doi:10.1111/j.1365-2591.2012.02031.

Antibacterial activity of dentine and pulp extracellular matrix extracts

J. G. Smith, A. J. Smith, R. M. Shelton & P. R. Cooper

Oral Biology, School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK

Abstract
Smith JG, Smith AJ, Shelton RM, Cooper PR. Antibacterial activity of dentine and pulp extracellular matrix extracts. International Endodontic Journal, 45, 749755, 2012.

Aim To determine whether extracellular matrix (ECM) preparations from pulp (pECM) and dentine (dECM) possess antimicrobial activity. Methodology Dentine and pulp ECM preparations were isolated with 10% ethylenediaminetetraacetic acid (EDTA), pH 7.2 and sequential use of 0.5 mol L)1 NaCl, pH 11.7 and 0.1 mol L)1 tartaric acid, pH 2.0, respectively, with protease inhibitor inclusion throughout. Antimicrobial activity against Streptococcus mutans, Streptococcus oralis and Enterococcus faecalis was assessed using turbidity as a measure of bacteria growth. The cytotoxicity of the extracts on primary pulp cells was also determined by

lactate dehydrogenase (LDH) release. Statistical analysis of data was performed using paired students t-tests. Results Extracellular matrix extracts from the pulp and dentine showed antibacterial activity against three types of anaerobic bacteria associated with dental disease (P < 0.05). The ECM extracts demonstrated no signicant cytotoxic effect on pulpal cells at the concentrations used for antibacterial activity. Conclusions The bacteriostatic antibacterial activity of pECM and dECM indicates that the release of these matrix molecules from pulp and dentine may contribute to defence responses during dental disease, treatment and repair. Keywords: antimicrobial, matrix, pulp. dentine, extracellular

Received 29 August 2011; accepted 9 February 2012

Introduction
Pulp and dentine are protected from the harsh oral environment by the physical barriers of enamel and cementum. However, dentine can become exposed owing to caries, wear, trauma or restorative procedures (Tronstad & Langeland 1971, Pashley 1990, Peters et al. 1995, Love 1996) following which dentine tubules become invaded by bacteria leading to infection and disease progression within the dentinepulp complex (Love & Jenkinson 2002). The innate defence responses of the tooth involve recruitment and activation of a range of immune cell types by complex signalling networks involving cell- and tissue-derived

Correspondence: Dr Paul Cooper, Department of Molecular Biology, 7th Floor Main Laboratory, School of Dentistry, The University of Birmingham, St. Chads Queensway, Birmingham B4 6NN, UK (Tel.: +44 0 121 237 2895; fax: +44 0 121 237 2882; e-mail: p.r.cooper@bham.ac.uk).

cytokines and chemokines (McLachlan et al. 2003, 2004, Hahn & Liewehr 2007). Toll-like receptors expressed on the surface of many immune and host structural cells play a key role in the regulation of the innate immune responses by the recognition of pathogen-associated molecular patterns (Cooper et al. 2010). These host tissue-derived responses can subsequently slow or halt the bacterial invasion. However, if the microora continues to ourish, infection and inammation will ensue which may become uncontrolled leading to local tissue necrosis and more extensive apical disease (Trowbridge 1981). Despite the extraordinary diversity of the oral microora, only a relatively limited group of bacterial species are described as being involved in carious invasion of the dentinepulp complex (Ozaki et al. 1994, Preza et al. 2009). Anaerobic species including Eubacterium, Propionibacterium, Bidobacterium, Peptostreptococcus microorganisms and Veillonella show high prevalence amongst the carious bacteria

2012 International Endodontic Journal

International Endodontic Journal, 45, 749755, 2012

749

Antibacterial activity of extracellular matrix extracts Smith et al.

