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 DOI: 10.1177/00220345000790011401

2000 79: 77J DENT RESH. Palosaari, J. Wahlgren, M. Larmas, H. Rönkä, T. Sorsa, T. Salo and L. Tjäderhane

1βThe Expression of MMP-8 in Human Odontoblasts and Dental Pulp Cells is Down-regulated by TGF-  

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H. Palosaaril, J. Wahlgren2, M. Larmas1'3,H. Ronka, T. Sorsa2, T. Salol,5*and L. Tjiderhanel 6*'Institute of Dentistry, University of Oulu, PO Box 5281,FIN-90401 Oulu, Finland; 2Institute of Dentistry, Universityof Helsinki, Finland; 3Oulu University Hospital, Oulu,Finland; 4Medix Biochemica Oy Ab, Kauniainen, Finland;and 5Department of Pathology, University of Oulu, Finland;6corresponding author, lst@cc.oulu.fi; *The last two authorscontributed equally to the supervision of this work.

J Dent Res 79(1): 77-84, 2000

ABSTRACTRecent findings show that matrix metalloproteinase-8(MIMP-8) is expressed, in addition to neutrophils, byhuman chondrocytes, cultured fibroblasts, andendothelial cells. We investigated the expression ofMMP-8 in other human mesenchyme-derived cells,odontoblasts, and pulp tissue. Odontoblasts and pulptissue were collected from extracted human teeth forMMP-8 mRNA analysis with reverse-transcription/polymerase chain-reaction (RT-PCR)and Southem blot. The expression, localization, andsecretion of MMP-8 protein were studied withWestern blot, immunohistochemistry, andimmunofluorometric assay. The effect of TGF-f31 (10ng/mL) on the expression, secretion, andconcentration of secreted MMP-8 was studied byodontoblast and pulp tissue culture methods(Tjaiderhane et al., 1998a). RT-PCR demonstratedMMP-8 mRNA expression in native and culturedodontoblasts and pulp tissue and cultured pulpfibroblasts, with a 522-bp transcript comparable withthat of bone marrow cells. The specificity of PCRwas confimed with Southem blot. Westem blot withMMP-8-specific antibody detected 65- and 50-kDaproteins in native samples, representing latent andactive forms of mesenchymal-type MMP-8, and inthe conditioned odontoblast culture media, 50-kDaprotein was observed. TGF-1 down-regulated theMMP-8 mRNA and concentration of secreted proteinin both cultures. Immunohistochemical stainingdetected MMP-8 in odontoblasts. These findingsindicate that mesenchyme-derived cells of the dentin-pulp complex express, synthesize, and activate MMP-8, which may, in concert with odontoblast-derivedgelatinases, participate in organization of dentinorganic matrix prior to mineralization.

KEY WORDS: MMP, collagenase, odontoblast,dental pulp, dentin.

Received June 26, 1998; Last Revision November 30, 1998;Accepted February 25, 1999

The Expression of MMP-8in Human Odontoblastsand Dental Pulp Cellsis Down-regulated byTGF-31

INTRODUCTIONM atrix metalloproteinases (MMPs) are a family of endopeptidases which in

concert are capable of degrading almost all, if not all, extracellular matrixproteins, including different collagens in native and denatured forms (Birkedal-Hansen, 1995). Human collagenases exist in three distinct molecules, namely,MMP-1 (interstitial collagenase; collagenase-1), MMP-8 (human neutrophilcollagenase; collagenase-2), and MMP-13 (collagenase-3) (Birkedal-Hansen,1995). Among them, MMP-8 is the most effective in hydrolyzing type Icollagen, while MMP-1 prefers type III and MMP-13 type II collagen (Hasty etal., 1987; Knauper et al., 1996).

The information of the presence and function of MMPs in dental tissues islimited. Animal studies show gelatinolytic activity in predentin (Betti andKatchburian, 1982; Smith and Smith, 1984) and unidentified collagenase indentin organic matrix (Fukae et al., 1991). A collagenase and a collagenaseinhibitor are present in bovine pulp (Kishi and Hayakawa, 1982; Kishi et al.,1985), and cloned rabbit odontoblast-like cells produce latent collagenase(Sakamoto et al., 1985).We have recently demonstrated the presence of MMP-8, MMP-2

