Cell Tissue Res (1998) 292:143±149
REGULAR ARTICLE
Nagako Yoshiba ´ Kunihiko Yoshiba ´ Daniel Aberdam
Guerrino Meneguzzi ´ Fabienne Perrin-Schmitt
Corinne Stoetzel ´ Jean Victor Ruch ´ HervØ Lesot
Expression and localization of laminin-5 subunits in the mouse incisor
Received: 26 August 1997 / Accepted: 9 October 1997
Abstract Laminin-5 is associated with several epithelial
tissues and forms part of the anchoring filaments of hemi-
desmosomes. Recent data have shown that the expression
of laminin-5 subunits is impaired in junctional epidermo-
lysis bullosa (JEB), and, in these patients, enamel hypo-
plasia is commonly observed. Rodent incisors are contin-
uously growing teeth with an asymmetry between their la-
bial and lingual sides. Enamel matrix formation is restrict-
ed to the labial side. We have analyzed the changes in the
expression and localization of laminin-5 subunits (a3, b3,
and g2) in lower incisors of the mouse. The apical loop
located at the end of the labial side contained stem cells
and showed expression for all laminin-5 subunits. In the
anterior direction, the inner dental epithelial cells (IDE)
transiently lost the immunoreactivity for all subunits,
whereas the transcripts for the b3 subunit remained in
the IDE. All subunit mRNAs and proteins were expressed
in ameloblasts facing predentine and also in secretory and
maturation stage ameloblasts. Enamel matrix contained
laminin-5. On the lingual side, the expression of lami-
This work was supported by INSERM and by the International
Human Frontier Science Program (grant RG-558/95 M). N. Yoshiba
was the recipient of a travelling fellowship from the Taro Yamashita
Foundation, and K. Yoshiba was supported by a research fellowship
from the Ministry of Education, Science, Sports, and Culture of
Japan.
N. Yoshiba ´ K. Yoshiba ´ J. V. Ruch ´ H. Lesot
INSERM U424, Institut de Biologie MØdicale, FacultØ de MØdecine,
11 rue Humann, F-67085 Strasbourg Cedex, France
D. Aberdam ´ G. Meneguzzi
INSERM U385, FacultØ de MØdecine,
Av. Valombrose, F-06107 Nice Cedex 2, France
F. Perrin-Schmitt ´ C. Stoetzel
LGME-CNRS, Institut de Chimie Biologique, FacultØ de MØdecine,
11 rue Humann, F-67085 Strasbourg Cedex, France
)
N. Yoshiba ( ) ´ K. Yoshiba
Department of Operative Dentistry and Endodontics,
Niigata University School of Dentistry,
5274, Gakkocho-dori 2-bancho, Niigata 951±8514, Japan
Tel.: +81±25±223±6161, ext. 4342; Fax: +81±25±222±2290;
e-mail: nagako@dent.niigata-u.ac.jp
 Springer-Verlag 1998
nin-5 subunits was continuous from the epithelial root
sheath to the epithelial rests of Malassez in the periodon-
tal ligament. These results suggest that spatial and tempo-
ral regulation of laminin-5 subunits correlates with the
histogenesis of the dental organ, ameloblast differentia-
tion, and enamel formation and also that laminin-5 plays
a role in the adhesion between dental epithelial cells and
the extracellular matrix (enamel or dentine) in areas
where the dental basement membrane is absent.
Key words Incisor ´ Basement membrane ´ Laminin-5 ´
Ameloblasts ´ Enamel proteins ´ Mouse
Introduction
Rodent incisors are continuously growing teeth with an
asymmetry between their labial and lingual sides. Dentine
is present on both sides, whereas enamel formation is re-
stricted to the labial side. The compartment located at the
labial-apical end is considered to consist of stem cells for
all the epithelial types of the continuously erupting incisor
(Smith and Warshawsky 1975, 1976). On the labial side,
histomorphogenesis leads to the differentiation of the in-
ner dental epithelium, the stratum intermedium, the stel-
late reticulum, and the outer dental epithelium. The cells
of the dental papilla that are in contact with the basement
membrane differentiate into predentine/dentine-secreting
odontoblasts. The cells of the inner dental epithelium dif-
ferentiate into functional ameloblasts, which secrete
enamel proteins. Gradients of odontoblast and ameloblast
cytodifferentiation are seen on the labial side of the tooth.
In contrast, histogenesis of the epithelium on the lingual
side leads to the formation of the epithelial root sheath
consisting only of inner and outer dental epithelium.
