Basic Research—Biology

Evaluation of Gelatinases, Tissue Inhibitor of MatrixMetalloproteinase-2, and Myeloperoxidase Proteinin Healthy and Inflamed Human Dental Pulp TissueThais Accorsi-Mendonca, DDS, MSc, PhD,* Emmanuel Jo~ao Nogueira Leal Silva, DDS, MSc,

†

Andrea M�arcia Marcaccini, DDS, MSc, PhD,‡Rachel Fernanda Gerlach, DDS, MSc, PhD,

‡

Keila Maria Roncato Duarte, MS, PhD,§Ana Paula Souza Pardo, DDS, MSc, PhD,

k

S�ergio Roberto Peres Line, DDS, MSc, PhD,kand Alexandre Augusto Zaia, DDS, MSc, PhD

†

Abstract

Introduction: The aim of this study was to compare thegelatinolytic activity of matrix metalloproteinase(MMP)-2 and MMP-9 and the expression of tissue inhib-itor of matrix metalloproteinase (TIMP)-2 and myeloper-oxidase protein (MPO) in clinically healthy human pulpand inflamed pulp tissue specimens. Methods: Twentydental pulps clinically diagnosed as inflammatorytissues and 20 healthy pulp tissues from enclosed thirdmolars were harvested and evaluated. The gelatinolyticactivity for MMP-2 and MMP-9 was assessed by usingthe zymography technique, TIMP-2 gene expressionwas evaluated using the enzyme-linked immunosorbentassay, and MPO was determined using the MPO assay.Results: Data showed increased levels of MMP-9,active MMP-2, TIMP-2, and MPO in inflammatory pulptissues compared with healthy tissues (P < .05). Nostatistical difference could be observed for pro-MMP-2(P > .05). Conclusions: Although all samples wereassociated with MMP-2 expression, the active form ofthis MMPwas observed only in inflamed pulps. Inflamedpulps showed an up-regulation of MMP-9, TIMP-2, andMPO. (J Endod 2013;39:879–882)

Key WordsGelatinases, inflammation, pulp biology

From the *Department of Endodontology, Grande RioUniversity, Rio de Janeiro, Brazil; †Department of RestorativeDentistry, Endodontics Division, Piracicaba Dental School, StateUniversity of Campinas, Piracicaba, S~ao Paulo, Brazil;‡Department of Buco-Maxilo-Facial Surgery and Periodon-tology, Dental School of Riber~ao Preto, University of S~ao Paulo,Ribeir~ao Preto, S~ao Paulo, Brazil; §SAA/APTA/Institute ofAnimal Sciences and Pastures, Nova Odessa, S~ao Paulo, Brazil;and kDepartment of Morphology, Piracicaba Dental School,State University of Campinas, Piracicaba, S~ao Paulo, Brazil.

Address requests for reprints to Dr Alexandre Augusto Zaia,Department of Restorative Dentistry, Endodontics Division, Pi-racicaba Dental School, State University of Campinas, Av Lime-ira 901, Piracicaba-SP, CEP-13414-018, Brazil. E-mail address:zaia@fop.unicamp.br0099-2399/$ - see front matter

Copyright ª 2013 American Association of Endodontists.http://dx.doi.org/10.1016/j.joen.2012.11.011

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Extracellular matrix (ECM) protein destruction is thought to occur in pulpal inflamma-tion, andmatrix turnover requires the activity of many different endopeptidases acting

on a variety of compositionally distinct proteins. Matrix metalloproteinases (MMPs) arean important group of zinc enzymes responsible for the degradation of extracellularmatrix components such as collagen and gelatin. They are involved in normal remodelingprocesses such as embryonic development; postpartum involution of the uterus, bone,and growth plate remodeling; wound healing; and several disease processes such as jointdestruction in rheumatoid and osteoarthritis, tumor invasion, and periodontitis (1–3).MMP-2 andMMP-9, also referred to as gelatinases, are of particular interest because theyhave been shown to play an important role in the pathogenesis of the chronic inflamma-tory process and in periodontal and periapical tissue destruction (3–6).