(gelatinase A; 72-kDa gelatinase), and MMP-9 (gelatinase B; 92-kDagelatinase) in caries lesions in human dentin, along with the activation modelof these enzymes due to the exposure to pH changes typical for caries lesions(Tjaderhane et al., 1998b). These experiments suggest that host MMPs areinvolved in the degradation of dentin organic matrix during caries progression.Even though the major source for MMPs in carious dentin might be gingivalcrevicular fluid (Sorsa et al., 1990, 1995; Uitto et al., 1990; Ingman et al.,1994; Westerlund et al., 1996), from which they are distributed into wholesaliva, the possibility of the dentin-pulp complex as an alternative source ofMMPs in carious lesions cannot be excluded. Indeed, mature humanodontoblasts produce MMP-2 and MMP-9 in vitro (Tjaderhane et al., 1998a).Moreover, TGF-131, which is known to be a key factor in regulation of theresponse of the dentin-pulp complex in pathological conditions (Magloire etal., 1992; Smith et al., 1995), markedly induces MMP-9 production(Tjaderhane et al., 1998a). MMP-1 mRNA has been presented in culturedhuman pulp fibroblasts after interleukin-lot induction (Tamura et al., 1996).Also, the most recently discovered member of the MMP family, also known asenamelysin (MMP-20) because of its capability of degrading proteins presentin immature enamel, was found to be uniquely expressed in mature humanodontoblasts (Llano et al., 1997). Overall, these findings indicate that MMPshave a role in the modeling of adult human dental tissues. The role ofMMPs inphysiological conditions is further supported by the presence of a latentcollagenase in demineralized human dentin matrix (Dayan et al., 1983).

Previously, it was thought that MMP-8 is uniquely produced by developingneutrophils (PMN cells) in bone marrow, and that the MMP-8 activity isdependent on the release of the enzyme from PMN cells by degranulation(reviewed by Weiss, 1989; Mainardi et al., 1991). However, recent findingsdemonstrate the expression of MMP-8 in mesenchyme-derived, non-PMN-

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78 Palosaari et 0l. J Dent Res 79(1) 2000

lineage cell lines, including human cartilage chondrocytes(Chubinskaya et al., 1996; Cole et al., 1996), synovialfibroblasts, and endothelial cells (Hanemaaijer et al., 1997). Inthis study, we investigated the expression and localization ofMMP-8 in human odontoblasts and pulp tissue, both ofmesenchymal origin. To determine the basic level of the MMP-8expression, we used native samples of odontoblasts and pulptissue obtained from freshly extracted teeth. The odontoblastorgan culture method, with the pulp tissues cultured separately(Tjaderhane et al., 1998a), was used to study the effect of TGF-,Bon the expression of MMP-8. We used reverse-transcription/polymerase chain-reaction (RT-PCR) and Southernblot to detect the expression of MMP-8 mRNA, Western blot todemonstrate the expression and secretion of the enzyme protein,MMP-8 immunofluorometric assay to determine theconcentration of secreted protein in the culture media, andimmunohistochemistry to determine the localization of theenzyme in the dental tissues.

MATERIALS & METHODS

All teeth used for the experiments (total number of 166 teeth) werefreshly extracted caries-free third molars. All teeth were removed as apart of the normal treatment of patients and were used for the studieswith the patients' informed consent. The teeth were stored in sterilePBS and prepared for further handling immediately after extraction.

Cells and Culturing CondifionsThe teeth were swabbed with 70% alcohol, and soft tissue andcementum were removed with curettes. A horizontal groove wasgently cut around the root surface 2 mm deep about 5 mm apicallyfrom the cemento-enamel junction with a diamond cutting disk underwater cooling. Care was taken not to reach the pulp. The root wasdissected from the crown with pliers, and the crowns were placed intosterile culture medium. The other procedures were performed in alaminar-flow tissue-culture hood under sterile conditions.

To collect the native odontoblast and pulp tissue mRNA samples,we gently removed the pulps with forceps and placed them intoEppendorf tubes containing 60 jiL of Trizol® Reagent-solution(Gibco BRL, Roskilde, Denmark). The odontoblasts lining the pulpchamber walls were covered with a small amount of Trizol® Reagentto prevent RNA degradation, and the cells were immediately scrapedoff with a sterile excavator and stored into a final volume of 30 ,uLof Trizol® Reagent. Odontoblasts from two crowns were combinedfor each sample, and the samples were stored in -70°C untilanalyzed. For the analysis, the odontoblasts of20 teeth and the pulpsof five teeth were combined. The experiments were done induplicate and repeated three times.

Odontoblast and Pulp Tissue CulturesThe protocol for culturing the odontoblasts and pulp tissues has beenpreviously described in detail (Tjaderhane et al., 1998a). Serum-freeOPTI-MEM I Reduced Serum Medium (Gibco BRL, Grand Island,NY, USA), supplemented with 100 IU/mL penicillin G, 100 IU/mLstreptomycin, 50 IU/mL nystatin, 0.25 ,ug/mL amphotericin B(Fungizone®) (all from Gibco BRL, Grand Island, NY, USA), and 50,ug of ascorbic acid (Sigma, St. Louis, MO, USA), was used for thesecultures. The teeth were cut and pulps removed as described above.For odontoblast organ cultures, 24-well cell culture clusters (Costar@,Cambridge, MA, USA) and for pulp tissue cultures, 96-well cell

Agarose gel, 1.5% (w/v) (Agarose NA, Pharmacia Biotech AB,Uppsala, Sweden), was mixed into the culture medium and was usedas bedding material for odontoblast cultures. The crowns withodontoblasts lining the pulpal chambers were partially embedded inthe agarose gel, leaving from 2 to 3 mm of the tooth structure abovethe gel surface with the pulp chambers facing upward. The pulpchambers were gently washed 3 times at 10-minute intervals withculture medium to remove pulp tissue fluid, and were finally filledwith the culture medium. The crowns were covered with glasscoverslips to prevent evaporation from the medium, and a 200- to300-,uL quantity of medium was also placed on top of thesurrounding gel to prevent dehydration.

