antibacterial activity of dentine and pulp

8/13/2019 Antibacterial Activity of Dentine and Pulp

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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. Antibac-

terial 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 Entero-

coccus 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 anal-

ysis 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 significant cytotoxic effect on pulpal cells at the

concentrations used for antibacterial activity.

Conclusions The bacteriostatic antibacterial activ-

ity of pECM and dECM indicates that the release of these

matrix molecules from pulp and dentine may contrib-

ute to defence responses during dental disease, treat-

ment and repair.

Keywords:antimicrobial, dentine, extracellular

matrix, pulp.

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 com-

plex (Love & Jenkinson 2002). The innate defence

responses of the tooth involve recruitment and activa-

tion of a range of immune cell types by complex

signalling networks involving cell- and tissue-derived

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 path-

ogen-associated molecular patterns (Cooper et al.

2010). These host tissue-derived responses can subse-

quently slow or halt the bacterial invasion. However, if

the microflora continues to flourish, infection and

inflammation will ensue which may become uncon-

trolled leading to local tissue necrosis and more

extensive apical disease (Trowbridge 1981).

Despite the extraordinary diversity of the oral

microflora, 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, Bifidobacterium,

Peptostreptococcus microorganisms and Veillonella

show high prevalence amongst the carious bacteria

Correspondence: Dr Paul Cooper, Department of Molecular

Biology, 7th Floor Main Laboratory, School of Dentistry, The

University of Birmingham, St. Chads Queensway, Birming-

ham B4 6NN, UK (Tel.: +44 0 121 237 2895; fax:

+44 0 121 237 2882; e-mail: [email protected]).

doi:10.1111/j.1365-2591.2012.02031.x

2012 International Endodontic Journal International Endodontic Journal, 45, 749755, 2012 749


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(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, noncol-lageneous proteins, glycosaminoglycans and proteogly-

cans (Linde 1985). Some of these molecules regulate

cellular behaviour in vivo, and this feature has led to

them being studied for tissue engineering and regen-

erative 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 bio-

logical 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 differ-

ently charged ECM components, as well as degradation

products from liver and bladder, also have demonstra-

ble 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)includ-

ing neuropeptides substance P (SP), neurokinin A (NKA),

calcitonin gene-related peptide (CGRP), neuropeptide Y

(NPY), vasoactive intestinal polypeptide (VIP) and adre-

nomedullin (ADM) (Awawdehet al.2002, El Karimet al.

2003, 2006, Tomsonet al.2007). These molecules mayplay an important role in host tissue defence following

infection, and therefore this study examined the hypoth-

esis that total ECM preparations from dentine and pulp

possess antimicrobial activity.

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 fluoride,

5 mmol L)1 N-ethylmaleimide) and 1.5 mmol L)1 so-

dium azide with gently agitation for 24 h at 4 C. The

extraction was repeated three times with isolation of

solubilized components by centrifugationfollowed bytwo

further extractions with 1 mL 0.1 mol L)1 tartaric acid

solution (pH 2.0) 24 h 4 C perpulp (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 extracellu-

lar matrix (dECM) was prepared as previously described

(Smith et al. 1979) from healthy teeth. Healthy teethwere cleaned and cut into 1 mm longitudinal sections

using a diamond-edged rotary disc saw (TAAB, Alderm-

aston, UK). Non-dentine tissue was removed from

sections using bone clippers, and the remaining dentine

was crushed into a fine 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-eth-

ylmaleamide (Sigma) and 5 mmol L)1 phenyl-methyl-

sulphonyl fluoride (Sigma). Extractions were performed

with constant agitation at 4 C for 14 days. Superna-

tants from each extraction day were pooled and dialysed

against water exhaustively for 14 days prior to lyoph-

ilization 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 ammo-

nium 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 ammo-

nium sulphate added to the supernatant to 50%

saturation. This was further repeated to 70% and 90%

ammonium sulphate saturations, and the four precip-

itated fraction pellets were washed and dialysed exhaus-

tively against water prior to lyophilization to produce

charge separated fractions of ECM that have previously

reported to differ in antibacterial activity when ex-

tracted from other tissues (Brennan et al.2006).

