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Regulation and Interactions of MT1-MMP and MMP-20 in Human Odontoblasts and Pulp Tissue in vitro
H. Palosaari1,*,
Y. Ding2,
M. Larmas1,3,
T. Sorsa4,
J.D. Bartlett2,5,
T. Salo1,3,6 and
L. Tjäderhane1,7,6
1 Institute of Dentistry, PO Box 5281, 90014 University of Oulu, Oulu, Finland;
2 Department of Cytokine Biology, Forsyth Institute, Boston, MA, USA;
3 Oulu University Hospital, Oulu, Finland;
4 Oral Pathology Unit and Biomedicum, University of Helsinki, Laboratory Diagnostics, Helsinki University Central Hospital (HUCH), Helsinki, Finland;
5 Harvard-Forsyth Department of Oral Biology & Department of Cytokine Biology, Forsyth Institute, Boston, MA, USA;
7 Department of Endodontics, Faculty of Dentistry, University of Toronto, ON, Canada;
Correspondence: *corresponding author, palosaar{at}csc.fi
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ABSTRACT
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MT1-MMP is a cell-membrane-bound metalloenzyme that activates other proMMPs such as proMMP-2 and -13. We studied MT1-MMP expression in mature human odontoblasts and pulp tissue, the regulation of MT1-MMP expression by growth factors TGF-β1 and BMP-2, and the activation of odontoblast-derived MMP-20 by MT1-MMP. MT1-MMP mRNA is expressed by native and cultured mature human odontoblasts and pulp tissue. Western blot analysis of human odontoblasts and pulp tissue detects 65- and 51-kDa pro- and active forms of MT1-MMP, and smaller truncated MT1-MMP forms. BMP-2 down-regulates MT1-MMP expression in odontoblasts and pulp tissue, while TGF-β1, alone or with BMP-2, decreases MT1-MMP mRNA levels only slightly. We also demonstrate that MT1-MMP is capable of converting proMMP-20 into a form corresponding to the active MMP-20. In conclusion, this study demonstrates the expression and differential regulation of MT1-MMP in human dentin-pulp complex cells, and the activation of MMP-20 by MT1-MMP.
Key Words: MMP • TGF-β • BMP-2 • odontoblasts • pulp tissue • activation
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INTRODUCTION
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Matrix metalloproteinases (MMPs) are a zinc-dependent endopeptidase family classified into collagenases, gelatinases, stromelysins, membrane-type MMPs (MT-MMPs), and other MMPs. They are involved in extracellular matrix (EM) degradation in both physiological and pathological conditions (Birkedal-Hansen et al., 1993). MMPs present in the mature human dentin-pulp complex include enamelysin (MMP-20) (Llano et al., 1997), gelatinases (MMP-2, -9) (Tjäderhane et al., 1998), and collagenase-2 (MMP-8) (Palosaari et al., 2000). Animal studies have demonstrated the presence of MT1-MMP (MMP-14) in developing porcine tooth odontoblasts (Caron et al., 1998). MT1-MMP is a known activator of proMMP-2 (Sato et al., 1994) and proMMP-13 (Knäuper et al., 1996). Therefore, MT1-MMP may play a role in the activation of the odontoblast-derived MMPs.
Transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMPs) are among the growth factors detected in dentin (Bessho et al., 1991; Cassidy et al., 1997) and are known to regulate tissue repair in teeth (Lesot et al., 1994). In response to an external irritation, these growth factors (especially TGF-β1) are thought to be liberated from the mineralized dentin and to activate odontoblast extracellular matrix secretion and reparative dentin formation (Sloan and Smith, 1999). However, TGF-β1 does not affect type I collagen synthesis in mature human odontoblasts and pulp tissue (Palosaari et al., 2001). Instead, TGF-β1 differentially regulates MMP expression in cells of the mature human dentin-pulp complex (Tjäderhane et al., 1998; Palosaari et al., 2000).
Here we show that MT1-MMP is expressed in mature human odontoblasts and pulp tissue and that the expression is regulated by TGF-β1 and BMP-2. We also demonstrate that, in addition to activating proMMP-2, MT1-MMP also converts proMMP-20 synthesized by the dentin-pulp complex cells into a form corresponding to active MMP-20.
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MATERIALS & METHODS
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Intact third molars (n = 94) of young patients (age 18-25 yrs) were removed as a part of the normal treatment at the University Student Health Care Centre and the Department of Oral and Maxillofacial Surgery, University of Oulu. The molars were used for the experiments after the patients' informed consent, following the Faculty of Medicine at the University of Oulu guidelines for the use of human samples.