(Love & Jenkinson 2002, Chu et al. 2005). Streptococci are one of the most commonly found bacterial species within carious teeth, and they express multiple surface protein adhesins (Hasty et al. 1992) that allow binding to a variety of substrates, including the extracellular matrix (ECM) components (Jenkinson & Lamont 1997). Dentine and pulp ECMs comprise collagens, noncollageneous proteins, glycosaminoglycans and proteoglycans (Linde 1985). Some of these molecules regulate cellular behaviour in vivo, and this feature has led to them being studied for tissue engineering and regenerative purposes (Cordeiro et al. 2008, Zhang et al. 2011). Recently, ECM derived from tissues such as small intestine and bladder has been shown to possess antimicrobial activity (Sarikaya et al. 2002), and biological scaffolds containing ECM molecules have shown enhanced resistance to bacterial infection (Badylak et al. 1994, 2003, Mantovani et al. 2003, Ruiz et al. 2005). Ammonium sulphate-derived fractions of differently charged ECM components, as well as degradation products from liver and bladder, also have demonstrable antibacterial activity against Staphyloccus aureus and Escherichia coli, indicating the presence of either a potent individual antibacterial constituent or a series of molecules acting synergistically (Brennan et al. 2006). Dentine and pulp are known to contain a range of naturally occurring antimicrobial peptides (AMPs) including neuropeptides substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and adrenomedullin (ADM) (Awawdeh et al. 2002, El Karim et al. 2003, 2006, Tomson et al. 2007). These molecules may play an important role in host tissue defence following infection, and therefore this study examined the hypothesis that total ECM preparations from dentine and pulp possess antimicrobial activity.

solution (pH 2.0) 24 h 4 C per pulp (Bellon et al. 1988). Supernatants from each extraction step were pooled to generate pulp ECM (pECM) samples and dialysed against water exhaustively prior to lyophilization. Lyophilized EDTA-soluble human dentine extracellular matrix (dECM) was prepared as previously described (Smith et al. 1979) from healthy teeth. Healthy teeth were cleaned and cut into 1 mm longitudinal sections using a diamond-edged rotary disc saw (TAAB, Aldermaston, UK). Non-dentine tissue was removed from sections using bone clippers, and the remaining dentine was crushed into a ne powder using a percussion mill (Spex 6700 Freezer/Mill; Glen Creston Ltd, London, UK) cooled with liquid nitrogen and sieved through a 60 lm mesh sieve. Powdered dentine was exposed to 10% EDTA (pH 7.2) (Sigma, Poole, UK) extraction solution containing the protease inhibitors, 10 mmol L)1 n-ethylmaleamide (Sigma) and 5 mmol L)1 phenyl-methylsulphonyl uoride (Sigma). Extractions were performed with constant agitation at 4 C for 14 days. Supernatants from each extraction day were pooled and dialysed against water exhaustively for 14 days prior to lyophilization to generate dentine ECM (dECM) samples.

Ammonium sulphate fractionation of ECM extracts

Fractionation of ECM extracts was performed by protein precipitation using increasing concentrations of ammonium sulphate between 30% and 90% saturation. Lyophilized ECM extracts were solubilized in 10 mL ice cold sterile PBS at a concentration of 2 mg mL)1 and ammonium sulphate added to reach a saturation of 30% (1.76 g). Following vortexing for 2 min and gentle agitation for 1 h at room temperature, the sample was centrifuged at 800 g for 15 min and further ammonium sulphate added to the supernatant to 50% saturation. This was further repeated to 70% and 90% ammonium sulphate saturations, and the four precipitated fraction pellets were washed and dialysed exhaustively against water prior to lyophilization to produce charge separated fractions of ECM that have previously reported to differ in antibacterial activity when extracted from other tissues (Brennan et al. 2006).

Materials and methods Preparation of pulp and dentine ECM extracts

Pulps were dissected from 6 month-old bovine freshly extracted mandibular incisor teeth and minced prior to homogenization in 0.5 mol L)1 NaCl (pH adjusted to 11.7) containing protease inhibitors (25 mmol L)1 EDTA, 1 mmol L)1 phenylmethylsulfonyl uoride, 5 mmol L)1 N-ethylmaleimide) and 1.5 mmol L)1 sodium azide with gently agitation for 24 h at 4 C. The extraction was repeated three times with isolation of solubilized components by centrifugation followed by two further extractions with 1 mL 0.1 mol L)1 tartaric acid

Antimicrobial assay
Tryptone soya agar plates (Oxoid, Basingstoke, UK) containing 5% horse blood (Oxoid) were inoculated with Streptococcus mutans (American Type Culture Collection 25175, Manassas, VA, USA), Streptococcus oralis (American Type Culture Collection 35037, Manassas, VA,