To study the effects of TGF-1I on the expression of MMP-8mRNA, we cultured odontoblasts and pulp tissues for two days in theserum-free culture medium described above with (n = 18) or without(n = 22) 10 ng/mL ofhuman recombinant transforming growth factorbeta-l (TGF-13) (Sigma, St. Louis, MO, USA) for two days,collecting and replacing the medium daily. Then the cells werecollected, and total RNA was isolated as described below. Theculture period oftwo days was chosen, since the effect of TGF-13 onMMP mRNA in human fibroblasts has previously been shown to beobserved after 24 hours and to remain basically unaltered for 72hours (Overall et al., 1991).

Pulp Fibroblast CulturesThe pulp samples for the human pulp fibroblast (PF) isolation werecollected as described above. After extraction of the pulp from thepulpal chamber, the tissue was cut into pieces and placed into six-well cell culture clusters (Becton Dickinson, Franklin Lake, NJ,USA). The tissue pieces were allowed to attach for 30 min before theaddition of Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% (v/v) fetal bovine serum (FBS) (both fromGibco BRL, Grand Island, NY, USA), and an antibiotic-antimycoticcombination and ascorbic acid as above. The explants were thenincubated at 37°C in humidified atmospheric mix of air/CO2 (19:1,v/v), and the medium was collected and replaced twice a week. Thecells were examined daily, and when the outgrowth of fibroblasts wasobserved, the pulp tissue pieces were removed. After reachingconfluence, the fibroblast cell lines were established by subsequenttrypsinization with trypsin-EDTA (0.05% trypsin, Gibco BRL,Paisley, Scotland; 0.004% EDTA, Oy FF-Chemicals, Yli-Ii, Finland)and culturing through several passages. The cells were alwaysallowed to reach confluence before being subcultured. To analyze theMMP-8 mRNA production, we washed PF cells in passage two (n =2) three times with sterile PBS, and total RNA was isolated asdescribed below.

RNA Isolation, cDNA Synthesis, and PCR AmplificationTotal RNA was isolated from odontoblasts and pulp tissue (nativeand cultured) and pulp fibroblast cultures by means of the total RNAisolation method (Trizol®, Gibco BRL, Roskilde, Denmark). TheRNA yield was measured by absorbance at 260 nm, and the sampleswere stored at -70°C until used. In addition, RNA isolated from bonemarrow cells (BM) (Hanemaaijer et al., 1997) served as positivecontrols.

For reverse transcription (RT), a 20-p,L reverse-transcriptionreaction was carried out on 1.7 to 11 ,Ig of total RNA. The reactionmixture contained 0.5 mM dNTPs (Promega, Madison, WI, USA), 1x first-strand buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCI, 3 mMMgCl2), 10 mM dithiothreitol (DTT), random hexamer primers, and200 units of Superscript' II RnaseH- Reverse Transcriptase (Gibcoculture clusters (Nunclon Nunc A/S, Roskilde, Demnark) were used.

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MMP-8 Expression

BRL, Roskilde, Denmark), and was incubated at 42°C for 50 min.cDNA was used for PCR amplification.

Specific primers for amplifying MMP-8 were the same asdescribed in Hanemaaijer et al. (1997). The ,B-actin, constitutivelyexpressed housekeeping gene, was used as a control for the RNAintegrity, with primers described previously (Himaliiinen et al.,1995). PCR for MMP-8 was performed in a final concentration of 1 xExpand HF buffer (Boehringer Mannheim GmbH, Mannheim,Germany), 0.2 mM dNTPs, 17 pmol ofboth primers, 1 x Master PCREnhancer (Epicentre Technologies Corp., Madison, WI, USA), and2.6 units of Expand' High Fidelity PCR System enzyme mix(Boehringer Mannheim GmbH, Mannheim, Germany) in a totalvolume of 30 pL. PCR amplification for ,B-actin was performed in afinal concentration of 1 x PCR buffer (Finnzymes Oy, Espoo,Finland), 0.2 mM dNTPs, 10 pmol of both primers, and 1 unit ofthermostable DNA polymerase (Dynazyme®, Finnzymes Oy, Espoo,Finland) in a total volume of 30 ,uL. To find a linear area of PCRreaction for both MM-8 and 3-actin primers, we serially diluted theRT reaction mixture ofMMP-8 to one-half and 1/10, and for 3-actinto 1/10 and 1/100 in sterile water, and the PCR amplification wasdone at 30, 40, 45, 50, and 55 cycles. During the MMP-8 mRNA and,B-actin amplification reactions, each cycle ofPCR included 30 sec ofdenaturation at 95°C, 90 sec of annealing at 59°C, and 90 sec ofextension at 72°C. The size of the PCR product was 522 bp forMMP-8 and 392 bp for 1-actin transcripts, respectively, amplifiedfrom total RNA. The PCR products were analyzed on a 1.5% agarosegel containing 1 p,g/mL ethidium bromide, which attaches to nucleicacids, allowing the PCR products to be visualized under UV light.The gel was photographed to allow the products to be quantitated asdescribed below.