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(Amer-

ican Type Culture Collection 35037, Manassas, VA,

Antibacterial activity of extracellular matrix extracts Smith et al.

International Endodontic Journal, 45, 749755, 2012 2012 International Endodontic Journal750


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USA) and Enterococcus faecalis (American Type Culture

Collection 29212, Manassas, VA, USA). Bacterial iden-

tities were confirmed 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. Followinggrowth, 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) contain-

ing various concentrations (0.1100 lg mL)1) of ECM

extracts in 100lL tryptone soya broth. A negative

control containing tryptone soya broth alone and a

positive control containing the antibiotics penicillin and

streptomycin (Sigma) at a final concentration of

0.5 lg mL)1 within the broth were used as these

controls have previously been used in studies investi-

gating 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. Follow-

ing a further 24 h growth, bacteria were quantified as

described earlier.

Lactate dehydrogenase (LDH) cytotoxicity assay

Enzymatically isolated rodent primary pulpal cells

(5 103) were seeded in 200lL 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 a-

MEM 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 Micro-

plate reader; Bio-Tex Instruments INC).

Statistical analysis

Data were expressed as means +/) standard deviation;

statistical differences between experimental groups were

determined using the students t-test with P < 0.05

deemed as statistically significant 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

Absorbance

(OD)

Concentration dECM(g mL1)

Time (h)

Absorbance

(OD)

(a)

(b)

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

concentration reduces bacterial growth at 24 h. Absorbance

values are all statistically significant for Streptococcus mutans

compared with negative control (PBS). Only 10lg mL)1

dECM demonstrated statistically significant difference of activ-

ity against Streptococcus oralis and Enterococcus faecalis,

P < 0.05. (b) Removal of dECM from bacterial growth

environment at 24 h removed inhibitory growth effect. At24 h, dECM demonstrated a statistically significant decrease

(P < 0.05) from negative control and no statistically signifi-

cant difference from positive control. At 48 h, dECM demon-

strated no statistically significant decrease in bacterial growth

compared to negative control (PBS) and showed a statistically

significant increase in growth compared to the positive control

(penicillin/streptomycin).P < 0.05, (n = 5).

Smith et al. Antibacterial activity of extracellular matrix extracts



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S. mutansat each concentration (1, 5 and 10 lg mL)1)

with statistically significant decreases (P < 0.05) in

bacterial growth demonstrable. The inhibition of bac-

terial growth was lifted when the bacteria were

resuspended and cultured further in unsupplemented

broth, suggesting a bacteriostatic rather than bacteri-

cidal effect of the dECM (Fig. 1b).All ammonium sulphate fractions of the pECM

preparation also demonstrated statistically significant

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 significant differences were only

detected between the 30% and other fractions.

To assess whether ECM-derived antibacterial activity

represented a general cytotoxic effect on both prokary-

otic and eukaryotic cells, an LDH assay was applied to

assess the effects of pECM and dECM on pulp cells. No

significant cytotoxic effects were observed at the

concentrations of the extracts used, which had previ-

ously 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 agree-

ment 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 includingS. mutansand E. faecalis(El Karim

et al.2008). ADM is a multifunctional peptide also with

antibacterial function directed against both Gram-

positive 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 significant 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 per-

formed to produce molecular groupings with differing

charge profiles. 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 significant decrease in bacterial growth compared with negative control (PBS) with P < 0.01, (n = 5).

pECM = pulp extracellular matrix.




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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

significance for the defence of the dentinepulp after

infection of the tissues.

Whilst both streptococci species studied here are

found in the plaque microflora, S. mutansis commonly

associated with the highly acidic carious environment,

whereasS. oralisis acid-sensitive (Marsh 1994). E. fae-

calis has been most commonly associated with root

canal infections (Portenier et al. 2003). Notably, the

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

difficult 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 reflect a defence

response of the dentinepulp complex to relatively

early or slowly progressing disease.

The present study used a salinetartaric acid extrac-

tion 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 benefit 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


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