750

International Endodontic Journal, 45, 749755, 2012

2012 International Endodontic Journal

Smith et al. Antibacterial activity of extracellular matrix extracts

Lactate dehydrogenase (LDH) cytotoxicity assay

Enzymatically isolated rodent primary pulpal cells (5 103) were seeded in 200 lL a-MEM in 96-well plates and cultured for 16 h at 37 C in 5% CO2 to enable cell adherence. Cells were then washed and exposed to concentrations of dECM or pECM in 200 lL of a-MEM for 48 h. The negative control comprised aMEM alone and the positive control included the addition of 5 lL kit lysis buffer to cultures containing a-MEM 30 min prior to the end of the 48 h incubation period. Following culture, supernatant from each well was analysed as per kit protocol (Roche Applied Sciences, Burgess Hill, UK) and the optical densities measured at 490/630 nm (ELX800 Universal Microplate reader; Bio-Tex Instruments INC).

Absorbance (OD)

USA) and Enterococcus faecalis (American Type Culture Collection 29212, Manassas, VA, USA). Bacterial identities were conrmed using molecular, cellular and biochemical phenotype tests (data not shown). Colonies of cultured bacteria were used to inoculate 10 mL of tryptone soya broth (Oxoid) and grown in an anaerobic chamber at 37 C with gentle agitation. Following growth, bacterial suspensions were diluted to 2 105 mL)1 and 100 lL of suspension (pH 7.3) added to 96-well plates (Corning, Loughborough, UK) containing various concentrations (0.1100 lg mL)1) of ECM extracts in 100 lL tryptone soya broth. A negative control containing tryptone soya broth alone and a positive control containing the antibiotics penicillin and streptomycin (Sigma) at a nal concentration of 0.5 lg mL)1 within the broth were used as these controls have previously been used in studies investigating the antibacterial activity of ECM (Sarikaya et al. 2002, Brennan et al. 2006). Bacterial growth in each well was recorded after a 24 h period for turbidity measured at 570 nm (ELX800 Universal Microplate reader; Bio-tex Instruments INC, Potton, UK). To determine whether the antimicrobial effects were bacteriostatic or bacteriocidal, after the initial 24 h growth in test solutions, the bacteria were isolated by centrifugation and resuspended in fresh tryptone soya broth in the absence of the ECM preparations. Following a further 24 h growth, bacteria were quantied as described earlier.

determined using the students t-test with P < 0.05 deemed as statistically signicant from control.

Results
The dECM preparation demonstrated antibacterial activity against S. mutans, S. oralis and E. faecalis (Fig. 1a). The greatest activity was observed against
(a)

Absorbance (OD)

Concentration dECM (g mL1)

(b)

Time (h)

Figure 1 (a) Increasing dentine extracellular matrix (dECM)

Statistical analysis
Data were expressed as means +/) standard deviation; statistical differences between experimental groups were

concentration reduces bacterial growth at 24 h. Absorbance values are all statistically signicant for Streptococcus mutans compared with negative control (PBS). Only 10 lg mL)1 dECM demonstrated statistically signicant difference of activity against Streptococcus oralis and Enterococcus faecalis, P < 0.05. (b) Removal of dECM from bacterial growth environment at 24 h removed inhibitory growth effect. At 24 h, dECM demonstrated a statistically signicant decrease (P < 0.05) from negative control and no statistically signicant difference from positive control. At 48 h, dECM demonstrated no statistically signicant decrease in bacterial growth compared to negative control (PBS) and showed a statistically signicant increase in growth compared to the positive control (penicillin/streptomycin). P < 0.05, (n = 5).

2012 International Endodontic Journal

International Endodontic Journal, 45, 749755, 2012

751

Antibacterial activity of extracellular matrix extracts Smith et al.