After finding the linear portion for each set of primers forquantitation of the products, we quantitated them with an imageprocessing and analyzing program (ScionImage PC, ScionCorporation, Frederick, MD, USA) to analyze the effect of TGF-,1on the expression ofMMP-8 and 3-actin mRNA.

Southern Blot AnalysesPCR products were fractionated on 1.5% agarose gels, transferred tonylon filter (Amersham, Buckinghamshire, UK), and hybridized asdescribed elsewhere (Ausubel et al., 1988). The specific cDNA probefor MMP-8 was prepared. Briefly, the Southern filter was hybridizedto a 32P-labeled MMP-8 cDNA probe in ExpressHyb hybridizationbuffer (Clontech, Palo Alto, CA, USA) for 1 hr at 600C. The filterswere washed for 20 min in 2x saline-sodium citrate (SSC)/0.05%sodium dodecyl sulphate (SDS) at room temperature (RT) and twicein 0.1 x SSC/0.1% SDS at 50°C for 40 min. The filters were exposedto x-ray film (Kodak X-OMAT) in -700C for 20 min.

Western Blot AnalysisThe teeth were handled as described above, until the removal of thepulp. The pulp tissue was placed into Laemmli sample buffer (SB),and the tissue fluid remaining in the pulpal chamber was gentlycollected into separate SB-containing Eppendorf tubes with thepipette. The pulp chamber containing the odontoblasts was coveredwith SB, and odontoblasts were scraped into SB-containingEppendorf tubes. The samples were stored in a final volume of 30 pL1of SB in 4°C until being handled further.

To investigate the secretion of MMP-8 into the odontoblast andpulp tissue culture medium and the effect of TGF-,B1 on the amountof secreted protein, we used conditioned serum-free OPTI-MEMmedium with and without TGF-I3 for Western blot analysis. Medium

J Dent Res 79(1) 2000

proteoglycans, we pre-treated the sections with 2 mg/mL bovinetesticular hyaluronidase (Sigma, St. Louis, MO, USA) in PBS for 2hrs in 37°C. After being washed with PBS, the sections were treated

in Dental Tissues 79

samples of three odontoblast cultures in both groups were collectedafter 24 and 48 hrs in the culture and were pooled within the groups,and 5 ,uL was used for the Western blot analysis as described below.Quantities (200-,uL) of the respective conditioned pulp tissuemedium samples were freeze-dried, and the proteins were diluted into10 ,uL ofSB buffer and used for the Western blot as described below.

The samples were run on 10% sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) and transferred to nitrocellulosefilter (Schleicher & Schuell, Dassel, Germany) in non-reducingconditions. Non-specific binding was blocked by incubation withPBS supplemented with 5% non-fat dry milk (Difco Laboratories,Detroit, MI, USA) for 60 min. After being washed, the filters wereincubated overnight at RT with rabbit polyclonal antibody againstMMP-8 (Michaelis et al., 1990), which has been shown not torecognize MMP-1 or other MMPs (Lauhio et al., 1994), at a 1:500dilution. After being washed, the filters were incubated withperoxidase-conjugated swine anti-rabbit immunoglobulins (1:200;DAKO A/S, Glostrup, Denmark) for 1 hr at RT. For detection, theblots were directly developed by use of a solution of 6 mg/mLdiaminobenzidine tetrahydrochloride (DAB; 3,3'4,4',-tetraaminobiphenyl) in 50 mmol/L Tris-HCl (pH 8) and 0.003%H202, or by means of the ECL Chemiluminescence Western Blottingdetection kit (Amersham, Buckinghamshire, UK) as described in theproduct protocol.

MMP-8 Immunofluorometric AssayConcentrations of MMP-8 secreted into the odontoblast and pulptissue culture media were determined by a time-resolvedimmunofluorescence assay as described previously (Hanemaaijer etal., 1997). Conditioned culture medium samples, both with andwithout TGF- 1, were collected from the odontoblast organ and pulptissue cultures (n = 10 in each group) after 24 and 48 hrs and werepooled within the groups. The monoclonal MMP-8 specific 8708 and8706 antibodies were used as a catching antibody and tracer antibody,respectively. The tracer antibody was labeled by europium chelate.The assay buffer contained 20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 5mM CaCl2, 50 mM ZnCl2, 0.5% bovine serum albumin, 0.5%sodium azide, and 20 mg/L DTPA. Samples were diluted in assaybuffer and incubated for 1 hr, followed by incubation for 1 hr withtracer antibody. Enhancement solution was added, and after 5 min,fluorescence was measured by means of a 1234 Delfia ResearchFluorometer (Wallac, Turku, Finland). The specificity of themonoclonal antibodies against MMP-8 corresponded to that ofpolyclonal MMP-8 (Hanemaaijer et al., 1997).

