The Localization of Matrix Metalloproteinase-20 (MMP-20,

Enamelysin) in Mature Human Teeth

1. M. Sulkala1

2. M. Larmas1,2
3. T. Sorsa3

4. T. Salo1,5,*

5. L. Tjderhane4,*

1.
2.
3.

Institute of Dentistry, University of Oulu, PO Box 5281, FIN-90014 University of Oulu, Finland;
Oulu University Hospital, Oulu, Finland;
3Oral Pathology Unit and Biomedicum Laboratory Diagnostics (HUCH), University of Helsinki, Helsinki, Finland;
and
4. 4Faculty of Dentistry, University of Toronto, Toronto, ON, Canada;
1
2

1. 5corresponding author, Tuula.Salo@oulu.fi;

Abstract
MMP-20 (enamelysin), the matrix metalloproteinase family member discovered in the enamel organ, has also been

detected in odontoblasts during dentin formation. We studied the presence and localization of MMP-20 in mature
human teeth in health and disease. In immunohistochemistry, MMP-20-positive staining was observed most

intensively in the radicular odontoblastic layer and also in dilated dentinal tubuli of caries lesions. By Western

blotting, MMP-20 was detected in odontoblasts and pulp tissue of both sound and carious teeth, in dentinal fluid
and dentin of sound teeth, but not in soft carious dentin. We conclude that MMP-20 produced during primary
dentinogenesis is incorporated into dentin and may be released during caries progression. The main cellular source
of MMP-20 in the dentin-pulp complex is the odontoblasts, which secrete MMP-20 into the dentinal fluid.

MMP-20

dentin

dentinal fluid

caries

INTRODUCTION
Matrix metalloproteinase20 (MMP-20, enamelysin) belongs to the family of zinc-dependent endopeptidases, which

can degrade almost all extracellular matrix proteins (Birkedal-Hansen et al., 1993). MMP-20 has been cloned from

porcine and bovine enamel organs (Bartlett et al., 1996; DenBesten et al., 1998) and mature human odontoblasts
(Llano et al., 1997). It degrades amelogenin, the major component of the enamel matrix, and thus may have an

important role during initial developmental stages of enamel (Llano et al., 1997; Fukae et al., 1998; Ryu et al.,

1999). MMP-20 has also been detected in some odontogenic tumors (Takata et al., 2000) and in human tongue

carcinoma cells (Vnnen et al., 2001). In odontoblasts, MMP-20 is first expressed in functional odontoblasts at the
onset of predentin secretion, continuing after dentin mineralization in fully mature odontoblasts in mouse (Bgue-

Kirn et al., 1998). However, the biological roles of MMP-20 in human odontoblasts during dentin development and
in mature teeth remain unclear.

In addition to MMP-20, odontoblasts synthesize other MMPs, at least gelatinases (MMP-2, -9) (Heikinheimo and
Salo, 1995; Tjderhane et al., 1998a), stromelysin-1 (MMP-3) (Hall et al., 1999), collagenase-2 (MMP-8) (Palosaari et

al., 2000), and membrane-type 1 MMP (MT1-MMP) (Caron et al., 1998; Palosaari et al., 2002). Their roles in the
dentin-pulp complex are not known, but they may participate in the organization of dentin organic matrix before
mineralization (Caron et al., 1998; Tjderhane et al., 1998a). Some MMPs are incorporated into dentin, since sound

human dentin contains latent mammalian collagenase (Dayan et al., 1983) and MMP-2 (Martin-De Las Heras et al.,

2000). In dentin caries progression, salivary- and/or pulp-derived host MMPs may have a role in organic dentin
matrix degradation (Tjderhane et al., 1998b; Sulkala et al., 2001).

MMP-2 expression is high in differentiating and secretory odontoblasts during dentin formation (Heikinheimo and

Salo, 1995; Caron et al., 2001), and MMP-2 has been found in sound dentin (Martin-De Las Heras et al., 2000).

Therefore, we set a hypothesis that because odontoblasts begin to synthesize MMP-20 at the onset of dentin
development, MMP-20 remains in mineralized dentin, and the protein could be detected in mature dentin. Since

mature odontoblasts synthesize MMP-20, we investigated whether MMP-20 is secreted into dentinal fluid of sound
teeth. To study the presence and localization of MMP-20 in mature teeth in states of both health and disease, we

examined soft carious dentin and odontoblasts and pulp tissue of sound and carious teeth by Western blotting and
immunohistochemistry.