The inner epithelium of the root sheath induces dentine
formation, but lacks the ability to form enamel matrix
(Warshawsky and Smith 1974; Smith and Warshawsky
1975, 1976).
Enamel is the only ectodermally derived hard tissue.
Enamel development consists of three main stages: (1)
144
presecretory, (2) secretory, and (3) maturation (reviewed
in Warshawsky 1985). During the presecretory stage,
ameloblasts differentiate and develop the organelles nec-
essary for protein synthesis and secretion. During the se-
cretory stage, organic matrix is deposited and a partially
mineralized enamel matrix is formed until almost the en-
tire thickness of enamel has been formed. Finally, the ma-
trix undergoes a process of maturation, where the previ-
ously deposited organic matrix is lost and the enamel be-
comes fully mineralized.
Laminins are major components of basement mem-
branes and mediate cell adhesion, growth, migration,
and differentiation (Timpl and Brown 1994). At least
eleven different heterotrimeric laminin isoforms have
been described (Timpl 1996; Miner et al. 1997), and
structural diversity amongst the laminin family members
allows for specialization in function (Ryan et al. 1996).
Laminin-1 (a1b1g1) is expressed ubiquitously in base-
ment membranes, whereas laminin-5 (a3b3g2) is associ-
ated with several epithelial tissues with secretory or pro-
tective functions, such as skin and mucous membranes
(Carter et al. 1991; Rousselle et al. 1991; Rousselle and
Aumailley 1994; Jones et al. 1994). It forms part of the
anchoring filaments of hemidesmosomes (Rousselle et
al. 1991; Green and Jones 1996). Recent data have shown
that the expression of laminin-5 subunits is impaired in
junctional epidermolysis bullosa (JEB) (Aberdam et al.
1994b; Pulkkinen et al. 1994), which is characterized clin-
ically by wide-spread blistering with erosion of skin and
mucous membranes and ultrastructurally by disorganiza-
tion of hemidesmosomes. In these patients, enamel hypo-
plasia is commonly observed (Wright et al. 1993, 1996).
The purpose of the present study was to analyze the
changes in the expression and localization of the lami-
nin-5 subunits in mouse lower incisors by in situ hybrid-
ization and indirect immunofluorescence using riboprobes
and antibodies specific for all three subunits (a3, b3, and
g2) and to compare the expression of laminin-5 between
the labial and lingual sides of the tooth.
Materials and methods
Tissue preparation
All animals were handled according to the ªPrinciples of laboratory
animal careº of the National Institute of Health (publication no. 86±
23, revised 1985). Embryonic day (E) 19.5 and postnatal day (P) 8
mice were sacrificed by decapitation. Heads were dipped in 2-meth-
yl butane pre-cooled in dry ice. The E19.5 heads were cut in 8-mm
sagittal sections to observe the gradients of the inner dental epithelial
cells and odontoblast cytodifferentiation on the labial and lingual
sides. P8 heads were cut in 8-mm frontal sections to observe the more
advanced stages on both sides of the tooth. The sections were mount-
ed on gelatin-coated glass slides. On cryosections predentine, den-
tine, and enamel were identified by phase-contrast microscopy.
In situ hybridization
In order to synthesize cRNA probes, MN97 (1.5 kb), MN9.1E
(2.0 kb), and MN21 (3.0 kb) cDNAs corresponding to the mouse
a3, b3, and g2 laminin-5 subunits (Aberdam et al. 1994) were used.
The [a-35S]CTP-labeled sense and antisense riboprobes were pre-
pared by in vitro transcription and used at a final concentration of
50000 cpm/ml in the hybridization buffer. In situ hybridization
was performed as described by DØcimo et al. (1994). Briefly, the
sections were dipped in cold acetone for 3 min and fixed with 4%
paraformaldehyde in phosphate-buffered saline (PBS) for 15 min.
After washing in PBS, they were acetylated and then rinsed in
2”standard saline citrate (SSC). The tissue RNAs were denatured
by heating in 50% formamide, 1”SSC at 60C for 10 min. The slides
were then transferred to 50% and 70% cold ethanol (±20C) and fi-
nally dehydrated in absolute ethanol.
Hybridization was carried out at 52C for 16 h in hybridization
buffer and followed by high-stringency washes with 50% form-
amide, 1”salts solution, and 10 mM DTT and RNase A (20 mg/
ml) treatment. The slides were dipped in Kodak NTB-2 emulsion
and exposed for 10 days at 4C. After development the slides were
counterstained with Harris haematoxylin and examined under
bright-field and dark-field illumination.