ImmunohistochemistryTo prevent the disturbance of enamel during the sectioning of thefrozen samples, we cut off the enamel with a thin diamond discmounted into a handpiece under constant flow of water to preventheating of the samples. Immediately after being cut, the teeth wereimmersed in liquid nitrogen, embedded into aqueous Na-carboxymethylcellulose (Fluka, Buchs, Switzerland), and seriallycut into longitudinal sections about 15 ,um thick on a piece ofadhesive tape. The sections were air-dried in -20°C overnight, fixedwith 96% cold methanol for 30 min, and again air-dried in -20°Covernight. The sections were demineralized with 0.33 Methylenediaminetetra-acetic acid (EDTA) overnight in 4°C andwashed with dH2O. To overcome the masking effect of matrix

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J Dent Res 79(1) 2000

with 0.3% H202 in methanol for 5 min to inhibit endogenousperoxidase activity and washed with PBS.

The staining was performed according to the protocol of theVectastain Elite kit (Vector Laboratories, Burlingame, CA, USA).We incubated the sections in normal goat serum to prevent the non-specific adsorption of antibodies, then incubated them with the sameMMP-8 antibody as used in Western blot (1:50) overnight in 4°C.For the control sections, the primary antibody was replaced withnormal rabbit serum. After being washed with PBS, the sectionswere treated with biotinylated anti-rabbit IgG, washed with PBS,and incubated with reagents A and B of the Vectastain kit. Thesections were colored with 3-amino-9-ethylcarbazole (Sigma, St.Louis, MO, USA) diluted into N,N-diethylformamide (Merck,Darmstadt, Germany), or DAB staining was performed by means ofa DAB Kit, and the color was enhanced with the DAB Enhancingsolution (both from Vector Laboratories, Burlingame, CA, USA).The sections were washed in running tap water, counterstained withhematoxylin, and mounted with Aquamount (Gurr BDH ChemicalsLtd., Poole, England).

RESULTS

Expression of MMP-8 mRNAA RT-PCR amplification method and Southern blot revealed a522-bp MMP-8 transcript amplified from total RNA inodontoblasts and pulp tissue in both native and cultured forms,as well as in cultured pulp fibroblasts. The transcripts wereidentical to those obtained from BM cells, serving as a positivecontrol (Fig. IA). The amplification of 3-actin demonstrated the

A5td-bhOhCPN..PC,PF.RMu NC

<- 522 bp

<-P-actinB

C

D

1*-< MP-8

MMP-8 f3-actin-3

Ob Ob p p Ob Ob P P

TGF-0 - + - +

12

14104-H a80

016

00 j0OOOb Ob P Ob Ob p pp

TGF-P + + + - +

good integrity of the RNA isolated from different samples (Fig.I B). Southern blot analysis, with specific MMP-8 cDNA probe,confirmed the specificity of the PCR amplification (Fig. IC).

TGF-1I down-regulated the MMP-8 mRNA toapproximately 10% and 25% of the levels of control samples inthe odontoblast and pulp tissue cultures, respectively. No effectwas observed in the 1-actin expression (Fig. I D).

Expression, Secretion,and Concentration of Secreted MMP-8In Western blot analysis with MMP-8-specific polyclonalantibody, all native odontoblast, pulp, and pulpal fluid samplesfrom sound teeth demonstrated 65-kDa immunoreactivity withvarious staining intensities (Fig. 2A). In all pulp and pulpal fluidsamples, a fainter 50-kDa band was also detected (Fig. 2A).Furthermore, high-molecular-weight (> 100 to 112 andapproximately 200 kDa) forms of MMP-8 immunoreactivitieswere consistently detected in all the native samples (Fig. 2A).

In the Western blot analysis from the conditioned odontoblastculture medium, a distinct 50-kDa band was also detected (Fig.2B), along with fainter 112-kDa bands, with no apparentdifferences between the TGF-)-induced and control samples. NoMMP-8 was detected in the area of 75 to 80 kDa, characteristicof PMN-MMP-8. With the conditioned pulp tissue medium, anextensive concentration of the samples (40-fold concentrationcompared with the odontoblast medium samples) was neededbefore the respective, but faint, 50-kDa bands could be observed(Fig. 2B). Also in the pulp tissue media samples, the bandscharacteristic ofPMN-MMP-8 were absent (Fig. 2B).