MATERIALS & METHODS

Teeth used in the experiments were removed as part of normal treatment at the University Student Health Care
Center and the Department of Oral and Maxillofacial Surgery, University of Oulu. They were used for the
experiments with the patients informed consent, following the guidelines of the Faculty of Medicine at the

University of Oulu for the use of human samples in research. Teeth of young patients (20-27 yrs) were stored in

sterile phosphate-buffered saline (PBS) at room temperature (RT) and either prepared for further handling
immediately after extraction or stored at 70C for dentin protein extraction.

Dentin Protein Extraction

Sound human third molars (n = 4) were used for dentin protein extraction, which has previously been described in
detail (Martin-De Las Heras et al., 2000). Briefly, the dentin pieces were crushed, and the powder was first subjected

to protein extraction with 4 M guanidine HCl, 65 mM Tris-HCl (pH 7.4) (G1 extraction). Subsequently, dentin powder
was demineralized with 0.5 M EDTA (pH 7.4) (E1-E5 extractions), and after demineralization, guanidine-HCl
extraction was repeated (G2 extraction).

Collection of Dentinal Fluid

An occlusal cavity through enamel and 2-4 mm into dentin was made with a turbine drill under water-cooling in
extracted sound human third molars. The smear layer was removed by being etched with 35% phosphoric acid gel

(3M Scotchbond 1 Etchant, St. Paul, MN, USA) for 20 sec, and the cavity was carefully washed with PBS and thereafter
filled with PBS (20-40 L) for 20 min. After incubation at RT, PBS was collected and stored at 20C. For Western

blotting, the dentinal fluids of 68 teeth were collected, pooled, divided into three samples, and concentrated with
the use of concentration tubes (Millipore Corporation, Bedford, MA, USA).

Collection of Odontoblasts, Pulp Tissue, and Carious Dentin

For Western blotting, odontoblast-predentin layers of 1-6 teeth and pulp tissues of 1-2 teeth were dissected and
diluted into 30 L of 1x Laemmli buffer (Laemmli, 1970). Tissue samples obtained from sound (n = 8) and carious
(n = 12) teeth were included in the experiment. Soft, active carious dentin was scraped from the carious teeth (n =
5) with a dental excavator and diluted into 1x Laemmli buffer.

ECL-Western Blotting
The Laemmli-buffer-diluted samples were prepared at +60C for 20 min and then run on 12% sodium dodecyl

sulphate-polyacrylamide gels (SDS-PAGE). The total protein loaded was visualized with 0.5% Coomassie Brilliant Blue

staining. The proteins were transferred to ImmobilonTM P PVDF Transfer membrane (Millipore Corporation, Bedford,
MA, USA) in non-reducing conditions. We blocked non-specific binding by incubating the filter with TBS

supplemented with 5% non-fat dry milk (Difco Laboratories, Detroit, MI, USA) for 60 min. After being washed, the
filter was incubated overnight in RT with monoclonal antibody against human recombinant MMP-20 (1 g/mL) (a

kind gift from Dr. Kazushi Iwata; Fuji Chemical Industries Ltd., Toyama, Japan). Following washings, the filter was

incubated with biotinylated secondary antibody (1:1000; DAKO, A/S, Glostrup, Denmark) and with the avidin-biotin-

peroxidase complex (DAKO, A/S, Glostrup, Denmark) for 1 hr. For detection, the filter was developed by means of
an ECL Chemiluminescence Western Blotting detection kit (Amersham, Buckinghamshire, UK).

Immunohistochemistry
The staining of the sound (n = 4) and carious (n = 4) third molars was performed on 6-m-thick, formalin-fixed

demineralized paraffin-embedded tissue sections, with the use of a VECTASTAIN Elite ABC Kit PK 6101 (Vector

Laboratories Inc., Burlingame, CA, USA) and polyclonal antibody for MMP-20 (Sigma, St. Louis, MO, USA). After
deparaffinization, the tissue sections were incubated in 0.3% H2O2 in methanol for 3 hrs to quench the endogenous

peroxidase activity, pre-treated with 0.4% pepsin for 60 min in 37C, and washed with PBS in between. Non-specific
binding was blocked with normal goat serum (diluted 1:20 in 2% BSA/PBS) for 30 min. Thereafter, the sections were

incubated overnight with the primary antibody (diluted 1:2500 to 1:1000 in 2% BSA/PBS) in 4C in a humid chamber.
We determined specificity of the staining by replacing the primary antibody with buffer alone and with bufferdiluted normal rabbit serum (DAKO, A/S, Glostrup, Denmark). The following day, the sections were washed in PBS,

incubated consecutively with biotinylated secondary antibody solution (Anti-Rabbit IgG) and with ABC reagent for 60

min, and washed with PBS in between. The tissue sections were stained with 3-amino-9-ethylcarbazole (DAKO
Corp., Carpinteria, CA, USA), counterstained with Mayers hematoxylin, and mounted in Aquamount (Gurr BDH
Chemicals Ltd., Poole, UK).

