Basic Research—Biology

Down-regulation of Inflammatory Mediator Synthesisand Infiltration of Inflammatory Cells by MMP-3in Experimentally Induced Rat PulpitisKoyo Takimoto, DDS,* Nobuyuki Kawashima, DDS, PhD,* Noriyuki Suzuki, DDS, PhD,*Yu Koizumi, DDS, PhD,* Mioko Yamamoto, DDS,* Misako Nakashima, DDS, PhD,†

and Hideaki Suda, DDS, PhD*

Abstract

Introduction: Matrix metalloproteinase (MMP)-3 is amember of the MMP family that degrades the extracel-lular matrix. Application of MMP-3 to injured pulp tissueinduces angiogenesis and wound healing, but its anti-inflammatory effects are still unclear. Here, we evalu-ated the anti-inflammatory functions of MMP-3in vitro and in vivo. Methods: Nitric oxide and in-flammatory mediator synthesis in macrophages acti-vated by lipopolysaccharide (LPS) was measured in thepresence or absence of MMP-3. The mouse Mmp3(mMmp3) expression vector containing full lengthcDNA sequence of mMmp3 or cDNA sequence ofmMmp3 missing the signal peptide and pro-peptide re-gions was transfected to RAW264, a mouse macro-phage cell line, and NO synthesis and inflammatorymediator expression were evaluated. Pulpal inflamma-tion was histologically and immunohistochemically eval-uated in a rat model of incisor pulpitis induced by theapplication of LPS for 9 hours in the presence or absenceof MMP-3. Results: NO and pro-inflammatory mediatorsynthesis promoted by LPS was significantly down-regulated by MMP-3 in vitro. The full length ofmMmp3 down-regulated the LPS-induced NO synthesisand chemical mediator mRNA expression, however themMmp3 missing the signal peptide failed to block theNO synthesis induced by LPS. The numbers of major his-tocompatibility complex class II+ and CD68+ cells,which infiltrated into the rat incisor pulp tissues inresponse to the topical application of LPS, were signifi-cantly decreased by the application of MMP-3 in vivo.Conclusions: These results indicate that MMP-3 pos-sesses anti-inflammatory functions, suggesting its po-tential utility as an anti-inflammatory agent for pulpalinflammation. (J Endod 2014;-:1–6)

From the *Department of Pulp Biology and Endodontics, DivisionUniversity, Tokyo, Japan; and †Department of Dental Regenerative Mand Gerontology, Research Institute, Aichi, Japan.

Address requests for reprints to Dr Nobuyuki Kawashima, Departand Dental Sciences, Tokyo Medical and Dental University, 1-5-45 B0099-2399/$ - see front matter

Copyright ª 2014 American Association of Endodontists.http://dx.doi.org/10.1016/j.joen.2014.04.001

JOE — Volume -, Number -, - 2014

Key WordsCytokine(s), inflammation, matrix metalloproteinase-3, pulp biology, pulpitis

Pulpitis is an inflammatory disease after the development of caries or dental traumaticlesions, and it is caused by the immunological host defense reaction activated by

bacterial infection (1). Bacterial invasion in the pulp tissue activates resident immuno-competent cells and induces infiltration of immunocompetent cells. These cells pro-duce large amounts of chemical mediators that typically characterize pulpal pathosis.The initial and fundamental response against exogenous stimuli in the pulp tissue isinnate immunity, and macrophages are typical members of this innate immunity (2).After the development of carious lesions, the amount of exogenous stimuli overcomesthe removal capacity of resident macrophages, leading to infiltration of activated mac-rophages and/or neutrophils, which characterizes the pathogenesis of acute pulpitis.Various proinflammatory mediators, such as nitric oxide (NO), interleukin (IL)-1,IL-6, and tumor necrosis factor alpha (TNF-a), are produced by macrophages inthe pulpitis (1, 3). Prostaglandins synthesized by cyclooxygenase 2 (Cox 2) are alsoproduced in the inflamed pulp (4). These mediators characterize pulpal pathosisand determine the establishment of pulpitis. Overproduction of these proinflammatorymediators further stimulates their production, which induces a vicious cycle. Pulp tissueis encased in dentin hard tissue, resulting in low compliance (5), and it is essential toregulate these overexpressed chemical mediators to avoid pulpal necrosis. In addition,the control of inflammation is very important for pulp regeneration (6) and regener-ative endodontics (7).