The biological activities of MMPs can be regulated post-transcriptionally, includingtheir interaction with specific tissue inhibitors of matrix metalloproteinases (TIMPs)(7). At least 4 members of the TIMP family are characterized, and TIMP-2 is themost effective against gelatinases (8). In the pulp inflammatory process, the regulationof host MMPs by their endogenous inhibitors (TIMPs) is not well understood. OnlyTIMP-1, which is present in parotid and submandibular saliva (9), gingival crevicularfluid (10), and human dentine (11), has been shown to have a regulatory homeostaticeffect on activities of MMPs in dental tissues and during odontogenesis but not duringdisease. Moreover, a study on the effect of synthetic MMP inhibitors (chemically modi-fied tetracyclines and zoledronate) showed a markedly reduced progression of dentinalcaries in rats (12). The factors that regulate MMP/TIMP synthesis and secretion maybe important in the pathogenesis of pulp inflammation. Information on TIMP expres-sion and role in the extracellular matrix degradation of pulp tissue is limited.

Neutrophil-derived myeloperoxidase (MPO) is found in primary granules fromneutrophils, and these cells are regarded as one of the main sources of MMPs (13).It has been established that the level of MPO activity is directly correlated with thenumber of polymorphonuclear leukocytes (PMNs) in the tissues, and, therefore,MPO activity is used in studies to assess the degree of inflammatory reaction (14,15). Some dental studies on the use of MPO in gingival cervical fluids have shownthat MPO is increased in the gingival cervical fluid collected from inflamed sites(14–16). Because elevated MPO levels represent the extent of PMN infiltration, thedetermination of MPO might be a valuable tool to assess the degree of pulpinflammation. Thus, the aim of this study was to investigate the gelatinolytic activity ofMMP-2 and -9, TIMP-2, and MPO expression in healthy and inflamed dental pulps.The null hypothesis tested was that the pulp condition has no effect on the gelatinolyticactivity and the expression of TIMP-2 and MPO.

Materials and MethodsApproval for conducting the study was granted by the Institutional Review Board

(protocol 040/2005). The current investigation did not in any way alter the treatmentplan of any patient. Written consent to participate in the study was obtained from allparticipants.

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Basic Research—Biology

Human Subjects and Sample SelectionTwenty pulpal tissue specimens (10 clinically healthy and 10 in-

flamed pulps) obtained from 20 individuals at the Department ofEndodontics, Piracicaba Dental School, Campinas State University, Pira-cicaba, S~ao Paulo, Brazil, were used in this study. Medical histories of allindividuals in this study were noncontributory, and they were notreceiving any long-term anti-inflammatory or antibiotic medication.The inflamed pulps were obtained from teeth caused by carious expo-sure of the pulp and showing spontaneous pain and/or lingering pain inresponse to cold and/or heat stimulus and no radiographic periapicalpathology. Whole pulps were collected during endodontic treatment asemergencies with a diagnosis of symptomatic pulpitis. Asymptomaticpulps from impacted third molars were used as normal controls. Theteeth were grooved longitudinally with a fissure bur immediately afterextraction and then split in half.

Sample PreparationThe dental pulp samples were individually weighed. After that, the

pulps were directly transferred to microtubes containing 500 mL Dul-becco modified Eagle medium (Gibco, Grand Island, NY) and incu-bated at 37�C for 24 hours. The samples were centrifuged at 9000 gfor 10 minutes in a centrifuge (Eppendorf, Westbury, NY), and super-natant was removed and stored at�20�C for further analysis (17). Thetotal protein concentrations in pulp supernatants were determined bythe Bradford technique (18). The absorbance of the pulp sampleswas compared with that of a dilution series of bovine serum albumin.There was no statistically significant difference between groups (P =.16, unpaired t test) in dental pulp weight (data not shown).