The concentrations of secreted MMP-8 protein wereanalyzed from the serum-free OPTI-MEM culture medium usedto culture odontoblasts and pulp tissue after 24 and 48 hrs of theculture with and without TGF-3I-induction. The concentrationof MMP-8 was higher in the odontoblast organ cultures than inthe pulp tissue cultures, in spite of the markedly lower numbersof cells in the odontoblast cultures. The MMP-8 protein level inthe odontoblast culture medium was lower after 48 hrs than after24 hrs both with and without TGF-j in the medium, and in theTGF-3-containing samples, the levels were lower than in therespective control samples at both time points. In pulp tissue

Figure 1. MMP-8 and 3-actin mRNA in native and culturedo0[ontoblasts and pulp tissue and cultured pulp fibroblasts. RNAsamples were transcribed into cDNA, and MMP-8 and p-actintranscripts were amplified by RT-PCR and subjected to gelelectrophoresis. DNA molecular-mass standard (Std) was used as a sizemarker. Bone marrow cells (BM), known to express MMP-8 mRNA,were used as positive controls. (A) Native and cultured odontoblasts(ObN and ObC, respectively) and pulp tissues (PN and PC,respectively) and cultured pulp fibroblasts (PF) expressed a 522-kbtranscript respective to the MMP-8 mRNA from BM cells. In negativecontrol (NC), no cDNA was added. (B) 1-actin transcript confirmed thegood integrity of RNA in each sample. Abbreviations as in Fig. IA. (C)Southern blot analysis of PCR-amplified DNA. PCR transcripts, asdemonstrated in Fite 1A, were transferred to nylon filters andhybridized with 32P-labeled MMP-8 DNA probe, confirming the identityof MMP-8 mRNA in the transcript. Abbreviations as in Fig. 1A. (D)MMP-8 and 3-actin mRNA PCR transcripts in the pooled samples ofcultured odontoblasts and pulp tissue after 48 hrs in culture with (n =18) and without (n = 22) TGF-p1 in the culture medium. The bars(mean and standard deviation) represent quantitation of the bands offive separate PCR reactions. Down-regulation to approximately 10%and 25% in the MMP-8 mRNA levels with TGFI was observed inodontoblast and pulp tissue cultures, respectively. No apparent effectwas observed in the p-actin levels between the samples.

80 Palosoari eta/.

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J Dent Res 79(1) 2000 MMP-8 Expression in Dental Tissues

culture media, the concentrations were also lower in the samplescultured with TGF-f3 in the medium after 24 hrs, while bothTGF-4 and control samples were below the detection level after48 hrs in the culture (Fig. 2C).

Immunohistochemical StainingDistinct staining with MMP-8-specific antibody in severalodontoblasts demonstrated the expression of MMP-8 in theodontoblast cell layer (Fig. 3A). The staining was localized to thebody of odontoblasts. In the untreated control sections, no

staining could be observed in the odontoblasts (Fig. 3B) or pulpcells. Some endothelial cells lining the walls of the vessels in thepulp were detected to express MMP-8 (Fig. 3C), even thoughmost of the endothelial cells remained unstained.

DISCUSSION

Even though previous experiments have suggested the presence

of mammalian collagenase in dental tissue, the results of our

study provide the first data demonstrating the expression andsynthesis of MMP-8 collagenase in mature human odontoblastsand pulp. Detection of 522-bp transcript with RT-PCR andverification of the MMP-8 mRNA with Southern blot totallycorrespond with previous results in rheumatoid synovialfibroblasts and cultured endothelial cells (Hanemaaijer et al.,

1997), in which the sequencing revealed 100% homology withthe published human MMP-8 sequence (Hasty et al., 1990). Thesynthesis and secretion of the enzyme were further confirmedwith Western blot, and the concentration of the secreted enzyme

was confirmed with the immunofluorometric assay.

The expression of MMP-8 in pulp and odontoblasts issignificant because the data demonstrating the expression ofMMP-8 in cells other than neutrophils are currently ratherlimited (Hanemaaijer et al., 1997). Since all of the non-PMN-lineage cells so far shown to express MMP-8 are ofmesenchymal origin (Chubinskaya et al., 1996; Cole etal., 1996;Hanemaaijer et al., 1997), these results further demonstrate thepossibility of mesenchyme-derived cells expressing andsynthesizing MMP-8. The absence of the PMN-type MMP-8further confirms that the enzyme expressed by odontoblasts andpulpal cells is the mesenchymal-type MMP-8.

The published molecular sizes of MMP-8 protein vary but are

usually between 85 and 50 kDa, obviously depending on the

Fi ure 2. (Al MMP-8 protein expression. Native samples ofocantoblasts, pulp tissue fluid, and pulp tissue were subjected to