Cells and Culture Conditions
The collection of the native (uncultured, n = 3) odontoblasts and pulp tissue, with the use of Trizol® Reagent solution (Gibco BRL, Roskilde, Denmark) for total RNA extraction from the odontoblasts and pulp tissue separately, has been described previously in detail (Palosaari et al., 2000). In addition, native odontoblasts (n = 6) and pulp tissue (n = 3) were suspended into a 1x Laemmli buffer for MT1-MMP analysis by Western blot.
The procedures for odontoblast and pulp tissue cultures have been previously described in detail (Tjäderhane et al., 1998). For analysis of the effects of TGF-β1 and BMP-2 on MT1-MMP expression, odontoblasts and pulp tissue were cultured for 24 hrs in serum-free OPTI-MEM I culture medium with antibiotics (Gibco BRL, Life Technologies Inc., Grand Island, NY, USA) (Tjäderhane et al., 1998) with 1 ng/mL TGF-β1, 100 ng/mL BMP-2, 1 ng/mL TGF-β1 combined with 100 ng/mL BMP-2, or without any mediator (n = 18 in each group). Recombinant human TGF-β1 (Sigma, St. Louis, MO, USA) and recombinant human BMP-2 (Genetics Institute, Cambridge, MA, USA) were reconstituted according to the manufacturer's directions. Total RNA for MT1-MMP mRNA analysis was isolated as described below to produce 6 independent samples. Conditioned culture medium, without mediators, was used for the MMP-20 and MMP-2 activation analysis as described below. In addition, odontoblasts and pulp tissues (n = 3) cultured without mediators were suspended into a 1x reduced Laemmli buffer for MT1-MMP protein analysis by Western blot.
RNA Isolation, cDNA Synthesis, and PCR Amplification
Total RNA was isolated by means of Trizol® Reagent isolation. Before cDNA synthesis, genomic DNA was digested with DNase enzyme (1 U/2.0 µg total RNA) for 15 min at 37°C. The first-strand cDNA was synthesized from 2.0 µg of total RNA with the use of 200 units of SuperscriptTM II RnaseH- Reverse Transcriptase (Gibco BRL, Roskilde, Denmark) and random hexamer primers.
MT1-MMP mRNA expression in mature human odontoblasts and pulp tissue was analyzed by PCR procedures. Specific primers for amplifying MT1-MMP were: sense 5' CTGGCTACAGCAATATGGC 3' and antisense 5' TCACGGATGTAGGCATAGG 3'. Nested MT1-MMP sense primer was 5' AGTCACTCTCAGCGGCCAT 3' and antisense 5' AACGCCTTGCGAATGGCCT 3' (Sato et al., 1994). 18S ribosomal RNA was used as a control for RNA integrity. Primers were: sense 5' GGTTGATCCTGCCAGTAGCATATGCTTG 3' and antisense 5' GCGAGCGACCAAAGGAACCATAACTGAT 3' (McCallum and Maden, 1985). PCR amplification of 1 µL of cDNA consisted of 40 cycles (25 for nested PCR or 20 for 18S ribosomal), including 30 sec at 95°C, 90 sec at 54°C for MT1-MMP/56°C for nested PCR/68°C for 18S ribosomal, and 90 sec at 72°C followed by a 10-minute extension at 72°C. The sizes of the PCR products were 395 bp for MT1-MMP, 267 bp for nested MT1-MMP, and 125 bp for 18S.
We verified the identity of amplified product by sequencing the PCR product directly with MT1-MMP sense primer, using a DNA Sequencing Kit (Applied Biosystems, Warrington, UK). The data were analyzed with the use of NCBI's BLAST search tool.
Ribonuclease Protection Assay (RPA)
The effect of TGF-β1 or BMP-2 on the expression of MT1-MMP mRNA in cultured odontoblasts and pulp tissue was assessed by RPA according to RPA IIITM Ribonuclease Protection Assay Kit instructions (Ambion Inc., Austin, TX, USA). A 3-µg quantity of the odontoblast (n = 6) and a 5-µg quantity of pulp tissue (n = 6) total RNA, respectively, was hybridized with 8 x 104 cpm [ -32P]UTP-labeled MT1-MMP antisense RNA probe (nucleotides 218-638). The hybridized RNA and probe were treated with 1:100 dilution of RNase A/RNase T1 mixture for 30 min at 37°C, and the protected fragment was analyzed by a 5% denaturing PAGE gel.