Matrix metalloproteinase (MMP)-3 is a member of the MMP family, typical pro-teinases that degrade the extracellular matrix. MMPs affect migration, differentiation,growth, inflammatory processes, angiogenesis, and apoptosis (8). Furthermore,MMPs are involved in various physiological and pathological processes such as jointdisorders, cancer, and coronary heart disease (9). However, MMP-3 is also reportedto inactivate proinflammatory mediators (10), enhance clearance of inflammatorycells, and regulate inflammatory conditions (11, 12). These reports indicate thatMMPs are involved in both tissue destruction and wound healing.

MMP-3 expression has been observed in healthy pulp tissue (13), and it isinvolved in remodeling of the dentin matrix (14), suggesting that MMP-3may be relatedto the maintenance of pulpal homeostasis. Furthermore, MMP-3 expression has beenobserved in inflamed (13) and injured (15) pulp tissue. Recently, dental pulp stem

of Oral Health Sciences, Graduate School of Medical and Dental Sciences, TokyoMedical and Dentaledicine, Center of Advanced Medicine for Dental and Oral Diseases, National Center for Geriatrics

ment of Pulp Biology and Endodontics, Division of Oral Health Sciences, Graduate School of Medicalunkyo-ku, Tokyo, 113-8549 Japan. E-mail address: kawashima.n.endo@tmd.ac.jp

Inflammatory Mediator Synthesis 1

Basic Research—Biology

cells were reported to synthesize large amounts of MMP-3 (16). More-over, the application of MMP-3 to injured pulp tissue induces angiogen-esis (17). These reports suggest that MMP-3 may be involved in woundhealing of pulp tissue. However, the anti-inflammatory effects have notbeen fully evaluated for MMP-3. In this study, we evaluated the anti-inflammatory properties of MMP-3 in pulpal inflammation in vitroand in vivo.

Materials and MethodsNitric Oxide Assay and Bead Array

All animal experiments were approved by the Institutional AnimalCare and Use Committee of Tokyo Medical and Dental University(#0120156A). Six-week-oldmaleWistar rats (N=2; Clea Japan, Tokyo,Japan) was used as the source of macrophages. Resident peritonealmacrophages were obtained after intraperitoneal injection of a 20 mLcold saline solution. Macrophages were cultured in RPMI-1640(Wako Pure Chemical Industries, Osaka, Japan) supplemented with10% FBS (60�C heat-inactivated; Thermo Scientific HyClone, Logan,UT, USA) and a 1% penicillin-streptomycin-amphotericin B solution(Wako).

Macrophages (3 � 104 cells/well in 96-well plate; NO synthesis,1.6 � 105 cells/well in 96-well plates; bead array) were stimulatedwith LPS (100 ng/mL; E.Coli 0111:B4; Sigma-Aldrich, St Louis, MO)in the presence or absence of MMP-3 (100 ng/mL; EMD Millipore; Bill-erica, MA) for 20 hours. Non-LPS-stimulated macrophages were usedas a control. NO synthesis was estimated by the accumulation of nitritethat was measured by the Griess reagent (1% sulfanilamide, 0.1% N-1-naphthylethylene-diamine dihydrochloride, and 2.5% phosphoric acid;Sigma-Aldrich). The Bradford protein assay was used to measure thetotal protein (Protein Quantification Kit-Rapid, Dojindo, Kumamoto,Japan). Cytokines in the culture medium were measured by Bio-PlexSuspension Array System (Bio-Rad Laboratories, Hercules, CA) usingRat Cytokine/Chemokine magnetic beads panel 96-well plate assay kit(EMD Millipore).