RESULTS
MMP-20 Western Blotting of Dentin Proteins and Dentinal Fluid of Sound Teeth, Soft
Carious Dentin, and Odontoblasts and Pulp Tissue of Sound and Carious Teeth
In the fractions obtained by EDTA demineralization (E1-E5) and the second guanidine extraction (G2) of dentin
powder, an MMP-20-immunoreactive double-band of 46 and 43 kDa, corresponding to the calculated molecular

masses of the human MMP-20 active form, was detected (Fig. 1A). Also, higher- (100-110 kDa) and several lower-

molecular-weight bands (20-32 kDa) were observed (Fig. 1A). In the first guanidine extraction fraction (G1), MMP20 was not detected (Fig. 1A).

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Figure 1.
MMP-20 is detected in sound dentin, dentinal fluid, and odontoblasts and pulp tissue of sound and carious teeth.

Western blottings were performed with monoclonal MMP-20 antibody (1 g/mL). Molecular-weight standards (kDa)
are marked on the left. (A) MMP-20 in different dentin protein fractions of sound tooth. Each dentin sample

contained 60 g of protein according to direct spectrophotometric analysis. In the first guanidine extraction (lane 1),
MMP-20 immunoblotting was negative. In subsequent EDTA extractions (lane 2; first EDTA extraction; lane 3, the
last EDTA extraction) and the second guanidine extraction (lane 4), a double-band of 46 and 43 kDa (arrows)

corresponding to the active form of human MMP-20 (Llano et al., 1997) with higher- and lower-molecular-weight
forms of MMP-20 was detected. As a positive control for MMP-20 detection, we used conditioned odontoblast

culture medium (lane 5) as described previously (Vnnen et al., 2001), in which a 46-kDa active form of the MMP20 band was observed. (B) In dentinal fluid (lane 1) concentrated from 26 teeth, a band of 46 kDa (arrow) as well as
a 57-kDa band (arrow) corresponding to the proform of human MMP-20 (Llano et al., 1997) were detected. Also,

complexed and truncated forms of MMP-20 were detected. In the conditioned odontoblast cell culture medium (lane
2), a 46-kDa band was observed, as in Fig. 1A. (C) MMP-20 in odontoblasts and pulp tissue. Tissues of two teeth
were combined to yield odontoblast (lanes 1 and 3) and pulp tissue samples (lanes 2 and 4). The results of the

MMP-20 Western blotting were similar in tissues obtained from carious (lanes 1 and 2) and sound (lanes 3 and 4)

teeth. In odontoblasts, in addition to a double-band of 46 and 43 kDa (arrows), complexed forms as well as several
truncated forms of MMP-20 were observed. In odontoblasts, a distinct band of approximately 110 kDa was

observed, which was not usually seen in the pulp tissue samples. Otherwise, the bands observed in pulp tissue were
similar to the bands of odontoblasts.

In dentinal fluid, a faint band of 46 kDa was observed as well as higher- (100-110, 85 kDa) and lower-molecularweight species (20-35 kDa) of MMP-20 (Fig. 1B). In one of three concentrated samples, a faint 57-kDa band
corresponding to the proform of human MMP-20 was also detected (Fig. 1B).

The result of MMP-20 Western blotting of soft carious dentin was usually negative. One of the carious dentin

samples analyzed (n = 5) showed a faint 43-kDa band (not shown). In odontoblast and pulp tissue Western blots, an
immunoreactive double-band of 46 and 43 kDa as well as complexed and truncated forms of 100-110, 60-85, and
20-40 kDa were observed, in both the sound and carious teeth (Fig. 1C).

Immunohistochemical Staining of Sound and Carious Teeth

MMP-20 immunoreactivity was observed most clearly in odontoblasts of sound and carious teeth. In both cases, the
density of stained odontoblasts was typically higher toward the root dentin, whereas, more coronally, the

odontoblastic layer was stained more weakly (Figs. 2A-2C). At the cellular level, the stain was positive in the
odontoblast cell body, mostly on the pulpal side (Fig. 2D). Occasionally, predentin was also MMP-20-positive (Figs.