S. mutans at each concentration (1, 5 and 10 lg mL)1) with statistically signicant decreases (P < 0.05) in bacterial growth demonstrable. The inhibition of bacterial growth was lifted when the bacteria were resuspended and cultured further in unsupplemented broth, suggesting a bacteriostatic rather than bactericidal effect of the dECM (Fig. 1b). All ammonium sulphate fractions of the pECM preparation also demonstrated statistically signicant antibacterial activity against S. mutans compared with the negative control (Fig. 2), suggesting that overall activity was associated with a variety of differently charged AMPs. Between the ammonium sulphate fractions, statistically signicant differences were only detected between the 30% and other fractions. To assess whether ECM-derived antibacterial activity represented a general cytotoxic effect on both prokaryotic and eukaryotic cells, an LDH assay was applied to assess the effects of pECM and dECM on pulp cells. No signicant cytotoxic effects were observed at the concentrations of the extracts used, which had previously demonstrated antimicrobial activity (Fig. 3).

Discussion
The present study demonstrated that dentine and pulp ECM preparations show antibacterial activity against three types of facultative anaerobic bacteria associated with infected dental tissues. These data are in agreement with previous studies which have demonstrated

that ECM extracts derived from other tissues possess antimicrobial activity (Sarikaya et al. 2002, Brennan et al. 2006). These antimicrobial properties are likely ascribed to a complex range of molecules, including AMPs that play a key role in innate immunity. Indeed, previous studies have demonstrated that SP, NKA, CGRP, NPY and VIP are present in dental pulp (Awawdeh et al. 2002, El Karim et al. 2003, 2006) and possess antimicrobial activity against several types of bacteria including S. mutans and E. faecalis (El Karim et al. 2008). ADM is a multifunctional peptide also with antibacterial function directed against both Grampositive and Gram-negative bacteria resident in the oral cavity (Allaker & Kapas 2003) and is present in dentine matrix (Tomson et al. 2007, Musson et al. 2010). The antibacterial action of ECM appeared to be bacteriostatic rather than bacteriocidal. The absence of any statistically signicant cytotoxic effects of the dentine and pulp ECM preparations on pulp cells precluded a general cytotoxic effect on all cell types. It is known that electrostatic interactions between AMPs and bacterial cell membranes are important in enabling the cellular association required for the peptides to exert their antibacterial action (Brogden 2005). Therefore, charge-dependent separation of ECM using ammonium sulphate fractionation was performed to produce molecular groupings with differing charge proles. Notably, antibacterial activity was observed in all of these pulp ECM fractions, indicating that the antibacterial activity was likely derived from a

Figure 2 Ammonium sulphate (A.S.) pECM fractions (10 lg mL)1) decreased Streptococcus mutans growth at 24 h. All fractions

demonstrated a statistically signicant decrease in bacterial growth compared with negative control (PBS) with P < 0.01, (n = 5). pECM = pulp extracellular matrix.

752

International Endodontic Journal, 45, 749755, 2012

2012 International Endodontic Journal

Smith et al. Antibacterial activity of extracellular matrix extracts

(a)

(b)

present study demonstrated that the ECM preparations possessed differing degrees of antibacterial activity against these three bacterial types. Because of the complex range of AMPs in these preparations, it is difcult to provide mechanistic insight as to the basis of the differing degrees of antibacterial activity, however, this may relate to degree and localization of infection. Indeed, it is interesting that activity was greatest against S. mutans, and this may reect a defence response of the dentinepulp complex to relatively early or slowly progressing disease. The present study used a salinetartaric acid extraction protocol, which has been previously used for the extraction of antimicrobial molecules from the ECM of other tissues (Bellon et al. 1988). EDTA is commonly used for the extraction of noncollagenous matrix molecules from dentine (Smith et al. 1979) and is also used clinically as a root canal irrigant (Haapasalo et al. 2010). It is possible that such clinical use of EDTA may have added benet by releasing AMPs that aid root canal disinfection. The concentration of dECM extract (10 lg mL)1) showing antibacterial activity would equate to less that 0.5 mg of intact dentine matrix (EDTA-soluble material represents <2% of intact dentine) dissolved in 1 mL, which might be anticipated to be within the range of concentrations encountered in situ. Further characterization of the extracted ECM components may also yield novel AMPs with improved targeted clinical antimicrobial applications.

Figure 3 Data demonstrating lack of cytotoxicity of (a)

dentine extracellular matrix (dECM) and (b) pulp extracellular matrix (pECM) on pulp cells. At both 1 and 10 lg mL)1, pECM and dECM preparations had no signicant effect on dental pulp cell viability compared with negative control (PBS). P < 0.01, (n = 4).