Construction of Eukaryotic Expression Vectorsand Transfection

The full-length (FL) open reading frame of mouse (m)Mmp3complementary DNA (cDNA) was subcloned into the pEF-Dest51, aneukaryotic expression vector for synthesis of C-terminally V5- and6xHis-tagged proteins (Life Technologies, Carlsbad, CA) to createpEF-mMmp3FL. mMmp3 cDNAs with the original signal sequenceand propeptide sequence removed was also subcloned into pEF-Dest51 to create pEF-mMmp3SP&PP-. An enhanced green fluorescentprotein (EGFP) expression vector was used as a control (pEF-EGFP).Protein expression was detected by Western blotting using an anti-V5antibody (1:2000, clone: V5005; Nacalai Tesque, Kyoto, Japan),which was probed with an HRP-conjugated anti-mouse IgG secondaryantibody (1:1000, Vector Laboratories, Burlingame, CA), andchemiluminescence HRP substrate (Immobilon, EMD Millipore).Chemiluminescent images were detected by LAS3000 (Fuji Film,Tokyo, Japan). RAW264, a mouse macrophage-like clonal cell line(Riken BRC, Tsukuba, Japan) cells were cultured in High Glucose D-MEM (Wako) supplemented with 10% FBS (60�C heat-inactivated;Thermo Scientific HyClone) and a 1% penicillin-streptomycin-amphotericin B solution (Wako) at 37�C with 5% CO2. Theexpression vectors were transfected into RAW264 cells using FuGENE(Roche, Basel, Switzerland). After 48 hours, LPS (100 ng/mL; E. coli0111:B4, Sigma-Aldrich) was added to the transfected cells (2 � 105

cells/well in 96-well plate; NO synthesis, 1 � 106 cells/well in 24-well plate; RT-PCR) which were cultured for 20 hours. NO synthesiswas measured by the Griess reagent as described previously.

2 Takimoto et al.

RT-PCRTotal RNA was extracted from RAW264 cells with Quick GeneMini-

80 (Fuji film). cDNA was synthesized from total RNA (300 ng) usingRevertAid H Minus Reverse Transcriptase (Thermo Fisher ScientificInc., Waltham, MA) and an oligo (dT) 18. Quantitative RT-PCR was per-formed using GoTaq qPCR Master Mix (Promega, Madison, WI) on aCFX96 (Bio-Rad). Quantitative RT-PCR amplification was performedat 95�C for 15 seconds, and 60�C for 60 seconds for 45 cycles.Specific primers and product sizes (in parentheses) were as follows:b-actin: 50-AGGGAAATCGTGCGTGACAT-30 and 50-AACCGCTCGTTGC-CAATAGT-30 (130 bp); IL-1b: 50-ACCCAAGCACCTTCTTTTCC-30 and50-GTTTGGGATCCACACTCTCC-30 (351 bp); IL-6: 50-ATGTTGTTGA-CAGCCACTGC-30 and 50-AAACGGAACTCCAGAAGACC-30 (314 bp);Cox2: 50-AGTATCAGAACCGCATTGCC-30 and 50-TAAGGTTTCAGGGA-GAAGCG-30 (310 bp). Primers for IL-1b, 6 and Cox2 were designedfor rat targets, and slight difference of nucleic acid sequences occurred.However, there was no difference at 3’ end of their sequence, and theywork well on mice samples.

Induction of PulpitisSix-week-oldmale Wistar rats (N=18; Clea Japan) were given free

access to food and water. After anesthesia induced by an intraperitonealinjection of a mixture of ketamine hydrochloride (2.5 mg/100 g bodyweight; Sankyo, Tokyo, Japan) and sodium pentobarbital (2.5 mg/100 g body weight [Somnopentyl; Kyoritsu Seiyaku Corp, Tokyo,Japan]), 2 mm of mandibular left and right incisor crowns wereremoved by a diamond disc to expose the pulp chamber. After disinfec-tion with 70% ethanol, the coronal pulp chamber was enlarged by ster-ilized K-files up to #40. LPS (10 mg/mL, 1 mL), sterile saline (1 mL), orLPS (10 mg/mL, 1 mL) plus MMP-3 (100 mg/mL, 1 mL) were applied tothe exposed upper incisor pulps with a sterile paper point (Johnson &Johnson, New Brunswick, NJ). The cavity was sealed with Caviton (GC,Tokyo, Japan). Rats were sacrificed under anesthesia at 9 hours post-operatively, and the upper incisors were extracted. Infiltration of mac-rophages in response to LPS has been reported to peak at 9 hours in aprevious study (18).