Western blot with MMP-8-specific antibody. Molecular-weightstandards(Std) were used to determine the molecular weight of theproteins detected (Mr expressed in kiloDaltons). A 65-kDa band wasdetected inpulp(P), odontoblast(Ob), and pulpal fluid(PF) samples,and pulpand pulpal fluid samples also demonstrated a 50-kDa band.Bands of 80 and 75 kDa, representing PMN-MMP-8, were not

detected. Separated lanes represent different samples used for theanalysis.(B) Western blot analysis of the conditioned serum-freeodontoblast(OB) and pulp tissue (P) culture media, with MMP-8-specific antibody. The conditioned odontoblast media samplesdemonstrated a 50-kDa protein in both the TGF-p-induced and controlsamples(+ and-, respectively).In the pulp tissue media, a faint 50-kDaband was observable only afer extensive concentration of the samples.(C) The concentrations (pLg/L) of MMP-8 secreted into conditionedserum-free odontoblast (OB) and pulp tissue(P) culture media after 24and 48 hrs in culture. The concentration of MMP-8 decreased after 24hrs, andulp tissue culture media samples were below detection levelsaher 48 rs in culture. In addition, TGF-f decreased the concentrationof enzyme at both time points in both cultures.

degree of enzyme glycosylation and the difference in sizesbetween latent and active forms of the enzyme (Hanemaaijer etal., 1997). The 50- and 65-kDa forms detected in the presentstudy most likely represent the different forms of themesenchymal-type MMP-8 species (Cole et al., 1996;Hanemaaijer et al., 1997). The high-molecular-weight (from 100to 112 kDa) MMP-8 immunoreactivities apparently representMMP-8 dimers or complexes with other proteins. Inchondrocytes, 200-kDa MMP-8 immunoreactivity has beenpresented in a non-reduced condition, suggested to representsimilar complex formation (Cole et al., 1996). The less intensedetection of MMP-8 in Western blot in odontoblast samples iscaused by the relatively small amount of the cells collected, sinceodontoblasts form a monocellular layer of cells lining the pulpalwalls (Linde and Goldberg, 1993). The immunohistochemicalstaining, however, confirms that MMP-8 is indeed present in theodontoblasts.

There are several possibilities for the functionial role ofMMPs in dental tissues. Prior to mineralization, the

AStd P Ob PF P Ob PF

112

84

53

35

28

+ 65

<-50

B112

84

TGF-f3

c

a

*50

- + - +

*24

048

P P POb Ob Ob Ob

TG- - + +

81

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J Dent Res 79(1) 2000

cOmponients ol oniganic maltrix of dentini are secreted, and thcmatrix is processed and organized in predenitini. DLurinig thisor-anli/ation, hreakdown anid renioxal of soImle ImlatrixComlpOnCInS OCCUIr (Lin(de and Goldberg, 1993). The pr-esCIeceoi collaLcriase (Day an et il.. 1983: FLukac et al., 1991) andeelati nase (Betti and KatchhuiIiaLn 1982 Smithi and Smiti,1984) in predenitin and dentlin imiiplies that MMPs are requireddLtiilnc thl oicraaln/atioll ol matrix helfoic imineralization canoccur. Siice type I collagen is the iviaiii componeint of denitiniorganic maLtrix MMI'-8 ohviotisly has an ilmpor-tanit role in theorganli/ation process. Moreov er, the secretioni ol'tllcCtlzy me iinthe active loiori conFirimis the iunLctionial role of MMP-8 in themodelincg ol the organic matrix. Additionally, the latenit form-lmay he incor-porated into the organ ic matiix behloic or dUiringmineinaliiation, siice latent collaoenase has beeni obserxcvd inmineralized dent ini ( Dava etil.. 1 983).

Ocdontoblasts also express type Ill collageniimRNA(Likinimilaa e/ al., 1993). and type Ill procollagen (Becker- elal.1 986) andl col la-en (Kai-ja laininen ril., 1 986) have beendeimionisti-ated in predeinti n. 1loxever, in inineralized dentill,type Ill collagen is present in substantial amounlts only in casesol thic hereditary detiniii defect called dentirlogenesis imperfeeta(Kar alainen and Si5der-ling, 1984; Waltimo et a!., 1994), and inthe reparative denitini forimied as a response to stronc: externalirritationi hy caiics (Magloire el l., 1988). Degradation olftypeIll collagen dutrinc matr-ix or-ani7ati on, along with themodel ing ol type I collagen, mnay thus be required for thenormiially minrci-ali/ed dentin. I'ven thotigh typc Ill collagen istIle mainl substrate for- MMP-I (Hasty el 6l., 1987), MMP-8 isalso capable of decrading type IlI collagen (13irkedal-liiansei,1995). If the iolc of odontoblast-deriived MMPs indeed is tomodel the precenitini prior- to minieralizatiojn, the dowix -

reccilation olf MMP-8 by TGI -3 obserxved in this stuLy mightsilmulate tlhC situationi tindcer active caries lesions, in wlhicireparative cfeintiin witli poorly organ i/ed organic matrixcontaining type Ill colla-enr occurs (Magloire et a!.. 1988). It isinterestiriu to note that, in cin-gival fibroblasts andkeratinocytes. -(GF 1- has hecn shoxxr to up-regutlate MMP-2and MMP-9 (Ox erall el d., 1991: Salo et/ l.. 1991) and dowix-regIlate I MMIP- expiCSsiionI (Oxera I Cle L., 1991). W hIi le inodontoblasts, MMP-9 is Up- iCegulaled xw ith no eftfect oniMMP-2expression (Tjhdealiaie et l., 1998a), and MMP-8 is dowix-rcgulated as presented here.