The MT1-MMP bands and respective 28S ribosomal RNA, which allowed for the quantification of MT1-MMP mRNA in different samples, were visualized following exposure to the x-ray film. Bands were scanned with an image-processing and analyzing program (ScionImage PC, Scion Corporation, Frederick, MD, USA). We calculated relative amounts of MT1-MMP expression by dividing the MT1-MMP scanning unit value by the respective value of the 28S ribosomal RNA. Six samples were used to calculate the mean and standard error of the mean (SEM) for the odontoblast and pulp tissue cultures. One-way analysis of variance (ANOVA) with an LSD post hoc test was used for analysis of the statistical significance among the groups, with the SPSS 10 program (SPSS Inc., Chicago, IL, USA).
ECL-Western Blotting
We used Western blot analysis to assess the synthesis of MT1-MMP in native and cultured odontoblasts and pulp tissue. A 10-µL quantity of native and a 20-µL quantity of cultured samples were resolved by 12% SDS-PAGE and transferred onto a nitrocellulose filter (Hoefer Scientific Instruments, San Francisco, CA, USA). The filter was incubated with a polyclonal antibody against human MT1-MMP (0.9 µg/mL) (Chemicon International, Inc., Temecula, CA, USA), and immunoreactive bands were visualized by an ECL system (Amersham, Buckinghamshire, UK) according to the manufacturer's instructions. The pro-recombinant human soluble MT1-MMP (200 ng) (Lee et al., 2001) served as a control for MT1-MMP antibody specificity.
MMP-20 Activation by MT1-MMP and APMA
The conditioned culture media from 7 odontoblast cultures were pooled for the analysis. The odontoblast cells, collected from 3 intact native teeth, were suspended into 100 µL of 50 mM Tris-HCl, 0.2 M NaCl, 1 mM CaCl2, pH 7.8. These cells were used for study of the activation of secreted and intracellular MMP-20, respectively. Soluble pro- and catalytic domains of MT1-MMP and soluble proMT1-MMP (Invitek GmbH, Berlin, Germany) were activated with 2 µM tumor-associated trypsin-2 (TAT-2) for 30 min at RT. The reactions were terminated by the addition of 200 ng of tumor-associated trypsin inhibitor (TATI) (Sorsa et al., 1997). The odontoblast cell and medium samples were treated with or without soluble activated MT1-MMP catalytic domain (further referred to as MT1-MMP [A]) (100 ng), and soluble MT1-MMP catalytic and hemopexin domains (further referred to as MT1-MMP [B]) (100 ng) for 6 hrs and 24 hrs, and with 2.0 mM aminophenyl mercuric acetate (APMA) for 24 hrs, in a 20-µL volume at 37°C. APMA is a wide-spectrum MMP-activator and served as a control for MT1-MMP activation. MMP-20 processing was analyzed with 12% SDS-PAGE and Western blotting with polyclonal antibody specific for porcine recombinant MMP-20 (1 µg/mL) (Bartlett et al., 1996) or polyclonal anti-human MMP-20 hinge region (1 µg/mL) (Sigma, St. Louis, MO, USA).
Conversion of pro-recombinant human MMP-20 by MT1-MMP (A) was assessed as follows. The human enamelysin catalytic domain was point-mutated (a glutamic acid at position 235 was changed to an alanine [VAAHEFGHA to VAAHAFGHA]), so that catalytic activity would be eliminated. Point-mutated rhMMP-20 was collected from an E. coli expression system and was purified over a Ni column. The eluted pro-rhMMP-20 containing an N-terminal His-Tag was refolded by dialysis. The supernatant of refolded point-mutant rhMMP-20 was concentrated ten-fold by use of a microcon YM-10 (Amicon, Inc., Beverly, MA, USA), and 15 µL was incubated with 200 ng of MT1-MMP (A) at 37°C for 6 and 24 hrs, respectively. A 15-µL quantity of MMP-20 alone served as the control. The reaction was terminated by the addition of 15 µL of 2x reducing sample buffer; the solution was then heated at 100°C for 5 min. Conversion of pro-rhMMP-20 was assessed by 12% SDS-PAGE and Western blotting with the use of a monoclonal anti-human-MMP-20 antibody (Fuji Chemicals, Inc., Toyama, Japan).