ImmunohistochemistryPulp tissues were fixed with 4% paraformaldehyde at 4�C over-

night and then decalcified with 15% EDTA at 4�C for 4 weeks. Sampleswere embedded in Tissue-Tek O.C.T. Compound (Sakura, Tokyo,Japan) and quickly frozen in hexane (Wako) cooled by dry ice. Immu-nohistochemical staining was performed on sections (7-mm thickness)with mouse antirat major histocompatibility complex (MHC) class IIRT1B (1:10,000, clone: OX6; Serotec, Oxford, UK) and mouse antiratCD68 (1:100, clone: ED1; Serotec) antibodies by incubation at 4�Covernight. Sections were then incubated with biotinylated horse anti-mouse IgG (1:1000; Vector Laboratories) at room temperature for30 minutes followed by avidin-biotin-peroxidase complex(VECTASTAIN Elite ABC Reagent, R.T.U, Vector Laboratories) at roomtemperature for 30 minutes. Colorization was performed with 3,30-diaminobenzidine (DAB; ImmPACT DAB Peroxidase Substrate, VectorLaboratories). Sections were counterstained with methyl green (MutoPure Chemicals, Tokyo, Japan).

Positively stained inflammatory cells on 3 typical sections fromeach sample were counted in 10 high-power fields (�20:objective, �10: ocular) of the dental pulp tissue excluding abscesses,blood vessels, and dentin. The 3 typical sections were selected at inter-vals of 10 serial sections containing the majority of the dental pulp tis-sue. The average number of cells in the 10 visual fields of selectedsections was regarded as representative.

JOE — Volume -, Number -, - 2014

Figure 1. The in vitro effects of MMP-3 on NO and inflammatory mediator synthesis in rat peritoneal macrophages. MMP-3 blocked NO synthesis in LPS-stimulatedrat peritoneal macrophages. LPS induced NO synthesis rat peritoneal macrophages, which was down-regulated by treatment with MMP-3. (A) NO concentration wasnormalized by total protein amount (mg). Mean � standard deviation (n = 10). Synthesis of IL-6, IL-17A, and MCP-1 was significantly up-regulated by LPS, buttreatment with MMP-3 down-regulated their synthesis (B, C, and D). Mean � standard deviation (n = 5). *P < .05, **P < .01.

Basic Research—Biology

Statistical AnalysisNO and inflammatory mediator synthesis, inflammatory mediator

synthesis, inflammatory mediator expression, and the number of posi-tively stained cells were evaluated statistically using the Tukey-Kramerpost hoc test. Statistical significance was set at P < .05.

ResultsEffects of MMP-3 on NO and Inflammatory Synthesis

NO and inflammatory mediator synthesis was up-regulated in LPS-stimulated rat peritoneal macrophages, but MMP-3 treatment with LPSsignificantly down-regulated NO and inflammatory mediator synthesis(Fig. 1A–D). There was no effect of MMP-3 on mediator synthesis innormal rat peritoneal macrophages.

Enforced Expression of mMmp3The sizes of the original mMmp3 peptide (pEF-mMmp3FL) and

the mMmp3 peptide missing the signal sequence and propeptidesequence (pEF-mMmp3SP&PP-) were approximately 60 and 55 kDa,respectively (Fig. 2A). NO synthesis was significantly down-regulatedby pEF-mMmp3FL, but not pEF-mMmp3SP&PP- (Fig. 2B). mRNAexpression of pro-inflammatory mediators (IL-1b, IL-6, and Cox2)was highly up-regulated in LPS stimulated RAW264 cells, whichwas significantly down-regulated by pEF-mMmp3FL transfection(Fig. 2C–E).

JOE — Volume -, Number -, - 2014

Evaluation of MHC Class II+ and CD68+ CellsDilatation of blood vessels and severe infiltration of MHC class II+

and CD68+ cells were induced by LPS application in the pulp tissue(Fig. 3A and B). Most of the infiltrating cells showed a round or ovalmorphology, indicating active phagocytizing macrophages expressingclass II antigens. MHC class II+ cells removed exogenous materialsand presented the antigens on their cell surfaces. MMP-3 drasticallyblocked the infiltration of macrophages into pulp tissues induced byLPS application (Fig. 3A and B). The numbers of MHC class II+ andCD68+ cells in the LPS +MMP-3 group were similar to that in the sterilesaline group (Fig. 3C and D). Histologically, rat dental pulp treated withthe same dose of MMP-3 was similar to control pulp tissues treated withsterile saline.

