similar enamel rod arrangement in both groups.
Decreased mineral content was observed in some specimens by polarized light microscopy,
and microhardness values were much lower in the experimental teeth. FTIR analysis
showed that higher amounts of protein were found in most experimental teeth, compared
with the control teeth. Enamel proteins could not be resolved on 15% SDS-PAGE gels,
suggesting that most of them were below 5 kDa. These results suggest that the enamel
matured in the absence of ameloblasts has increased protein content and a much lower
mineral content, suggesting that maturation stage ameloblasts are essential for proper
enamel mineralization.
# 2009 Elsevier Ltd. All rights reserved.
* Correspondingauthorat:DepartamentodeMorfologia,EstomatologiaeFisiologia,FaculdadedeOdontologiadeRibeira ˜oPreto,FORP/USP,
Avenida do Cafe ´, S/N, CEP: 14040-904 Ribeira ˜o Preto, SP, Brazil. Fax: +55 16 3633 0999.
E-mail address: rfgerlach@forp.usp.br (R.F. Gerlach).
0003–9969/$ – see front matter # 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.archoralbio.2009.01.007
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/arob

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possible ones. The first is that maturation stage ameloblast is
essential for organic matrix removal. The second is that these
cells create the conditions for the enamel to achieve its very
high mineral content, and this role would be independent of
organic matrix removal.
Maturation stage ameloblasts undergo cyclic morphologi-
cal transitions between cells with ruffled and smooth apical
borders4–8and some studies have correlated these ameloblast
transitions with the loss of organic content.9,10Since the
advent of molecular cloning, the characterization of some
enamel proteins has demonstrated the presence of protei-
nases in the enamel, which are capable of degrading most of
theenamel structural proteins intosmallpolypeptide chains11
that would leave the enamel by a paracellular route.12Normal
proteolytic activity seems to be necessary for normal
amelogenesis, as supported by knockout model studies.
Altered enamel is formed in MMP-20 null mice.13,14Further-
more,thereis anamelogenesisimperfectafamilyin whichthe
defective enamel phenotype is linked to an amelogenin gene
defect15that possibly causes reduced hydrolysis of amelo-
genin by MMP-20.16,17A mutation in the MMP-20 gene results
in autosomal recessive hypomaturation amelogenesis imper-
fecta.18A mutation in the gene that encodes KLK-4 also leads
to a human enamel phenotype associated with amelogenesis
imperfecta.19All these evidences support the view that
enamel proteins are degraded by proteinases resident in the
matrix. Thus, proteolysis and organic matrix removal may be
an extracellular event independent of the presence of the
maturation stage ameloblasts. Enamelysin, also known as
MMP-20, is a specific enzyme secreted into the enamel matrix
duringthesecretorystage,anditwasshownthatitspecifically
cleaves amelogenin,20and amelogenins comprise 90% of the
total protein content of secretory stage dental enamel.3
Kallikrein 4 (KLK-4) is a protease secreted into the enamel
in the transition phase, being active in the maturation stage,
when its non-specific catalytic action is supposed to be
important for the final removal of proteins from the enamel
matrix.11
Regarding the second role proposed for maturation stage
ameloblasts, these cells might create adequate conditions for
mineral precipitation in the forming enamel or exert a direct
control over calcium influx into the enamel. Less is known
about this possible role, and no direct proof exists so far that
maturation stage ameloblasts are essential for the enamel to
achieveitsvery highmineralcontent. Enamelmineralcontent
(weight%) increases from around30% and 48% in the secretory
andearlymaturationstages,respectively,toaround95%atthe
end of maturation stage.3Calcium and phosphate concentra-
tions increase from 17% and 8%, respectively in the secretory
stage to about 37% and 17.5%, respectively at the end of the
maturation stage.21Phosphate taken up during maturation
stage is firmly trapped within the crystals and becomes
inaccessible for exchange in the surface.Therefore,the crystal
grows in width during this stage.22As the mineral content
increases, the enamel hardness also increases.21The cyclic
modifications between smooth- and ruffle-ended cells may be
linked to the calcium influx into the enamel. In ruffle-ended
ameloblasts, epithelial tight junctions are located near the
enamelsurfaceandcalciumissupposedtotakeatranscellular
route. In smooth-ended ameloblasts, there is a permeability
breach, allowing calcium to take a paracellular route.23,24
Ameloblasts start to express calcium storage and transport
proteins,25and they also double their mitochondrial content
during maturation.26Increases in the number of mitochondria
and in the membrane surface area are typical of epithelia that
carry out active ion transport.27Thus, there are biochemical