Activation of proMMP-2 by APMA or MT1-MMP served as positive controls for the activation studies of proMMP-20. ProMMP-2 activation by odontoblasts and conditioned odontoblast media was assessed by zymography as described previously (Tjäderhane et al., 1998).
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RESULTS
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Expression of MT1-MMP by Mature Human Dissected Odontoblasts and Pulp Tissue
MT1-MMP 395-bp cDNA fragments were amplified from uncultured (ObN) and cultured (ObC) mature human odontoblasts plus uncultured (PN) and cultured (PC) pulp tissue (Fig. 1A ). The specificity of the product was confirmed by nested-PCR of the cDNA fragments (Fig. 1A, 267 bp), and by sequencing. The endogenous control 18S ribosomal RNA was amplified by RT-PCR to the expected size of 125 bp from all cells assayed (Fig. 1A).
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Figure 1. Expression and regulation of MT1-MMP by TGF-β1 and BMP-2 in odontoblasts and pulp tissue. (A) Total RNA from native and cultured odontoblasts (ObN and ObC, respectively) and native and cultured pulp tissue (PN and PC, respectively) were transcribed into cDNA. MT1-MMP mRNA and 18S ribosomal RNA were amplified by PCR. Products were fractionated on a 1.5% agarose gel containing 1 µg/mL ethidium bromide and were photographed. PCR was repeated 4 times. A 395-bp MT1-MMP transcript was amplified from native and cultured odontoblasts and pulp tissue, respectively. In nested PCR amplifications, a 267-bp product was amplified from all samples, confirming the identity of MT1-MMP. In the negative control (NCtr), where template was not added, no product was amplified. 18S ribosomal transcripts, amplified from the samples, served as the endogenous control. (B) Odontoblasts and pulp tissue (n = 6 for all samples) were treated without (Ctr) or with TGF-β1 (T), BMP-2 (B), or both growth factors combined (T+B). MT1-MMP and 28S ribosomal RNA were analyzed by RPA. The bands were scanned, and the relative MT1-MMP levels in each group were calculated based on the 28S standard values. (C-D) A mean and standard error of the mean (SEM) of the relative MT1-MMP mRNA expression in odontoblasts (C) and pulp tissue (D) (n = 6 in all groups). 100 ng/mL BMP-2 decreased the expression of MT1-MMP mRNA by 66% when compared with controls (**: p = 0.006, ANOVA with LSD test), while with 1 ng/mL TGF-β1, alone or in combination with BMP-2, only a moderate 36-40% decrease was detected, and this difference was not statistically significant (C). In the pulp tissue, a similar but less marked non-significant reduction in MT1-MMP expression was observed (D).
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Regulation of MT1-MMP mRNA Expression by TGF-β1 and BMP-2
RPA demonstrated specific MT1-MMP mRNA and 28S ribosomal RNA from both the cultured odontoblasts and pulp tissue (Fig. 1B ). In the odontoblasts, BMP-2 significantly down-regulated MT1-MMP mRNA expression by 66% (Figs. 1B, 1C, p = 0.006), while with TGF-β1, alone and in combination with BMP-2, only modest down-regulation was observed. In the pulp tissue, similar effects were observed, but the reduction was not statistically significant (ANOVA, p = 0.231) (Figs. 1B, 1D).
MT1-MMP Protein in Odontoblasts and Pulp Tissue
On Western blots, immunoreactive 65-kDa and 51-kDa bands, corresponding to latent and active forms of MT1-MMP, were detected (Figs. 2A, 2B). Also, smaller 45-, 42-, 35-, and 20-kDa forms were present, especially in the pulp tissue (Figs. 2A, 2B). The specificity of the antibody was confirmed by localization of pro-recombinant human MT1-MMP to the expected size of 62 kDa on a Western blot (Fig. 2B).
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Figure 2. MT1-MMP protein synthesis in odontoblasts and pulp tissue detected by Western blot analysis. (A) A 20-µL quantity of native odontoblasts (ObN) and native pulp tissue (PN) in 1x Laemmli buffer was subjected to Western blot analysis with the use of an MT1-MMP-specific polyclonal antibody. ProMT1-MMP (65 kDa) and MT1-MMP (51 kDa) bands were detected in all samples. In addition, truncated MT1-MMP (45, 42, 35, and 20 kDa) forms were also observed. (B) Odontoblasts (ObC) and pulp tissue (PC) were cultured for 24 hrs without growth factors and were subjected to Western blotting with the use of MT1-MMP-specific polyclonal antibody. ProMT1-MMP (65 kDa) and MT1-MMP (51 kDa) bands were detected. In addition, truncated MT1-MMPs (42, 35, and 20 kDa) were seen in the pulp tissue. Recombinant human soluble proMT1-MMP was used as a positive control for antibody specificity. The expected 62-kDa immunoreactive band was observed (rhMT1-MMP). This control strongly suggests that the 65-kDa band observed in the odontoblasts and pulp tissue samples represents the latent form of MT1-MMP.