DiscussionIn the present study, MMP-3 significantly down-regulated NO syn-

thesis and inflammatory cytokine expression in LPS-stimulated macro-phages in vitro. MMP-3 is reported to degrade active cytokines such asIL-1b (19, 20) and chemokines (21), suggesting direct proteolysis ofmediators. MMP-3 may function as a nuclear factor (22), but the non-secretory type of MMP-3, which was produced by pEF-mMmp3SP&PP-,did not down-regulate NO synthesis induced by LPS applicationin RAW264 cells (Fig. 2B). Furthermore, MMP-3 did not affect prolif-eration or apoptosis of RAW264 cells (data not shown). The precisemechanisms of the regulatory functions of MMP-3 are still unclear,

Inflammatory Mediator Synthesis 3

Figure 2. Enforced expression of mMmp3 in RAW264 cells. mMmp3 peptides synthesized by pEF-mMmp3FL and pEF-mMmp3SP&PP- cells were approximately60 and 55 kDa, respectively (A). NO synthesis induced by LPS was down-regulated by enforced expression of mMmp3 (pEF-mMmp3FL), but not nonsecretorymMmp3 (pEF-mMmp3SP&PP-) in RAW264 cells. NO concentration was normalized by total protein amount (mg). (mean � standard deviation, n = 5) (B).mRNA expression of IL-1b, IL-6, and Cox2, which was up-regulated by LPS, was significantly down-regulated by pEF-mMmp3FL transfection in RAW264 cells(C, D, and E). Mean � standard deviation (n = 5). EGFP, pEF-EGFP; FL, pEF-mMmp3FL; DSP&PP, pEF-mMmp3SP&PP-. **P < .01.

Basic Research—Biology

but MMP-3 may act mainly on mediators in the extracellular matrix,which are secreted from cells as paracrine or autocrine factors.

Inflammation is characterized by proinflammatory mediators suchas NO and cytokines. NO is a free radical gas and plays important roles ininflammation. Previously, we reported the involvement of NO in the pro-gression of experimentally induced rat pulpitis (18). NO and variousother mediators, including IL-1, IL-6, IL-10, IL-12, interferon-g,TNF-a, and Cox2, are highly expressed in rat pulpitis (4). This mediatornetwork regulates the progression of lesions (4), indicating that theproduction of proinflammatory mediators further induces these proin-flammatory mediators. This vicious cycle exacerbates pulpal inflamma-tion, and its blockade and removal of the infection are the targets torelieve pulpal inflammation. The blockade of NO synthesis by an induc-ible NO synthase (iNOS)-specific inhibitor drastically down-regulates

4 Takimoto et al.

proinflammatory mediator production and infiltration of immunocom-petent cells into the rat pulpitis (18). The present study showed thatMMP-3 down-regulated the synthesis of various proinflammatory medi-ators. However, it is unclear whether there are different inhibitory ac-tions of MMP-3 on each mediator although MMP-3 may regulate theproinflammatory mediator network globally. In vivo, a severe accumu-lation of macrophages in the incisor pulp in response to LPS was in-hibited by MMP-3 application, suggesting its partial function in thesuppression of chemokine synthesis by activated macrophages.Down-regulation of MCP-1 (Figs. 1C), a typical chemokine for macro-phages (23), may contribute to the reduction of macrophage infiltra-tion. MMP-3 is also reported to produce antagonistic factors bycleaving the N-terminus of MCP-1 (24), suggesting anti-inflammatoryfunctions of MMP-3. We applied MMP-3 to rat pulp tissue with LPS to

JOE — Volume -, Number -, - 2014

Figure 3. In vivo effects of MMP-3 on experimentally induced rat pulpitis. MMP-3 blocked the infiltration of immunocompetent cells into experimentally inducedrat pulpitis. The application of LPS to rat incisors induced infiltration of (A) MHC class II+ and (B) CD68+ cells into the pulp tissue, which was blocked by theapplication of MMP-3 (A and B). The numbers of (C) MHC class II+ and (D) CD68+ cells were significantly reduced by MMP-3. The numbers of MHC class II+ andCD68+ cells in LPS-treated rat incisors applied with MMP-3 were similar to those in the saline-treated samples (C and D). Mean � standard deviation (n = 6).*P < .05. **P < .01.