and morphological evidences suggesting that ruffle-ended
ameloblasts actively transport ions. However, the question
remains whether ruffle-ended ameloblasts transport ions
from the enamel or to the enamel. After
calcium phosphate was shown to be deposited directly into
the enamel matrix and no labelled calcium was detected in
ameloblasts.28The high-affinity calcium binding protein,
calbindin 28 kDa is extremely down-regulated during matura-
tion, contradicting the concept that calbindins might act as
transcellular calcium ferries during the maturation phase.29
Therefore, it is still uncertain whether the proteins described
to be part of the calcium transporting apparatus of maturation
ameloblasts are essential for the latter to actively transport
calcium into the enamel or whether they are an evolutionary
acquisition that allows these cells to cope with a calcium-rich
extracellular environment. In addition to active calcium
transport, maturation stage ameloblasts might create a
physical barrier that would create microenvironments ade-
quate for mineral precipitation in the enamel matrix. It is also
possible to speculate that such microenvironments do exist in
the forming enamel since tight junctions in adjacent amelo-
blasts are formed and recreated at least three times a day in
the rat incisor model.30,3In the light of these facts, a direct
proof that maturation stage ameloblasts are important for
proper enamel mineralization would contribute to a better
understanding of the enamel mineralization process.
We have developed a surgical procedure that removes
maturation stage ameloblasts and other enamel organ cells
from the continuously growing rat incisors. By means of such
procedure,theenamelformedintheabsenceofthesecellscan
be obtained and studied.
The aim of this study was to test whether the surgical
removal of maturation stage ameloblasts affects the protein
and/or the mineralization degree of the enamel in the
continuously growing rat incisor model.
45Ca injection,
2.Materials and methods
Male Wistar rats with an average weight of 250 g (240–260 g)
were used. Throughout this study, animals were kept under
controlled conditions of lighting (12-h light, 12-h dark) and
temperature (25–30 8C), with food and water ad libitum. The
experimental protocol was approved by the University’s
Ethical Committee for Animal Research (CEEA – IB – UNICAMP,
Protocol number: 556-1).
The left incisor was used as the experimental tooth from
which the maturation stage ameloblasts were surgically
removed. The surgical procedures were described by Merzel
et al.31Briefly, under anaesthesia with ketamine (90 mg/kg)
and xylasine (8 mg/kg), the lower border of the left hemi-
mandible was exposed. Using a small osteotome, 4 mm of
bone were removed starting from the insertion of the anterior
bellyofthedigastricmuscleuptothelevelofthemesialrootof
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thesecond lowermolar, thusexposingthelabial surfaceof the
incisor. The exposed area comprised the transition and the
initial maturation zones of the enamel organ.32The enamel
was scrubbed with a piece of gauze, to remove the enamel-
related periodontium (dental follicle) and the enamel organ.
An excavator was employed to remove the same tissues at the
lateral surfaces. A number 15 endodontic file was introduced
into the incisal part of the labial periodontal space until its tip
appeared through the gingival margin, in order to damage the
samesofttissuescoveringtheenamel-relatedtothediastema.
Two notches were made in the exposed enamel, to mark the
extension of the main lesion. To avoid the possibility of new
cells growing to repopulate the exposed enamel, the odonto-
genic area of the tooth was resected. An incision on the skin
following a plane between the angle of the mouth and the
external auditory meatus and a second incision of the anterior
half of the masseter muscle, above the facial nerve, exposed
the bluish bone elevation related to the odontogenic organ.
This bone was removed and the socket was thoroughly
curettedwith a dental excavator, to eliminatethe odontogenic
organ.
The right lower incisors were kept intact and used as
control. Both lower incisors were marked with notches at the
gingival margin, so that changes in eruption rates could be
followed.
2.1. Histology
Fiveratswerekilledat each ofthefollowing timeintervals: 1,7,
14 and 21 days after surgery and when the incisal notch of the
left incisor appeared in the oral cavity. The hemimandibles
weredissectedand,afterremovalofalltheexternalsofttissues,
theywereimmersedinto10%bufferedformaldehydepH7.4for
48 h. After being radiographed, the hemimandibles were
demineralized with 5% nitric acid in 10% formol. The radio-
graphs helped to localize the area of the incisor between the
notches, and guided the preparation of three 4 mm-long,
transversal blocks named: test, between the marks; incisal,
anterior to the incisal mark and related to diastema; and basal,
posteriortobasalmarkandrelatedtothesecretoryameloblasts.