Conclusions
The present study has demonstrated that ECM preparations derived from pulp and dentine possess antimicrobial activity. Both pECM and dECM extracts demonstrated bacteriostatic activity against S. mutans, S. oralis and E. faecalis. This indicates that the release of matrix molecules from pulp and dentine may contribute to defence responses during dental disease.

range of differently charged AMPs. Although all the fractions possessed antibacterial activity, the potency differed between the fractions, with 50% and 70% fractions showing the greatest level of activity. It is anticipated that this complex cocktail of AMPs will require extensive research to fully characterize, but their presence in the ECM will likely be of clinical signicance for the defence of the dentinepulp after infection of the tissues. Whilst both streptococci species studied here are found in the plaque microora, S. mutans is commonly associated with the highly acidic carious environment, whereas S. oralis is acid-sensitive (Marsh 1994). E. faecalis has been most commonly associated with root canal infections (Portenier et al. 2003). Notably, the

Acknowledgments
The authors deny any conicts of interest. This research was supported by the School of Dentistry, University of Birmingham.

References
Allaker RP, Kapas S (2003) Adrenomedullin and mucosal defence: interaction between host and microorganism. Regulatory Peptides 112, 14752.

2012 International Endodontic Journal

International Endodontic Journal, 45, 749755, 2012

753

Antibacterial activity of extracellular matrix extracts Smith et al.

Awawdeh LA, Lundy FT, Linden GJ, Shaw C, Kennedy JG, Lamey PJ (2002) Quantitative analysis of substance P, neurokinin A and calcitonin gene-related peptide in gingival crevicular uid associated with painful human teeth. European Journal Oral Sciences 110, 18591. Badylak SF, Coffey AC, Lantz GC, Tacker WA, Geddes LA (1994) Comparison of the resistance to infection of intestinal submucosa arterial autografts versus polytetrauoroethylene arterial prostheses in a dog model. Journal of Vascular Surgery 19, 46572. Badylak SF, Wu CC, Bible M, McPherson E (2003) Host protection against deliberate bacterial contamination of an extracellular matrix bioscaffold versus Dacron mesh in a dog model of orthopedic soft tissue repair. Journal of Biomedical Material Research. Part B, Applied Biomaterials 67, 64854. Bellon G, Wegrowski J, Perreau C et al. (1988) A parallel between two techniques of extraction of connective tissue macromolecules. Analytical Biochemistry 175, 26373. Brennan EP, Reing J, Chew D, Myers-Irvin JM, Young EJ, Badylak SF (2006) Antibacterial activity within degradation products of biological scaffolds composed of extracellular matrix. Tissue Engineering 12, 294955. Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nature Reviews Microbiology 3, 23850. Chu FC, Tsang CS, Chow TW, Samaranayake LP (2005) Identication of cultivable microorganisms from primary endodontic infections with exposed and unexposed pulp space. Journal of Endodontics 31, 4249. Cooper PR, Takahashi Y, Graham LW, Simon S, Imazato S, Smith AJ (2010) Inammation-regeneration interplay in the dentine-pulp complex. Journal of Dentistry 38, 68797. Cordeiro MM, Dong Z, Kaneko T et al. (2008) Dental pulp tissue engineering with stem cells from exfoliated deciduous teeth. Journal of Endodontics 34, 9629. El Karim I, Lundy FT, Linden GJ, Lamey PJ (2003) Extraction and radioimmunoassay quantitation of neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) from human dental pulp tissue. Archives of Oral Biology 48, 24954. El Karim IA, Lamey PJ, Ardill J, Linden GJ, Lundy FT (2006) Vasoactive intestinal polypeptide (VIP) and VPAC1 receptor in adult human dental pulp in relation to caries. Archives of Oral Biology 51, 84955. El Karim IA, Linden GJ, Orr DF, Lundy FT (2008) Antimicrobial activity of neuropeptides against a range of microorganisms from skin, oral, respiratory and gastrointestinal tract sites. Journal of Neuroimmunology 200, 116. Haapasalo M, Shen Y, Qian W, Gao Y (2010) Irrigation in endodontics. Dental Clinics of North America 54, 291312. Hahn CL, Liewehr FR (2007) Innate immune responses of the dental pulp to caries. Journal of Endodontics 33, 64351. Hasty DL, Ofek I, Courtney HS, Doyle RJ (1992) Multiple adhesins of streptococci. Infection and Immunity 60, 214752.