Indirect immunofluorescence
The antibodies used in this study were affinity-purified rabbit poly-
clonal-antibody SE513 (diluted 1:300) specific for laminin a3, SEt-
dc3 (diluted 1:200) specific for laminin b3, and SE144 (diluted
1:200) specific for laminin g2 (Aberdam et al. 1994a). A rabbit
polyclonal antibody against laminin (EHS-laminin) (a kind gift from
Dr. D. Hartmann, Institut Pasteur, Lyon, France) (diluted 1:1000)
was used as a control to label the basement membrane. The cryosec-
tions were fixed in acetone for 10 min at 4C and washed in TRIS-
buffered saline (TBS). They were pre-incubated with 1% bovine se-
rum albumin (BSA) for 15 min and thereafter incubated with prima-
ry antibody for 1 h at room temperature. After being rinsed with
TBS for 5 min, the sections were incubated with Cy3-conjugated
goat anti-rabbit IgG (Jackson Immunoresearch, West Grove, Pa.,
USA) as a secondary antibody (diluted 1:600) for 50 min. The spec-
imens were mounted with PBS-buffered glycerol containing 0.1% p-
phenylenediamine and then examined using a fluorescent micro-
scope. As a negative control, the primary antibody was omitted.
Results
mRNA expression and immunolocalization
of laminin-5 subunits in developing incisors (E 19.5)
The labial side protrudes in a posterior direction in the
form of a bulbous structure, the apical loop. In contrast,
the lingual side consists of a thin epithelial sheath, the
epithelial root sheath (Fig. 1A).
Labial side
The outer dental epithelium contained mRNAs for all sub-
units of laminin-5 (Fig. 1B±D), and the corresponding
proteins were distributed along the entire outer basement
membrane (Fig. 1F±H). At the apical loop, signals for all
subunit mRNAs were observed. b3 and g2 subunits cov-
ered the entire loop area (Fig. 1C,D), while a3 was con-
fined to the outer region (Fig. 1B). The immunoreactivity
was particularly strong along the basement membrane of
the external part of the apical loop (Fig. 1F±H).
The inner dental epithelium demonstrated differential
expression patterns for the three laminin-5 subunits

Fig. 1A±H mRNA expression and immunolocalization of laminin-5
subunits in developing incisor (E19.5). A shows a bright-field image
of B. B±D On the labial side, expression of a3 mRNA (B) is absent
from the anterior part of the apical loop (al, large arrowhead) to the
preameloblasts (pa, arrowheads). A constant expression of b3
mRNA (C) is seen from the apical loop to the secretory ameloblasts
(am). Expression of g2 mRNA (D) is restricted to the apical loop and
disappears in an anterior direction (arrowheads). Re-expression of
a3 (B) and g2 (D) subunits is observed before the secretory stage
of amelogenesis (arrow). On the lingual side, the epithelium of
the root sheath (rs) continues to express mRNA for all laminin-5
subunits. E On the labial side, basement membrane stained with an-
ti-EHS-laminin antiserum disappears from under the ameloblasts
with the onset of dentine mineralization (arrow). On the lingual side,
an immunopositive line along the inner dental epithelium becomes
indistinct in the area of dentine deposition (double arrows). F±H
Immunofluorescence staining for a3 (F), b3 (G), and g2 (H) sub-
units shows a similar distribution pattern. On the labial side, the
staining is found within the apical loop and along its basement mem-
brane, where the strongest staining is found along the outer area (ar-
rowhead). With advancing development, immunofluorescence dis-
appears from basement membrane facing the dental mesenchyme
(arrowheads). A faint staining is found in the region facing the pre-
dentine matrix and is strongest in the secretory ameloblasts. Along
the epithelial root sheath on the lingual side, the immunostaining
for all laminin-5 subunits is superimposed on that of EHS-laminin
until dentine formation. Lb Labial side, Li lingual side, af apical fo-
ramen, d dentine, dp dental papilla, od odontoblast, ode outer dental
epithelium, pa preameloblast; bar 100 mm
145
(Fig. 1B±D). b3 mRNA was seen continuously from the
apical loop to the secretory ameloblasts, which showed
a high level of signal (Fig. 1C). In contrast, transcripts
for the a3 and g2 subunits were confined to the apical
loop area (Fig. 1B,D). The expression of both subunits
disappeared temporarily in an anterior direction and were
then re-expressed in differentiating ameloblasts facing
predentine (Fig. 1B,D). The most prominent signals were
detected in secretory-stage ameloblasts (Fig. 1B,D). In
spite of differential mRNA expression in the inner dental
epithelium, the immunostaining showed a similar distri-
bution pattern within the apical loop area and along the
surrounding basement membrane (Fig. 1F±H). The stain-
ing then transiently disappeared from the basement mem-
brane in an anterior direction. However, differentiating
ameloblasts recurrently showed positive staining for all
subunits and strong staining was observed in the secretory
ameloblasts (Fig. 1F±H). In contrast, immunostaining for
EHS-laminin was continuously observed along the base-
ment membrane until it disappeared at the stage when
dentine mineralized (Fig. 1E).