Three blocksfromthe controlincisorweresectionedparallel to
thenotchesvisualizedintheexperimentalteeth.Thesegments
were embedded in paraplast and semiserial 5 mm-thick cross
sections were stained with haematoxylin and eosin.
2.2. Scanning electron microscopy (SEM)
Five rats were killed when the incisal notch was seen in the
oral cavity of the animals. The incisors were extracted and
fixed as described for histology. A thin line was painted at the
midsagittal plane of the incisors, on the labial face of the tooth,
so that the teeth could be cut and polished without missing
this plane. The incisors were cut using a hard tissue saw and
the sections were polished with sandpaper with decreasing
granulation, from 600 to 4000, using water. After cleaning the
sections in an ultrasound bath, they were etched with 37%
phosphoric acid for 30 s, dehydrated overnight at 37 8C, and
covered with a gold pellicle. The analysis of test region was
performed in a Jeol Scanning Electron Microscope, Model JSM-
5600LV (Tokyo-Japan).
2.3. Light microscopy analysis of mineralized enamel
Onemidsagittal section(100 mm-thick) from10rats killedwhen
the incisalnotch appearedinto theoral cavitywas preparedas
described above and mounted in water for analysis in bright
field (conventional), dark field and polarized light microscopy.
The sign of birefringence was determined with a Red I Plate
and the retardation colours were registered in photomicro-
graphs. The test, incisal and basal regions were analysed.
2.4. Microhardness testing
The lower incisors from 12 rats, killed when the incisal mark
appeared in the mouth, and another group of 10 rats, killed
when both notches were apparent, were used for enamel
cross-sectional microhardness testing, as described for rat
enamel.33The teeth, fixed as described above, were embedded
in acrylic resin with the mesial face positioned toward the
bottom of the block. Each block was ground as far as the
middle of the tooth and polished. Under a 10? eyepiece, the
blocks were oriented with the long axis of the indenter (Model
FMA-ARS, Future Tech Corp., Tokyo, Japan) parallel to the
dentine–enamel junction. Five indentations, in each of the
three named regions, were made on the outer 30 mm of the
enamel, 100 mm apart from each other, using a 25 g load and a
time of 5 s.
2.5. Fourier transform infrared (FTIR) spectroscopy
For FTIR analysis, enamel samples from the region between
the notches of five rats killed when the incisal notch appeared
in the mouth were powdered. The enamel powder was
separated from the dentine using a bromophorm/acetone
mixture34mixed with potassium bromide (KBr) and com-
pacted into a 13 mm-wide dry pellet. The pellets were
analysedinanFTIRSpectrometer(Nicolet380,ThermoNicolet,
USA) using the transmission mode. After the measurements,
the area under the absorption bands of the C–H bonds was
calculated using the Origin1software. For this procedure, we
subtracted the background and integrated the C–H bands with
the wavenumber ranging between 3000 and 2820 cm?1.
2.6. Protein analysis
Immature enamel was scraped from the test and basal
regions of experimental and control incisors of five animals
killedoneweekafterthesurgery.Theproteinswereextracted
from the enamel using the trichloroacetic acid (TCA)
method.35In short, samples were stored overnight at 0 8C
in TCA containing a cocktail of protease inhibitors, and they
were then centrifuged at 2500 ? g and 4 8C for 45 min. The
pellets were redissolved in 6 M urea. The amount of protein/
mg of enamel was determined by the Bradford method
(BioRad, Hercules, USA). For qualitative evaluation, 25 mg
protein from four control and four test samples were used for
separation by electrophoresis using the Laemmli discontin-
uous buffer system SDS-PAGE36carried out in 15% gels.