Basic Research—Biology

reveal the effects of MMP-3 on the acute phase of inflammation. Furtherstudy is necessary to evaluate the effects of MMP-3 on the chronic phaseof inflammation using other experimental pulpitis models of rat molarsbecause rat incisors grow continuously. In a canine irreversible pulpitismodel, similar effects of MMP-3 have been reported (25).

Generally, MMP-3 is classified as an inflammatory mediator, andits synthesis has been reported in chemically stimulated dental pulp

JOE — Volume -, Number -, - 2014

cells (26) and periapical lesions (27). However, MMP-3 knockoutmice show delayed wound healing compared with that of wild-typemice (28), suggesting protective functions of MMP-3. The anti-inflammatory effects of MMP-3 revealed in this study may contributeto such tissue repair.

Control of pulpal inflammation and removal of bacterial infectionsare essential to recover the integrity of pulp tissue from pulpitis

Inflammatory Mediator Synthesis 5

Basic Research—Biology

(29, 30). Our findings support the anti-inflammatory functions of MMP-3, suggesting the use of MMP-3 as a novel therapeutic for the treatmentof pulpal inflammation. In conclusion, MMP-3 possesses anti-inflammatory functions in vitro and in vivo, suggesting its potentialusefulness as an anti-inflammatory agent for pulpal inflammation.

AcknowledgmentsSupported by grants from the Japan Society for the Promotion

of Science (22390357 and 22659343) and the Global Center ofExcellence (GCOE) Program, International Research Center for Mo-lecular Science in Tooth and Bone Diseases.

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

References1. Kawashima N, Suda H. Immunopathological aspects of pulpal and periapical inflam-

mations. In: In Ørstavik D, Pitt Ford T, eds. Essential Endodontology, 2nd ed. Ox-ford: Blackwell Publishing; 2008:44–80.

2. Jontell M, Okiji T, Dahlgren U, et al. Immune defense mechanisms of the dentalpulp. Crit Rev Oral Biol Med 1998;9:179–200.

3. Fouad AF. Molecular mediators of pulpal inflammation. In: Hargreaves KM,Goodis HE, eds. Seltzer and Bender’s Dental Pulp, 1st ed. Chicago: QuintessencePublishing; 2002:247–79.

4. Kawashima N, Nakano-Kawanishi H, Suzuki N, et al. Effect of NOS inhibitor on cyto-kine and COX2 expression in rat pulpitis. J Dent Res 2005;84:762–7.

5. Kim S, D€orscher-Kim J. Haemodynamic regulation of the dental pulp. In: Inoki R,Kudo T, Orgart L, eds. Dynamic Aspects of Dental Pulp, 1st ed. London: Chapmanand Hall; 1990:167–98.

6. Nakashima M, Iohara K, Sugiyama M. Human dental pulp stem cells with highlyangiogenic and neurogenic potential for possible use in pulp regeneration. CytokineGrowth Factor Rev 2009;20:435–40.

7. Murray PE, Garcia-Godoy F, Hargreaves KM. Regenerative endodontics: a review ofcurrent status and a call for action. J Endod 2007;33:377–90.

8. Nagase H, Visse R, Murphy G. Structure and function of matrix metalloproteinasesand TIMPs. Cardiovasc Res 2006;69:562–73.

9. Murphy G, Nagase H. Progress in matrix metalloproteinase research. Mol AspectsMed 2008;29:290–308.

10. Murphy G, Nagase H. Reappraising metalloproteinases in rheumatoid arthritis andosteoarthritis: destruction or repair? Nat Clin Pract Rheumatol 2008;4:128–35.