146
Fig. 2A±F Immunolocalization of laminin-5 subunits at the more
advanced secretion and maturation stages of amelogenesis in the in-
cisor (P8). A Immunostaining for EHS-laminin is neither detectable
in secretory ameloblasts (am) nor over the enamel matrix (e). The
outer dental epithelium is positive for EHS-laminin (arrow). B In-
tense immunofluorescence for the b3 subunit is found within the se-
cretory ameloblasts and also over the enamel matrix and along the
dentino-enamel junction (arrowheads). The b3 subunit is present
along the outer dental epithelium (arrow). C,D D is higher magnifi-
cation of B, and C is the phase-contrast-microscopic image. The ar-
ea showing the strongest reaction in ameloblasts appears to corre-
spond to the Tomes processes. E An immunopositive line for
EHS-laminin (arrows) reappears along the apical surface of matura-
tion stage ameloblasts (am). F Intense immunofluorescence for the
a3 subunit is detected along the apical surface of the ameloblasts
(arrows). Immunostaining for the a3 subunit decreases in the matu-
ration stage enamel matrix (asterisk). d Dentine, od odontoblast; bar
in B 100 mm, in D 30 mm, in F 50 mm
Lingual side
The epithelial root sheath continued to express all laminin-
5 subunits (Fig. 1B±D). The corresponding immunostain-
ing (Fig. 1F±H) showed co-localization with EHS-laminin
(Fig. 1E) until dentine formation. In areas where dentine
had been deposited, the immunopositive line became in-
distinct (Fig. 1E±H). In a more anterior direction, cells
of the inner dental epithelium became positive for lami-
nin-5 subunits, but were negative for EHS-laminin. In con-
trast, the outer basement membrane was uniformly posi-
tive for EHS-laminin and laminin-5 subunits (Fig. 1E±H).
Immunolocalization of laminin-5 subunits
at the advanced secretion and maturation stages
of amelogenesis (P8)
Fluorescence for EHS-laminin was neither seen in the
secretory stage ameloblasts nor over the enamel matrix
(Fig. 2A). However, the ameloblasts showed intense im-
munostaining for all laminin-5 subunits (Fig. 2B indi-
cates b3 subunit). Immunofluorescence for laminin-5
subunits was also detected over the enamel matrix and
along the dentino-enamel junction (Fig. 2B). Examina-
tion with the phase-contrast microscope revealed that
the strongest immunostaining in ameloblasts appeared
to correspond to the Tomes processes (Fig. 2C,D). At
the maturation stage (Fig. 2E,F), an immunopositive line
for EHS-laminin reappeared along the apical surface of
the ameloblasts (Fig. 2E). Intense immunostaining for
laminin-5 subunits was also seen along the apical surface
of ameloblasts (Fig. 2F indicates a3 subunit). In con-
trast, the immunostaining for laminin-5 subunits de-
creased in the enamel matrix during the maturation stage
(Fig. 2F).
Immunolocalization of laminin-5 subunits on the lingual
side of the incisor (P8)
When predentine began to be mineralized, immunostain-
ing for the g2 subunit was detected along the inner dental
epithelium of the root sheath (Fig. 3C). Similar staining
was not detected for EHS-laminin (Fig. 3B). As dentine
formation advanced, the epithelial root sheath became
fenestrated and persisted as the epithelial rests of Malas-
sez within the periodontal ligament. At this stage, the epi-
thelial rests showed positive reaction for both EHS-lami-
nin (Fig. 3D) and laminin-5 g2 subunit (Fig. 3E). Similar
staining patterns were also observed for the a3 and b3
subunits (data not shown).