Enzyme-linked Immunosorbent AssayLevels of TIMP-2 were measured in supernatants of human dental

pulp tissue extracts using the enzyme-linked immunosorbent assay(ELISA). The absorbance of the pulp samples was compared with thatof a dilution series of bovine serum albumin. Data were standardized tothe amount of total protein per individual pulp. Polystyrene 96-wellmicro-plates were coated with 100mL protein extracts (10mg/mL) from healthyand inflamed pulps and incubated at 37�C for 1 hour. After incubation,plates were blocked with 200 mL 1% bovine serum albumin in phos-phate-buffered saline (PBS) per well for 1 hour at 37�C. The solutionwas discharged and samples of 1:200 anti-TIMP2 (PA1-21145; AffinityBioReagents, Golden, CO) were added to each well and incubated for 1hour at 37�C and then washed 3 times with PBS containing 0.05% Tween20 and 0.25% gelatine. After the washing step, plates were incubated for 1hour at 37�Cwith 50mL peroxidase-labeled affinity purified antibody anti-rabbit (041506; KPL, Gaithersburg, MD) diluted 1:5,000 in PBS. After thisperiod, the washing step was performed again, and 50 mL substrate solu-tion was added. The substrate solution was prepared immediately beforeuse by dissolving 0.4 mg o-phenylenediamine (Sigma-Aldrich, St Louis,MO) per milliliter in 0.05 mol/L citrate buffer (pH = 5.3). The plateswere incubated for 3 minutes in darkness. The reaction was stopped byadding 50 mL H2SO4 (6 N), and optical density at 492 nm was measuredwith an ELISA reader (550; Bio-Rad, Hercules, CA). The ELISA procedureswere performed according to established protocols (18, 19). Reactionswere performed in triplicate, and the results are expressed as anaverage optical density for each determination. The TIMP-2 concentra-tions in specimens were determined from a standard curve. Results areexpressed as nanograms per milligrams of the total protein.

ZymographyThe activities of MMP-2 and MMP-9 of pulp supernatants were

measured by a gelatin zymogram protease assay as previously described

880 Accorsi-Mendonca et al.

(20, 21). Briefly, aliquots of supernatants (15 mL) were mixed witha sample buffer (0.5 m Tris-HCl, pH = 6.8, glycerol, 10% SDS, and0.1%bromophenol blue) and loaded on the gel. Then, prepared sampleswere subjected to electrophoresis on 10% sodium dodecyl sulfate(SDS)–polyacrylamide gels containing 0.1% gelatin. After electropho-resis, the gels were washed twice in 2.5% Triton X-100 (Sigma-Aldrich)for 1 hour at room temperature to remove SDS. The gels were thenincubated at 37�C for 15 hours in substrate buffer containing 50mmol/L Tris-HCl and 10 mmol/L CaCl2 at a pH level of 8.0 and stainedwith 0.5% Coomassie blue R250 in 50%methanol and 10% glacial aceticacid. Protein standards (Bio-Rad) were run concurrently, and approxi-mate molecular weights were determined by plotting the relative mobil-ities of known proteins. Gelatinolytic activities were detected as unstainedbands against the background of Coomassie blue–stained gelatin.Enzyme activity was assayed by densitometry using a Kodak Electrop-horesis Documentation and Analysis System (Kodak, Rochester, NY).

MPO AssayMPO concentrations in pulp tissue were determined using the

supernatant. The amount of MPO in each sample was measured enzy-matically by suspending the material in 2.0 mL 0.5% hexadecyltrimethy-lammonium bromide (Sigma-Aldrich, St Louis, MO) in 50 mmol/Lpotassium phosphate buffer (pH = 5.4) to solubilize the MPO (16).After that, the MPO was assayed spectrophotometrically by the additionof 1.6 mmol/L tetramethylbenzidine (Sigma-Aldrich) diluted in d-diani-sidine dihydrochloride (Sigma-Aldrich) and 0.5 mmol/L hydrogenperoxide in a Costar 96-well plate (Corning, New York, NY). The color-imetric reading was accomplished at a wavelength of 450 nm after theaddition of 4 mol/L H2SO4, and the plates were read by using a mQuantmicroplate reader (Bio-Tek Instruments, Winooski, VT). A standardcurve was generated by using human PMNs. The PMNs were isolatedfrom blood by density-gradient centrifugation and suspended to 1 �106 cells per milliliter in PBS. MPO activity was expressed as PMNper milliliter in the supernatant of pulp samples.