0dontoblasts beai a sti-ong i esemblanice to anothel hard-tissie-forminii cell lie, namiely. ostcoblasts. Osteoblasts alsoexpress coll agcniase (Bo-rd e ci!., 1996) and gelatinases

82 Palosaari et a/.

A D;PD

14 ~,4 1thdv-4

P

(Lorcnieo et ;l., 992), sugLested( to beih soix ed rI hoth IIat1riXfortiation and the actix ation of hone remiodclinug (o30r1d / 11996). -1-he or-anic matrix of' norm-1Ial adl_llt hone, like dciltilnconsists almost exeCILsi\ Clx of type I collagcIn. huIt type' Ilicollagenl is present ill bone in cases of osteogclnCsis iliperfecta(MNilleI- ct a!.. 1977: Kirscli c/ !l., 1987: Sokolox c/ aL 1')9':Nei-licl clcila., 1993) aInd ill 1non1-nilittiI c fi attiLWr SitS (I sstonel al., 1997). The distiLibanices ill miatrix or'2aili/aiiol1in a\ tIlISbe at least partially i espolisible for the altered holne anldideitiiin ostco,crnesis inipelI'cta and( lentinioceinesis imnpei l.cta.

Anothier- possible role for MMIN s in dcitin m.ia be theregulatioll of pcritUibular dentin forilmitioll. C ollagcen is asignificant componIeIlt of' pcntInbh Lla SpICc (ID)a i (.L 1 91ITorneck. 1 994). and tylpe Iii collageen is also piCesent (Nagata c

a!.. 1992: Waltimo ci aTl.. i994). \cil tlhonIlt tIlc Mode ofperittubular delitini formnation is not cleai. it occrils oil1\ it thlpresenice of \ [able odontoblast process (Linde and( ioldbrcr.i993). Odontoblast piocess miay tIltLS piodUCC anI orgItllicinatrix that is acti\cIv rniincralized as a resmilt of odolntoblastactivity (llolland, 1985: Toi neck. 1994). SilIce tilC pCitulbLlardentini is practically de oid 1of ibrous strtictLic. tIlc olialiCmatr'ix tIllSt he degraided bCefloie oi dUnilc tilC 11inera I/iation.and MMPs may be essenitial in tllis process Also, duCiinc tIlecaries progression IMMNI 1snay also hc rIqtCiC,ed lfor tIldegradationi olfthie inLItrix derminciali/ed hevoi I the possibilityfor reincrinialization, thIls exposilng the iClmilneieia li/ble itlatrixtor the sali ary minicial ions. FinaIlly MMIN s ol thcdeltill-pillpcomplex may he nieeded lor tIlC iinCodCliCIc of pIllpalconnlecti e tisstc.

LVIen though1 the fulictiolI1i roles of IMMI s ini tilC deittillpulip coIlplex remaini to be elucidated, thcse Iirididits. tocetiherxxith the previons data (Tjhiderhane al. 1998a h), i itdicItethat MMI s inidecd has e an essential role in tIlc rCgulatioi of'physiological and, obxiously, pathological pi'Ocesses iil dentaltissuLCs. CuLr-rcntly, thiese qlueStiOnlS ar' Undei Lacti\sinivestigation in OuIr laboratoiiess The recenitly pi'esented orgmilCtiltule mcthlods lor olonitoblasts addIpullp tiSSUC (M.lajo1irC C/

fl., 1996. TJliderrhane el al., 1998a) of fei x\afiblhIC tools toovercome the problems prexviotisly calised hb the comnplexiti ofthle tooth stiSctLilC

ACKNOWLEDGMENTS

We gicatly appicciatc Di. Airi I insilnei and Itis stiff at tllcFinnishi Student leatlt C enltl'C OnIn.I flor pmrox idiltg tilc tCCtilor the cultul-cs. We thalik Ms. SLIIna Jl,tnnenCN,Ms. Sirpa

B D

PD

p

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P& 0

. , , 8.1~I1,.

P;~ f

Figure 3. Immunohistochemicalstaining. Using MMP-8-specificantibody, we stained frozen sections ofintact teeth to localize the presence ofMMP-8. (A) Some of the odontoblastsdemonstrated distinct staining(arrows). In the pulp, no markedstaining could be observed. Dmineralized dentin, PD predentin; P,pulp. Magnification, 250x. (B) Nostaining was observed in theodontoblast layer (OB) of the untreatedcontrol sections Magnification 250x.(C) Some but not all pulp capillaryendothelial cell pulp exhibited intensestaining. Magnification, 1 000x.

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MMP-8 Expression in Dental Tissues

Kangas, and Ms. Eeva-Maija Kiljander for their skillfullaboratory work. The study was financially supported by theAcademy of Finland and the Finnish Dental Society.

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