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Activation of MMP-20 by MT1-MMP
Western blotting with anti-MMP-20 demonstrated 57-kDa and 46-kDa immunoreactive bands which corresponded well to the calculated pro- and active forms of human MMP-20, respectively (Llano et al., 1997). As compared with controls, odontoblasts treated with APMA clearly converted the 57-kDa proMMP-20 form into the 46-kDa active MMP-20 (Fig. 3A). The active MT1-MMP catalytic domain (MT1-MMP [A]) induced the 57-kDa MMP-20 conversion into the 46-kDa form after only 6 hrs of incubation (Fig. 3A). In the conditioned culture media, the 57-kDa MMP-20 conversion to the 46-kDa form was observed with APMA, and with MT1-MMP (A) after a treatment of only 6 hrs, with no further changes after 24 hrs (Fig. 3B). MT1-MMP containing the catalytic plus hemopexin domain (MT1-MMP [B]) converted the MMP-20 57-kDa species to the 46-kDa form after a six-hour treatment, with no additional conversion after 24 hrs in the odontoblasts (not shown). In the conditioned culture media, the level of conversion was negligible even after 24 hrs (not shown).
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Figure 3. Activation of MMP-20 or MMP-2 with APMA or MT1-MMP. (A-B) Western blot analysis of proMMP-20 conversion by APMA or MT1-MMP in odontoblasts collected from intact mature human teeth (A) and conditioned odontoblast culture media (B). In the control samples from cells or media, 57-kDa proMMP-20 and 46-kDa active MMP-20 immunoreactive bands were observed. APMA and the soluble catalytic domain of MT1-MMP (MT1-MMP [A]) variable converted the 57-kDa proMMP-20 form into 46-kDa activated MMP-20 after incubation of 6 or 24 hrs. This occurred in both the odontoblasts (A) and conditioned odontoblast culture medium (B). (C-D) Conversion of recombinant human MMP-20 by the catalytic domain of active recombinant MT1-MMP. Western blot analysis (C) and SDS-PAGE with Coomassie Brilliant Blue staining (D) revealed that MT1-MMP converted proMMP-20 into an active MMP-20 after 6 hrs or 24 hrs of incubation. (E-F) MMP-2 activation assay with APMA or MT1-MMP analyzed by gelatin zymography. APMA clearly activated proMMP-2 in odontoblasts (E) and conditioned odontoblast culture medium (F). Soluble catalytic MT1-MMP (A) and soluble catalytic with hemopexin domain MT1-MMP (B) converted proMMP-2 into an active form of MMP-2 in the odontoblasts (E). No clear activation was seen in the conditioned odontoblast culture medium (F). (All samples were incubated for 24 hrs.)
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Treatment of pro-recombinant human MMP-20 by MT1-MMP (A) showed that proMMP-20 was converted into a smaller form corresponding to active MMP-20 after 6 and 24 hrs of incubation with MT1-MMP (Figs. 3C, 3D).
APMA activated proMMP-2 in the odontoblasts and conditioned odontoblast culture media, respectively (Figs. 3E, 3F). MT1-MMP (A) and (B) activated proMMP-2 present in the odontoblasts (Fig. 3E). Since MMP-2 was largely in active form in the conditioned odontoblast culture media (Fig. 3F), further proMMP-2 activation by MT1-MMP could not be observed in the conditioned culture media.
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DISCUSSION
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Previously, MT1-MMP has been detected in developing porcine tooth odontoblasts (Caron et al., 1998), and high-level MT1-MMP expression has been demonstrated in other mineralizing tissues, such as bone and cartilage (Buttner et al., 1997; Sato et al., 1997). This is the first study to demonstrate that MT1-MMP is constitutively expressed in mature human odontoblasts and pulp tissue. We also found that BMP-2 significantly down-regulates MT1-MMP mRNA expression in mature human odontoblasts, whereas TGF-β1, alone or with BMP-2, only slightly decreased MT1-MMP expression. In pulp tissue, similar effects were observed, but at markedly lower levels. These findings confirm the previously demonstrated differential regulation of dentin-pulp complex MMPs by specific growth factors (Tjäderhane et al., 1998; Palosaari et al., 2000).