Fig. 3A±E Immunolocalization of laminin-5 subunits on the lingual
side of the incisor (P8). A indicates a similar area of B and C. A
Mineralized dentine (d) is formed along the inner dental epithelium
(ide) of the root sheath area. B Immunofluorescence for EHS-lami-
nin is absent along the inner dental epithelium (arrowheads) of the
root sheath, while the outer dental epithelium (arrow) reacts posi-
tively. C Immunostaining for the g2 subunit is detected both along
the outer dental epithelium and along the inner dental epithelium
(arrowheads) of the root sheath. D,E The epithelial rests of Malas-
sez (er) in the periodontal ligament (pl) are stained positively for
both EHS-laminin (D) and the g2 subunit (E, arrowheads) am Am-
eloblast, bv blood vessel, dp dental papilla, od odontoblast, ode outer
dental epithelium; bar 100 mm
Discussion
Tooth histo-morphogenesis and cytodifferentiation are
controlled by a series of reciprocal interactions between
the epithelial and mesenchymal tissues (for reviews, see
Slavkin et al. 1981; Ruch et al. 1984; Ruch 1985, 1987;
Thesleff et al. 1995). An asymmetry in morphology, as
well as in the expression pattern for laminin-5, was ob-
served between the lingual and the labial sides of the
tooth epithelium. The lingual inner dental epithelium con-
tinued to express genes for laminin-5, and the distribution
patterns of EHS-laminin and laminin-5 molecules along
the inner basement membrane were superimposed until
they disappeared during dentine-mineralization initiation.
Immediately after dentine formation, immunostaining for
the laminin-5 subunits was again detected along the inner
epithelium and was maintained in the epithelial rests of
Malassez in the periodontal ligament. The transient disap-
pearance of laminin-5 seemed to correlate with the ex-
pression of the matrix metalloproteinase (MMP)-2
(72 kDa type IV collagenase), which may be involved
in the degradation of the dental basement membrane
(Sahlberg et al. 1992). On its lingual side, the dental organ
consists of two closely connected layers, the inner and
outer dental epithelium, and lacks the stratum intermedi-
um, which has been suggested to play a role in the control
of ameloblast differentiation (Nakamura and Ozawa
1990; Nakamura et al. 1995). In a pathological situation,
however, the epithelium of the root sheath has been
shown to differentiate into ameloblasts with the formation
147
of epithelial islands consisting of multiple cell layers in
rat molars (Hamamoto et al. 1996). Further investigations
are now required: (1) to identify whether lingual cell in-
teractions control the expression of laminin-5 in the inner
dental epithelium, and (2) to define the possible role of
MMP-2 in laminin-5 degradation. This collagenase has
been reported to be able to degrade laminin-5 g2 chains
(Giannelli et al. 1996). Our observations also highlight
the fact that, whether the basement membrane contains
laminin-5 (lingual side) or not (labial side), the differenti-
ation of odontoblasts occurs normally.
The apical loop demonstrated mRNA expression for
all laminin-5 subunits with slight differences in the local-
ization of their transcripts. This compartment is supposed
to correspond to the stem cells for all epithelial types of
the continuously erupting incisor (Smith and Warshawsky
1975, 1976). Significant levels of laminin-5 expression
have been found in epithelial cells of many embryonic tis-
sues (Kallunki et al. 1992; Aberdam et al. 1994a) and also
at the invasion front of cancer cells (Pyke et al. 1994). A
high level of expression for laminin-5 in the apical loop
may indicate the maintenance of embryonic characteris-
tics. The prominent immunostaining along the basement
membrane at the outer part of the apical loop, which cor-
responds to the multiple layer of outer dental epithelial
cells (Warshawsky and Smith 1974), could be associated
with stable anchoring to the surrounding tissue, suggest-
ing a supporting role in the continuous eruption in an an-
terior direction.
A transitory disappearance of a3 and g2 mRNAs and
immunoreactivity for all laminin-5 subunits was detected
in the labial inner dental epithelial cells in the prolifera-
tion zone. During mouse molar (Yoshiba et al. 1998)
and incisor (data not shown) development, the inner den-
tal epithelium showed a similar pattern of expression for
laminin-5 subunits from the cap to early bell stages. The
absence of laminin-5 has also been reported in the prolif-
erative cell compartment of intestinal epithelia (Leivo et
al. 1996; Orian-Rousseau et al. 1996). Tissue recombina-
tion experiments have suggested that the dental papilla
mesenchyme controls the unique expression pattern of
laminin-5 subunits in dental epithelium (Yoshiba et al.