11. Page-McCaw A, Ewald AJ, Werb Z. Matrix metalloproteinases and the regulation oftissue remodeling. Nat Rev Mol Cell Biol 2007;8:221–33.

12. Parks WC, Wilson CL, L�opez-Boado YS. Matrix metalloproteinases as modulators ofinflammation and innate immunity. Nat Rev Immunol 2004;4:617–29.

6 Takimoto et al.

13. Gusman H, Santana RB, Zehnder M. Matrix metalloproteinase levels and gelatinolyticactivity in clinically healthy and inflamed human dental pulps. Eur J Oral Sci 2002;110:353–7.

14. Palosaari H, Pennington CJ, Larmas M, et al. Expression profile of matrix metallo-proteinases (MMPs) and tissue inhibitors of MMPs in mature human odontoblastsand pulp tissue. Eur J Oral Sci 2003;111:117–27.

15. Yoshiba N, Yoshiba K, Ohkura N, et al. Expressional alterations of fibrillin-1 duringwound healing of human dental pulp. J Endod 2012;38:177–84.

16. Iohara K, Zheng L, Wake H, et al. A novel stem cell source for vasculogenesis inischemia: subfraction of side population cells from dental pulp. Stem Cells 2008;26:2408–18.

17. Zheng L, Amano K, Iohara K, et al. Matrix metalloproteinase-3 accelerates woundhealing following dental pulp injury. Am J Pathol 2009;175:1905–14.

18. Kawanishi HN, Kawashima N, Suzuki N, et al. Effects of an inducible nitric oxide syn-thase inhibitor on experimentally induced rat pulpitis. Eur J Oral Sci 2004;112:332–7.

19. Ito A, Mukaiyama A, Itoh Y, et al. Degradation of interleukin 1 beta by matrix metal-loproteinases. J Biol Chem 1996;271:14657–60.

20. Schonbeck U, Mach F, Libby P. Generation of biologically active IL-1 beta by matrixmetalloproteinases; a novel caspase-1-independent pathway of IL-1 beta processing.J Immunol 1998;161:3340–6.

21. McQuibban GA, Gong JH, Tam EM, et al. Inflammation dampened by gelatinase Acleavage of monocyte chemoattractant protein-3. Science 2000;289:1202–6.

22. Eguchi T, Kubota S, Kawata K, et al. Novel transcription-factor-like function of hu-man matrix metalloproteinase 3 regulating the CTGF/CCN2 gene. Mol Cell Biol 2008;28:2391–413.

23. Mukaida N, Harada A, Matsushima K. Interleukin-8 (IL-8) and monocyte chemo-tactic and activating factor (MCAF/MCP-1), chemokines essentially involved in in-flammatory and immune reactions. Cytokine Growth Factor Rev 1998;9:9–23.

24. McQuibban GA, Gong JH, Wong JP, et al. Matrix metalloproteinase processing ofmonocyte chemoattractant proteins generates CC chemokine receptor antagonistswith anti-inflammatory properties in vivo. Blood 2002;100:1160–7.

25. Eba H, Murasawa Y, Iohara K, et al. The anti-inflammatory effects of matrixmetalloproteinase-3 on irreversible pulpitis of mature erupted teeth. PLoS One2012;7:e52523.

26. Kim RH, Williams DW, Bae S, et al. Camphorquinone inhibits odontogenic differen-tiation of dental pulp cells and triggers release of inflammatory cytokines. J Endod2013;39:57–61.

27. Menezes-Silva R, Khaliq S, Deeley K, et al. Genetic susceptibility to periapical disease:conditional contribution of MMP2 and MMP3 genes to the development of periap-ical lesions and healing response. J Endod 2012;38:604–7.

28. Bullard KM, Lund L, Mudgett JS, et al. Impaired wound contraction in stromelysin-1-dificient mice. Ann Surg 1999;230:260–5.

29. Cooper PR, Takahashi Y, Graham LW, et al. Inflammation-regeneration interplay inthe dentine-pulp complex. J Dent 2010;38:687–97.

30. Fuks AB. Vital pulp therapy with new materials for primary teeth: new directions andtreatment perspectives. J Endod 2008;34:18–24.

JOE — Volume -, Number -, - 2014