Jenkinson HF, Lamont RJ (1997) Streptococcal adhesion and colonization. Critical Reviews in Oral Biology and Medicine 8, 175200. Linde A (1985) The extracellular matrix of the dental pulp and dentine. Journal of Dental Research 64, 5239. Love RM (1996) Bacterial penetration of the root canal of intact incisor teeth after a simulated traumatic injury. Endodontics and Dental Traumatology 12, 28993. Love RM, Jenkinson HF (2002) Invasion of dentineal tubules by oral bacteria. Critical Reviews in Oral Biology and Medicine 13, 17183. ` AL, Pisani Mantovani F, Trinchieri A, Castelnuovo C, Romano E (2003) Reconstructive urethroplasty using porcine acellular matrix. European Urology 44, 6002. Marsh PD (1994) Microbial ecology of dental plaque and its signicance in health and disease. Advanced Dental Research 8, 26371. McLachlan JL, Smith AJ, Sloan AJ, Cooper PR (2003) Gene expression analysis in cells of the dentine-pulp complex in healthy and carious teeth. Archives of Oral Biology 48, 27383. McLachlan JL, Sloan AJ, Smith AJ, Landini G, Cooper PR (2004) S100 and cytokine expression in caries. Infection and Immunity 72, 41028. Musson DS, McLachlan JL, Sloan AJ, Smith AJ, Cooper PR (2010) Adrenomedullin is expressed during rodent dental tissue development and promotes cell growth and mineralization. Biology of the Cell 102, 14557. Ozaki K, Matsuo T, Nakae H, Noiri Y, Yoshiyama M, Ebisu S (1994) A quantitative comparison of selected bacteria in human carious dentine by microscopic counts. Caries Research 28, 13745. Pashley DH (1990) Clinical considerations of microleakage. Journal of Endodontic 16, 707. Peters LB, Wesselink PR, Moorer WR (1995) The fate and the role of bacteria left in root dentineal tubules. International Endodontics Journal 28, 959. Portenier I, Waltimo T, Haapasalo M (2003) Enterococcus faecalis the root canal survivor and star in posttreatment disease. Endodontic Topics 6, 13559. Preza D, Olsen I, Willumsen T et al. (2009) Microarray analysis of the microora of root caries in elderly. European Journal of Clinical Microbiology and Infectious Diseases 28, 50917. Ruiz CE, Iemura M, Medie S et al. (2005) Transcatheter placement of a low-prole biodegradable pulmonary valve made of small intestinal submucosa: a long-term study in a swine model. Journal of Thoracic Cardiovascular Surgery 130, 47784. Sarikaya A, Record R, Wu CC, Tullius B, Badylak S, Ladisch M (2002) Antimicrobial activity associated with extracellular matrices. Tissue Engineering 8, 6371. Smith AJ, Price R, Leaver AG (1979) Components of the organic matrices of rabbit incisor and molar dentine isolated after digestion of the demineralized tissues with collagenase. Archives of Oral Biology 24, 95563.

754

International Endodontic Journal, 45, 749755, 2012

2012 International Endodontic Journal

Smith et al. Antibacterial activity of extracellular matrix extracts

Tomson PL, Grover LM, Lumley PJ, Sloan AJ, Smith AJ, Cooper PR (2007) Dissolution of bio-active dentine matrix components by mineral trioxide aggregate. Journal of Dentistry 35, 63642. Tronstad L, Langeland K (1971) Effect of attrition on subjacent dentine and pulp. Journal of Dental Research 50, 130.

Trowbridge HO (1981) Pathogenesis of pulpitis resulting from dental caries. Journal of Endodontics 7, 5260. Zhang R, Cooper PR, Smith G, No r JE, Smith AJ (2011) Angiogenic activity of dentine matrix components. Journal of Endodontic 37, 2630.

2012 International Endodontic Journal

International Endodontic Journal, 45, 749755, 2012

755