Statistical AnalysisAll assays were repeated 3 times to ensure reproducibility. For all

statistical analyses, the unit of observation was the patient individual.The mean patient MMP-2, MMP-9, TIMP-2, and total MPO levelsfrom the assays are presented as means � standard deviation. Theresults were subjected to the Kolmogorov-Smirnov test to evaluate thenormal distribution. All data were analyzed with parametric statisticsusing the unpaired t test. Differences were considered significantwhen P was <.05. Correlations between MPO, TIMP-2, and MMP-9were performed using the Pearson correlation test.

ResultsELISA

The ELISA was used to determine the levels of TIMP-2 in whole-pulp supernatants. Figure 1A shows the data for TIMP-2. There wasa statistically significant difference between the healthy (38.89 �25.22 ng/mL) and inflamed (244.8 � 193.9 ng/mL) groups (P =.0037, unpaired t test).

ZymographyA higher diversity of band degradation was observed in inflamed

pulp when compared with the healthy group. Figure 2 (A through C)shows 1 representative zymogram used for this quantification. Consid-ering descriptive analysis, high–molecular-weight bands ranging from95 to 86 kd with intense gelatinolytic degradation were found only in

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Figure 1. (A) TIMP-2 levels determined by ELISA from the supernatant of healthy (n = 10) and inflamed (n = 10) dental pulps. (B) MPO levels enzymaticallydetermined from the supernatant of healthy (n = 10) and inflamed (n = 10) dental pulps. *P < .05.

Basic Research—Biology

the samples from the inflamed group. The electrophoretic mobility ofthese high–molecular-mass bands was similar to that of a MMP-9 stan-dard of 92 kd. Bands with molecular masses of 72 kd consistent witha pro-MMP-2 standard and bands correspondent to active MMP-2(66 kd) were observed in both groups.

The amounts of the different molecular forms of MMP-2 werequantified in gelatin zymograms. There were no statistically significantdifferences between the healthy and inflamed groups with respect to the72-kd band of pro-MMP-2 (P = .06, unpaired t test, data not shown).

Figure 3 shows the quantification of the molecular forms of MMP-2 (active and inactive forms). Degradation bands of total MMP-2 weresignificantly more intense in the inflamed group (28,465� 8726 arbi-trary units) when compared with the healthy group (16,308 � 8872arbitrary units) as shown in Figure 3 (P = .048, unpaired t test).

MPO AssayFigure 1B shows that higher amounts of MPO concentrations were

found in the inflamed group (122.5� 153.6 PMNs/mL) compared withthe healthy group (13.94 � 2.74 PMNs/m), showing that inflameddental pulps weremore abundant and intense inMPO activity comparedwith healthy dental pulps (P = .038).

DiscussionThe proteolysis of ECM seems to be a key initiating event for the

progression of the inflammatory process. MMPs are expressed at lowlevels in the absence of inflammation, wounding, or other pathologicprocesses (7). MMP-2 and -9 are of particular interest because theyare synthesized by several pulp cell types and have been implicated inthe pathogenesis of periodontitis, oral carcinogenesis, and pulpalinflammation (1–8, 10, 11, 20–22). MMP-2 is frequently observedin radicular cysts, dentigerous cysts, and keratocystic odontogenictumors and eventually can contribute to bone resorption favoring the

Figure 2. A representative gelatin zymogram from the supernatant of healthy(lanes 2–5) or inflamed (lanes 7–11) dental pulp extracts. Lane 1: molecularweight standard for gelatinases. A, The 92-kd bands of MMP-9; B, the 72-kdbands of pro-MMP-2; and C, the 66-kd bands of active MMP-2 can beobserved.