The molecular masses for pro- and active MT1-MMP have been calculated to be 62.7 kDa and 53.8 kDa, respectively (Sang and Douglas, 1996). By Western blot, we detected 65- and 51-kDa immunoreactive bands, which most likely represent MT1-MMP latent and active forms. In addition, truncated forms were observed, especially in the pulp tissue. MMP-2 activation by MT1-MMP is accompanied by processing of MT1-MMP to a 43- to 44-kDa form (Lohi et al., 1996; Lehti et al., 1998; Hernandez-Barrantes et al., 2000), and active MT1-MMP can be shortened to a 43-kDa inactive membrane form and to a soluble 20-kDa fragment (Lehti et al., 2000). The presence of these truncated MT1-MMP forms may reflect the final phase of the proMMP-2 activation reaction (Stanton et al, 1998; Overall et al., 2000).
As previously described for tumor cells (Sato et al., 1994), we demonstrate that MT1-MMP activates proMMP-2 from odontoblasts. This may occur through the formation of the MT1-MMP, TIMP-2, and proMMP-2 complex (Cao et al., 1998; Kinoshita et al., 1998). However, the soluble catalytic domain of MT1-MMP can activate proMMP-2 without TIMP-2 (Will et al., 1996). Thus, we used the soluble catalytic domain for our experiments. In addition to proMMP-2, we demonstrate for the first time that MT1-MMP converts proMMP-20 into a form corresponding to active MMP-20 in odontoblasts. This was confirmed by the respective conversion of pro-recombinant human MMP-20 by MT1-MMP. Conversion of proMMP-2 or proMMP-20 in conditioned culture media was reduced compared with conversion by the cells. This likely occurred because, in the media, the enzymes are already mostly in an active form. This further indicates that the odontoblast-derived proMMPs may be activated at the cell surface during secretion.
The marked conversion of the MMP-20 57-kDa form into the 46-kDa form after APMA treatment strongly suggests that the 57-kDa species represents human proMMP-20, and that the 46-kDa species is an active form. The 57- and 46-kDa molecular weights are in accordance with the calculated 54.4-kDa and 42.6-kDa latent and active forms for human MMP-20 (Llano et al., 1997). Human MMP-20 was originally cloned from odontoblasts (Llano et al., 1997), but this is the first time it has been identified at the protein level as an enzyme activated by MT1-MMP.
In conclusion, this study demonstrates the expression and synthesis of MT1-MMP in human odontoblasts and pulp tissue, and demonstrates a possible role for MT1-MMP in the activation of other odontoblast-derived MMPs. Furthermore, TGF-β1 and BMP-2 were demonstrated to regulate MT1-MMP expression differentially in the dentin-pulp complex, which may indirectly affect the activity of other odontoblast-derived MMPs. Therefore, MT1-MMP may have a special regulatory role in the dentin biomineralization process by virtue of its ability to activate proMMPs.
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ACKNOWLEDGMENTS
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Dr. Ari Länsineva (DDS) and his staff at the Finnish Student Health Center, Oulu, are acknowledged for providing the teeth for the cultures. We thank the Genetics Institute (Cambridge, MA, USA) for kindly providing rhBMP-2. We thank Dr. K. Iwata of Fuji Chemical, Inc., Toyama, Japan, for providing the monoclonal antibody to MMP-20. We also thank Ms. Sanna Juntunen, Ms. Sirpa Kangas, and Ms. Eeva-Maija Kiljander for their skillful laboratory work. This study was financially supported by the Academy of Finland, Helsinki University Research Funds, the Finnish Dental Society, the Juliana von Wendt Foundation, and the Wilhelm and Else Stockmann foundation. Y. Ding and J. D. Bartlett were supported by NIDCR grant #DE13237.
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FOOTNOTES
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6 The last two authors contributed equally to the supervision of this work 
Received for publication June 11, 2001.
Revision received March 18, 2002.
Accepted for publication March 18, 2002.
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Journal of Dental Research, Vol. 81, No. 5,
354-359 (2002)
DOI: 10.1177/154405910208100513
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ABSTRACT
INTRODUCTION