2B-2D). In all the carious teeth, some, but never all, dilated dentinal tubuli were positively stained (Fig. 2E). The
pulp showed MMP-20 immunoreactive staining in capillary endothelial cells (Fig. 2F). There was no staining in the
sections incubated with non-immune serum (Fig. 2G).

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Figure 2.

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Immunohistochemical staining of mature human teeth demonstrating MMP-20 in odontoblasts and dilated dentin

tubuli of caries lesions. Key: Ob = odontoblasts, P = pulp, Cd = crown dentin, Rd = root dentin, Pd = predentin. (A)
Staining of a sound tooth. Positive staining in the odontoblast cell layer was detected in 3/4 of sound and 4/4 of

carious teeth, with increasing intensity in the apical direction. The sound tooth not demonstrating positive staining

in the odontoblast cell layer required extensive demineralization, which may have affected its immunoreactivity. The
rectangles show the area of higher magnification in (B) and (C). (B) Higher magnification of the coronal dentin-pulp
border showing positive odontoblasts and weaker staining in the predentin area. Positive staining in the predentin
area was observed in 3/4 of sound and 2/4 of carious teeth. (C) Higher magnification from the root dentin area

showing higher density of stained odontoblasts and stronger staining in the predentin area as compared with that

observed in the coronal part of the pulp chamber (B). (D) Higher magnification from the area in (C). Positive staining
is observed in the cell bodies of the odontoblasts, mostly in their pulpal side. (E) In all the carious teeth, some

dilated dentinal tubuli were positively stained (arrows). (F) In the pulp of all sound and carious teeth capillaries,

endothelial cells showed positive staining. Also, few pulpal fibroblasts were MMP-20-positive in 3/4 of sound and
3/4 of carious teeth. (G) Negative control, with non-immune serum instead of MMP-20 antibody, showing no
staining.

DISCUSSION
In dentinogenesis, odontoblasts secrete dentin organic matrix, which, after organization in the predentin area, is
mineralized. The fact that the onset of MMP-20 expression in odontoblasts coincides with the early onset of
predentin synthesis (Bgue-Kirn et al., 1998), and analysis of our data on the presence of dentin-bound MMP-20

suggests that MMP-20 may be involved in organic matrix organization during dentin formation. In dentin matrix

organization, the substrates for MMP-20 remain unknown, but in vitro it degrades casein and gelatin (Fukae et al.,
1998; Ryu et al., 1999), and fibronectin, type IV collagen, tenascin-C, and laminins-1 and 5 (Vnnen et al.,
2001).

We demonstrate that at least some of the MMP-20 synthesized by odontoblasts is incorporated into mineralized

dentin. In contrast to previous findings with MMP-2 (Martin-De Las Heras et al., 2000), no MMP-20 was found in the

first extraction fraction (G1) containing non-mineralized dentin matrix proteins. On the other hand, MMP-2-related
gelatinolytic activity could not be found in the mineralized part of dentin (Martin-De Las Heras et al., 2000), but we

found MMP-20 in these fractions (EDTA extractions). Like MMP-2, MMP-20 could also be detected in demineralized
dentin organic matrix (G2 fraction). The presence of the enzyme in the hydroxyapatite-bound and collagenous-

network-bound protein fractions, the molecular weights suggesting the active form of the enzyme (Llano et al.,
1997), indicates the activity of MMP-20 in dentin matrix organization before and during mineralization in vivo. It

can also be concluded that demineralization or degradation of demineralized organic matrix is required for MMP-20
release from dentin, which would therefore occur, e.g., during dentin caries progression.

Indeed, in immunohistochemical staining, we observed MMP-20 in some of the dilated dentinal tubuli. Tubuli of soft
carious dentin also contain a large pattern of other hydrolytic enzymes, including various endopeptidases (Larmas,

2001). However, in Western blotting, MMP-20 could be detected only occasionally in soft carious dentin. It is

possible that detection of MMP-20 in caries lesions by Western blotting would have required larger quantities of
tissue for the analysis. Alternatively, the enzyme may have been activated and fragmented into non-detectable
forms during earlier events of caries progression.