Samples were mixed with reducing sample buffer and heated
to100 8Cfor10 minpriortoelectrophoresis.Gelswerestained
with silver nitrate.37
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The protein profile of the mature enamel was obtained by
the same techniques described above, but using hard enamel
samples taken from the test and basal regions of the
experimental teeth of five animals killed when the incisal
notch reached the oral cavity. Corresponding samples of the
enamel from control animals were used. To obtain the
mineralized enamel samples for protein analysis, the labial
surface of the tooth was powdered, and the enamel/dentine
powder separation was performed using a bromophorm/
acetone mixture.34The enamel powder was then used for TCA
proteinextraction, as described above. The amount ofprotein/
mg enamel was determined by a fluorescence method, using
the NanoOrange protein quantification kit (Invitrogen, Mole-
cularProbes, Eugene,Oregon,USA). For qualitative evaluation,
0.3 mg proteinfromtwocontrolandtwoexperimental samples
wereusedforseparationbyelectrophoresisusingtheLaemmli
discontinuous buffer system SDS-PAGE36carried out in 15%
minigels. Samples were mixed with reducing sample buffer
and heated to 100 8C for 10 min prior to electrophoresis. Gels
were stained with silver nitrate.37Samples were kept on ice
during all the extraction procedures.Inhibitors of all classes of
proteinases were employed.
2.7. Statistical analysis
Comparison betweenexperimental and control data was done
by the t-test. The accepted level of significance was 5%.
3.Results
Experimental incisors erupted at a much slower rate than the
control ones, confirming previous observations.32When the
test area was visible (after the appearance of the basal mark),
the enamel was hard but whitish instead of being yellow as in
the control teeth, due to the absence of maturation amelo-
blasts which deposit iron on the enamel surface at the end of
the maturation.38
The enamel organ and the dental follicle were completely
removed from the test region as seen in the sections of rats
killed1dayaftersurgery(Fig.1A).Inthisregion,oneweekafter
surgery, the alveolar bone and the dental follicle were
regenerated, but not the enamel organ. The enamel was not
completely removed by acid and some remnants of the
enamel matrix were found (Fig. 1B). The same result was
observed at two (Fig. 1C) and three weeks (Fig. 1D) after
surgery. Control incisor sections showed normal maturation
stage ameloblasts covering the enamel space (Fig. 1F). Devel-
opment of a thin layer of cementum covering the enamel in
the test region was observed in some experimental incisors
(Fig. 1E). The incisal notch of the left incisors appeared in the
oral cavity in different times, ranging from 21 to 60 days after
surgery.
The orientation and thickness of the enamel rods were
similar in the test region from experimental and control
incisors, as revealed by SEM (Fig. 2A and B).
Bright field, dark field, and polarizing light microscopy
revealed heterogeneity in the mineralization pattern of the
test region in the analysed sections. Some sections (2/5) of the
experimental teeth were completely normal (Fig. 3J–L), being
similar to that of control incisors (Fig. 3A–C) with retardation
colours closeto thatofRedI Plate.Thisindicatesa lowpositive
birefringence. However, some sections (3/5) gave evidence of a
less mineralized enamel, seen as an opaque enamel by dark
field microscopy and a more positively birefringent enamel by
polarizingmicroscopy,identifiedbyhigherretardationcolours
(Fig. 3D–I).
A significant decrease (P < 0.001) in microhardness was
observed in all test regions of the experimental incisors
compared with the control incisors (Fig. 4).
Compared to the controls, the FTIR signals for C–H bonds
were higher in the test region of all the five experimental
incisors. However, fewer differences were observed as the
timetheincisorstooktoeruptincreased(Fig.5A).Anincreased
amount of protein in the enamel of the experimental incisors
was observed (Fig. 5B) (P = 0.03). C–H bonds are specific for
organic matter, being particularly high in aminoacids and
hydrocarbon chains. Since it is not expected to find hydro-
carbonsinamountshigherthan1.97 mglipids/100 genamel,39
most of the C–H signal probably originated from proteins.
One week after the surgery, no differences were observed
in the amount of protein/mg enamel matrix (100 mg protein/
mg enamel matrix in the test region of the experimental
incisor and 112 mg protein/mg enamel matrix in the control
incisor) or in the protein profile revealed in SDS-PAGE gels
when the experimental enamel from the test region was
compared with the corresponding enamel from control teeth.
In both groups, the majority of the proteins had molecular
masses ranging between 30 and 15 kDa (Fig. 6). When we
attempted to recover proteins from the mature enamel, much
less was recovered, as expected. Protein determination was
performed by a method that can determine 10 ng/mL of
protein and all our samples had protein levels within the
recommended curve. When we applied 0.3 mg of protein per
lane, however, no band profiles could be observed, suggesting
that most of the proteins were in the form of small
polypeptides that could not be resolved in a 15% gel (Fig. 7).