1998).
148
Concomitant with the predentine/dentine formation by
odontoblasts and the elimination of the basement mem-
brane stained with EHS-laminin, laminin-5 genes were
re-expressed by ameloblasts and the corresponding pro-
teins were also secreted. To our knowledge, laminin-5
is the only basement membrane component underlying se-
cretory ameloblasts. Two major groups of proteins have
been identified in enamel: amelogenins and enamelins
(Termine et al. 1980). In addition, novel enamel-related
proteins, ameloblastin (Krebsbach et al. 1996), amelin
(Cerny et al. 1996), and sheathlin (Hu et al. 1997), have
recently been reported to be secreted by functional amelo-
blasts. Amelin has been implicated in binding the amelo-
blast and the enamel matrix by means of a DGEA se-
quence, recognized by the a2b1 integrin (Cerny et al.
1996). Laminin-5 is also recognized as an adhesion mol-
ecule interacting with a6b4 integrin and the association is
suggested to be involved in hemidesmosome assembly
(Rousselle et al. 1991). During enamel formation, a6b4
integrins are present at the apical pole of secretory amelo-
blasts (Meyer et al. 1995). Therefore, the binding of a6b4
integrin to laminin-5 may play a role in adhesion between
the ameloblasts and the enamel matrix.
In addition, laminin-5 appears to be an enamel matrix
component. The other enamel-related proteins, ameloge-
nin, enamelin, ameloblastin, and sheathlin, are supposed
to play important roles in enamel-matrix formation and
mineralization; however, their exact functions are not
well established (reviewed in Zeichner-David et al.
1995). An antisense inhibition experiment suggested that
the secreted amelogenins form a supramolecular aggre-
gate and control the size and orientation of enamel crys-
tals (Diekwisch et al. 1993). Secretory ameloblasts
showed the most intense immunostaining for all lami-
nin-5 subunits, particularly in the Tomes processes.
These cell processes are considered to control the growth
in length and width of enamel crystals (Warshawsky
1968). Although the significance of laminin-5 staining
over the enamel matrix is unclear, laminin-5 might play
a role in enamel-matrix formation and mineralization as
one of the enamel proteins. However since laminin-5 is
not an enamel-specific protein, it is more likely to play
a fundamental role in epithelial cell (ameloblasts)-ECM
(enamel matrix) adhesion, which may be involved in the
maintenance of cell phenotype and function (Adams and
Watt 1993; Gumbiner 1996). Previous reports on the mi-
croscopical appearance of developing teeth in JEB have
shown that the ameloblasts develop normally until the
first dentine is formed, however, soon thereafter, they be-
come vacuolated and regress into short cells (Brain and
Wigglesworth 1968; Gardner and Hudson 1975).
A basement membrane-like structure has been reported
to be re-formed at the interface between the enamel ma-
trix and ameloblasts during the maturation stage (Takano
1979; Nanci et al. 1987, 1993; Sawada et al. 1992). The
present study also demonstrated that the immunostaining
for EHS-laminin reappeared along the apical surface of
the ameloblasts at this stage, which confirms previous ob-
servations. This structure seems to be different from the
ªtypicalº basement membrane. So far, laminin was the
only major basement-membrane component detected
(Nanci et al. 1987, 1993; Sawada et al. 1992). Numerous
hemidesmosome-like structures have been recognized on
the apical cell surface during the maturation stage (Nanci
et al. 1993), and our data suggest that laminin-5 observed
along the apical surface may be involved in the formation
of hemidesmosomes at this stage. The organic matrix of
enamel is removed before final mineralization. Two
groups of proteinases have been implicated in this process
(Overall and Limeback 1988); one group is represented
by serine proteinases, which show amelogeninolytic ac-
tivity, and the other is MMPs. However, as these enzymes
have relatively weak activity against amelogenins, their
function in enamel matrix degradation is still unknown
(DenBesten et al. 1989). A loss of laminin-5 subunits
from the enamel matrix was recognized during the matu-
ration stage, which is suggestive that some types of the
MMPs may degrade the laminin-5 subunits.
Acknowledgements The authors wish to thank Dr. A.J. Smith (De-
partment of Oral Biology, School of Dentistry, University of Bir-
mingham, Birmingham, UK) for critically reading this manuscript.
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