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growing and progression of these lesions (23). Several studies showedthat inflammatory mediators play an important role in the stimulation ofMMP production by different cell lines such as fibroblasts, neutrophils,and macrophages (2–7, 22). Recent studies showed that bondingagents can up-regulate MMP-2 expression contributing to extracellularcollagen matrix degradation in hybrid layers (24) and that EDTA mighthelp protect the hybrid layer by inhibiting the MMP activity (25). Bacte-rial products may also stimulate the production of MMPs, inducing pulpinflammation and tissue destruction (26).

The zymography assay technique distinguishes active and inactiveMMPs forms. In the present study, a high gelatinolytic activity of activeMMP-9 was observed in inflamed pulps. Previous studies using differentmethodologies also showed this up-regulation of MMP-9 in inflamedpulps (4–6). In some of the inflamed pulps, it was also possible toobserve bands of active MMP-2, coinciding with a previous study thatshowed that inflammation leads to the activation of pro-MMP-2 (27).No active MMP-2 was observed in healthy samples. The cause for thenonoccurrence of active MMP-2 in apparently inflamed pulp samplesremains unknown. One possible explanation is that some pulps werenot as heavily inflamed as it appeared clinically, a phenomenon thathas been described in earlier publications (28). In inflamed pulptissues, MMP-9 gelatinolytic activity also varied considerably, suggestingthat there is a difference in the amount or intensity of the stimulus thatcauses MMP production although the clinical symptoms presented bypatients were similar. These findings confirmed previous studiesshowing that MMP-9 plays a key role in inflamed pulps (3–6) andalso shows for the first time a possible involvement of MMP-2 in pulpinflammation.

Apparently, the presence of inflammation is contributing to pro-MMP2 activation as shown by the zymography assay. The activation ofpro-MMP-2 can occur with the formation of a trielements complex

Figure 3. The total MMP-2 levels determined from the supernatant of healthy(n = 10) and inflamed (n = 10) dental pulps using zymography. *P < .05.

Gelatinases, TIMP-2, and MPO in Pulp Tissue 881

Basic Research—Biology

involving the binding of TIMP-2 to the catalytic site of MMP-14 (29, 30).Therefore, in this scenery, TIMP-2 may act ambiguously, at timesfavoring the activation or inhibiting the MMP-2 (8). This modulatingaction of TIMP-2 over MMP-2 appears to be appropriate. As wasobserved in the present study, the increased expression of bothMMP-2 and TIMP-2 in inflamed samples was associated with the pres-ence of active MMP-2 in several samples.

The inflamed sample selection was based exclusively according toclinical criteria (ie, the presence of spontaneous pain without radio-graphic changes in the periapical region). However, it is not possibleto establish a direct relationship between clinical symptoms and inflam-mation observed in light microscopy. Using neutrophil marker proteinsto assess pulpal health seems to be a promising approach because theamount of these cells is positively correlated to the inflammation state ofa pulp as assessed histologically. In the present study, this assessmentproceeded an analysis of the MPO protein, which is found exclusivelyin neutrophils. Inflamed pulps showed a higher MPO activity thanhealthy pulps (P < .05). MPO was reported to oxidatively activate latentpro-MMP-9 in vitro (31, 32). Although an up-regulation of MPO wasnot observed in healthy samples, in some inflamed samples the analysisof MPOwas negative. Again, these results confirm the lack of correlationbetween clinical symptoms and the presence of inflammatory cells in thepulp. The pain is probably associated with intrapulpal pressure, whichoccurs in the early stages of inflammation because of increased capillarypermeability without having cell migration to the connective tissue.Another explanation is the direct stimulation of nerve endings by theintense secretion of specific chemical mediators of pain or the presenceof certain bacterial toxins.

Future studies with other MMPs and correlated proteins involvedin the tissue degradation process through different stimuli may help toclarify pulp inflammation process and regulation. In conclusion,although all samples were associated with MMP-2 expression, the activeform of this MMP was observed only in inflamed pulps. Inflamed pulpsalso showed an up-regulation of active MMP-9, TIMP-2, and MPO.

AcknowledgmentsThe authors deny any conflicts of interest related to this study.

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