No bands were compatible with collagen I alpha chains,
indicating an effective separation of dentine and enamel
proteins. This was also confirmed by staining the powders
(enamel,dentineandwholeincisorcrownpowders)spreadonaglass
slide with Haematoxylin and Sirius Red (a good stain for
collagen).
4. Discussion
The results shown here are consistent with the hypothesis
that the maturation stage ameloblasts are essential for proper
enamel mineralization, probably controlling mineral influx or
creating physical conditions for the precipitation of hydro-
xyapatite in a very controlled way. The capillary invasion of
theenamelorganduringtransitionstageformingthepapillary
layer1would also facilitate the mineral influx into the enamel.
The enamel formed in the absence of maturation stage
ameloblasts and papillary layer displayed decreased micro-
hardness (2/3 of control enamel), and microhardness is used
as a measure of the enamel mineral content.40,41Though low
for the enamel, the microhardness values obtained for the
experimental enamel were higher than the microhardness
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values described for dentine (Knoop microhardness values
described for the human dentine are around 70),42indicating
that some mineralization did occur. We may speculate that at
thepointwhenameloblastsandotherenamelorgancellswere
removed, mineral weight% was probably around 50%,3and at
the point when the experimental enamel reached the mouth,
in most teeth, enamel had mineralized to more than 70%,
which equals dentine mineral content. Therefore, it is
interesting to note that the enamel matrix itself is able to
gain mineral, but in the absence of the enamel organ cells this
mineral gain does not reach the very high mineral content
found in the normal enamel (95%).
EarlierstudiesalsoshowedthattheuptakeofCa45waslower
in rodent teeth in which the enamel organ was wiped from the
labial surface and kept in in vitro conditions compared with the
control teeth during the maturation stage.43–45Recently, a
decrease in the enamel mineral content has been described in
rats with an autosomal-recessive mutation that causes defects
resemblingthoseofamelogenesisimperfecta.Intheseanimals,
maturation stage ameloblasts were detached from the enamel
matrix, forming a cyst between ameloblasts and the enamel
matrix.46However, noprior study has tested in vivothe surgical
removal of ameloblasts and its effects on the physical proper-
ties of enamel to date.
Fig. 1 – Cross sections of the test region. (A–E) Experimental teeth. (F) Control tooth. (A) One day after surgery the enamel
organ and the dental follicle were completely removed. (B) One week after surgery, the alveolar bone and the dental follicle
were regenerated but the enamel organ was not. (C) Two weeks after surgery. (D) Three weeks after surgery. In (B), (C), and
(D), there are remnants of the acid insoluble enamel matrix, indicated by vertical arrows. (E) Killed when the incisal notch
appeared in the oral cavity, 30 days after surgery: a thin cementum layer (horizontal arrow) was observed. (F) Section of the
right hemimandible at the same level of the left hemimandible from the rat killed 21 days after surgery, showing the
normal aspect of the enamel organ, the dental follicle and the completely acid-soluble enamel. AB – alveolar bone; EO –
enamel organ; ES – enamel space; D – dentine; DF – dental follicle; P – pulp. Haematoxylin and eosin.
archives of oral biology 54 (2009) 313–321
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Fig. 2 – Scanning electron microscopy views of midsagittal sections of control (A) and experimental (B) incisors, in the test
region from an animal killed when the incisal notch appeared in the oral cavity. Orientation and thickness of the enamel
rods are similar in both teeth.
Fig. 3 – Ground sections of teeth in the test region of animals killed when the incisal notch appeared in the oral cavity,
photographed under bright field (bf), dark field (df) and polarized light (pol) using the Red I Filter. Control: control teeth; exp:
experimental teeth. The initial portion of the test region is on the right of the arrows (D–F). The arrow heads (G–I) indicate
the basal notch, showing, on the left, the final portion of the test region. (J–L) Surgical region from another animal. These
series of photographs illustrate the variation in the mineralization of the enamel formed in the test area. While (D–F) show
less mineralized areas than the more basal enamel (G–I) of the same tooth; (J–L) show a normal aspect of the enamel in the
test area.
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The histological analysis confirmed that enamel miner-
alizationwasdelayedintheexperimental teeth,asseenby the
amount of enamel matrix still observed in these teeth
compared with control decalcified teeth at the same stage.