The host MMPs have been suggested to be involved in dentin caries progression (Tjderhane et al., 1998b; Sulkala

et al., 2001). Since MMP-20 does not cleave type I or II collagen in vitro (Vnnen et al., 2001), dentin-bound
MMP-20 could contribute, e.g., to the early alterations in non-collagenous organic matrix during caries progression
in dentin (Lorme et al., 1986), rather than degradation of collagenous matrix per se. On the other hand, the
dentin-bound MMPs may have a defensive role during dentin caries progression by, e.g., releasing dentin-bound
growth factors, which in turn would participate in the regulation of dentin-pulp complex defensive reactions under
caries lesions (Tjderhane et al., 2002).

MMP-20 production was demonstrated here in both odontoblasts and pulp tissue by Western blotting. Previously,

MMP-20 has not been shown in pulp tissue, but a low level of MMP-20 expression has been detected in pulp

stromal cells of dental papilla (Fukae et al., 1998). However, in immunohistochemical staining, MMP-20-positive
pulpal fibroblasts were few and scattered, while endothelial cells demonstrated positive staining more uniformly.

Our results with Western blotting and immunohistochemistry suggest that MMP-20 synthesis is not markedly altered
in carious teeth when compared with sound teeth.

In immunohistochemical staining, the intensity of the MMP-20-positive staining in odontoblasts was not uniform
through the odontoblastic layer, but there was a gradient of more intensive staining toward the radicular dentin,

whereas, more coronally, fewer odontoblasts were stained. Primary dentinogenesis advances from the crown in the
apical direction, and it is likely that, in the third molars of young patients used in the experiment, primary
dentinogenesis proceeded in the root dentin area. The finding indicates that MMP-20 production of odontoblasts is

altered according to the stage of differentiation, and the level of synthesis may reflect the activity of the primary
dentinogenesis.

We also demonstrate MMP-20 in dentinal fluid, which is normal extracellular fluid with respect to its inorganic ion
content (Larmas et al., 1986). In vivo, there is a fluid flux toward the dentin-enamel junction (Vongsavan and

Matthews, 1991), and the content of dentinal fluid, at least without external irritation, has been suggested to be
regulated by the odontoblasts (Turner, 1992). Due to cavity preparation, the integrity of the odontoblast layer may

be disturbed (Turner et al., 1989), and some fluid components may have leaked from the pulp, since dentinal fluid
collected from cavities in vivo contains plasma proteins such as albumin (Knutsson et al., 1994). However, after

extraction of the tooth, the dentinal fluid flow is reversed (Vongsavan and Matthews, 1991). The immunostainings
demonstrated that the main source of MMP-20 in the human dentin-pulp complex is the odontoblasts. The results

imply that MMP-20 of dentinal fluid is most likely odontoblast-derived and is secreted into dentinal tubuli at least

during external irritation. Therefore, MMP-20 might be involved in defense reactions, e.g., secondary dentin
production, but may also be a component of normal human dentinal fluid.

In Western blots, the molecular weights observed (46 and 43 kDa) correspond to the calculated active form of

human MMP-20 (Llano et al., 1997). The 57-kDa band, corresponding to the proform of human MMP-20 (Llano et

al., 1997), was detected only in dentinal fluid. The absence of proform in dentin samples could be due to autolytic
fragmentation during extraction, or the enzyme may already have been in the 46/43-kDa active form in the
samples. Also, the 20- to 40-kDa bands detected in Western blots could represent autolytic cleavage products of

MMP-20. The high-molecular-weight bands, observed especially in the odontoblast and pulp tissue samples, but
also in other samples, most likely represent the complexed forms with other proteins, e.g., with the endogenous
tissue inhibitors of MMPs (TIMPs) of 70 to 80 kDa.

In conclusion, this study demonstrates that mineralized human dentin contains MMP-20, part of which is removed

during demineralization, while the rest is bound to the organic matrix. The results also indicate that the dentinbound MMP-20 might be released, and possibly activated, during dentin caries progression. The main cellular
source of MMP-20 in the dentin-pulp complex is the odontoblasts, which also produce MMP-20 in dentinal fluid.

Acknowledgments
Dr. Ari Lnsineva (DDS) and his staff at the Finnish Student Health Care Center, Oulu, and the staff of the

Department of Oral and Maxillofacial Surgery, University of Oulu, are acknowledged for providing the teeth for the
experiments. We thank Ms. Sanna Juntunen, Ms. Sirpa Kangas, and Ms. Eeva-Maija Kiljander for their skillful
laboratory work. The study was financially supported by the Academy of Finland, Helsinki University Research Funds,
the Finnish Dental Society, and the Juliana von Wendt Foundation