It is well established that acid soluble enamel has a very low
organic content (below 9.5%).47All experimental teeth also
showed decreased eruption rates throughout the study (not
shown), even after 7 days, when no signs of inflammation
were observed in the test region. Therefore, the delay in
eruption might have been due to the changes in the enamel
formed without maturation stage ameloblasts31and the
individual recovery of each rat from the surgery. It has been
shown that incisor eruption is delayed in rats exposed to lead,
and therefore a compensation mechanism might exist in
these teeth that increase the time for enamel mineralization
when amelogenesis is altered.48
Some studies showed that amelogenins control the
organization of the developing enamel crystallites.49–51The
self-assembly of amelogenin is very important in controlling
the oriented growth of apatite crystals.52Therefore, a logical
question was whether the large decrease in enamel micro-
hardness is associated with increased protein content.
In this study the enamel formed in the absence of
maturation stage ameloblasts displayed normal morphology,
and a slight increase in organic matter as observed by FTIR
analysis in some experimental teeth was detected. However,
we do not know whether lipids from cell membranes gained
access to the enamel. Interestingly, although FTIR data
suggested an increase in protein content, no increased protein
content was observed when the enamel matured in the
absenceofameloblastswasseparatedbyelectrophoresis.This
discrepancy suggests that the higher protein content may be
due to higher amounts of peptides or small proteins
(<5000 Da) that cannot be precipitated by TCA, and resolved
in an SDS-PAGE gel. Furthermore, analysis of each animal
shown in Fig. 5A suggests that the longer enamel remains
within the bone, the fewer C–H bonds are present. The one
animal with the slowest eruption rate (the incisal notch only
appeared in the oral cavity after 58 days) showed almost no
difference in the FTIR signals corresponding to C–H bonds in
the control and experimental enamel. This is a second
Fig. 4 – Mean (WS.D.) values of Knoop microhardness of the
enamel in the test region from rats killed when the incisal
notch (A) and the basal notch (B) appeared in the oral
cavity. *P < 0.001 for the indicated comparisons.
Fig. 5 – (A) FTIR C–H signal values of the enamel from the test region of the control teeth and the experimental teeth of five
animals killed when the incisal notch appeared in the oral cavity (39–58 days, indicated at the bottom of the columns). (B)
Mean (WS.D.) FTIR C–H signal values of the control and experimental enamel from the test region (n = 5). *P = 0.03.
Fig. 6 – Silver-stained 15% SDS-PAGE gel of enamel matrix
proteins from rats killed one week after surgery,
precipitated with TCA. Lane 1: molecular mass marker;
lanes 2 and 3: test region, experimental samples; lanes 4
and 5: test region, control samples; lanes 6 and 7: basal
region, experimental samples; lanes 8 and 9: basal region,
control samples. In both groups most of the proteins had
molecular masses between 30 and 15 kDa.
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evidence suggesting that the amount of time the enamel
remains within the bone may be important for the decrease in
the enamel organic content, and that this amount of organic
material (and not the amount of mineral) might somehow be
linked to the eruption rate in rodent teeth. A study showed
that ‘‘an increase in eruption rate increased the rate of
functional ameloblast production’’, another evidence that
eruption rate and ameloblasts are directly linked.53
In summary, we have presented a model in which
maturation stage ameloblasts and other enamel organ cells
were removed and the enamel formed without these cells
displayednormalmorphology of rods,as observed by SEM and
polarized light microscopy. Some changes in protein content
were observed, and a much lower mineral content character-
ized the enamel formed in the absence of maturation stage
ameloblasts (2/3 less than the normal enamel). These findings
strongly suggest that maturation stage ameloblasts are
essential for proper enamel mineralization, although enamel
mineralized to a certain extend in the absence of the enamel
organ.
Acknowledgements
We would like to acknowledge the help we received from the
staff of the Biochemistry Laboratory – FOP/UNICAMP. We are
grateful for the technical assistance of Mrs Maria Aparecida
Santiago Varella and Mrs Eliene Orsini Novaes Romani from
the Histology Laboratory–FOP/UNICAMP. This study was
supported by FAPESP and CNPq.
Funding: This study was supported by the State of Sao Paulo
Research Foundation (FAPESP) and the Brazilian Research
Council (CNPq).
Competing interests: None declared.
Ethical approval: This study’s protocol was approved by the
University of Campinas Ethical Committee for Animal
Research (CEEA – IB – UNICAMP, Protocol number: 556–1).
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