Antibacterial efficacy of silver-crosslinked hydrogel nanocomposite

IJBPAS, November, 2014, 3(11): 2316-2332

ISSN: 2277–4998

ANTIBACTERIAL EFFICACY OF SILVER-CROSSLINKED HYDROGEL NANOCOMPOSITE VERSUS SODIUM HYPOCHLORITE AND CHLORHEXIDINE ON ENTEROCOCCUS FAECALIS FOR USE IN ROOT CANAL INFECTION SAMIEI M1, DAVARAN S2, VALIPOUR F3*, DAVARI A1, GHIASIAN T1 AND LOTFIPOUR F 3, 4 1: Faculty of Dentistry, Tabriz University of Medical Sciences, Tabriz, Iran 2: Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 3: Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran 4: Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran *Corresponding Author: E Mail: [email protected]; Tel: +98 (41) 3392580; Fax: +98 (41) 3344798 ABSTRACT The aim of this study was to synthesize and evaluate the antibacterial properties of thermosensitivecross-linked poly(N-isopropylacrylamide-methacrylic acid-vinyl pyrrolidone) hydrogel (Poly (NIPAM-MAA-VP)containing silver nanoparticles (silvercross-linked hydrogel nanocomposites (SCHNC)) at a concentration of 30 ppm in dental root canals infected with Enterococcus faecalis and to compare its efficacy with two commonly used irrigants; 2.5% sodium hypochlorite and 2% chlorhexidine gluconate. Cross-linked

poly

(NIPAM-MAA-VP)

hydrogel

was

synthesized

by

free

radical

copolymerization of monomers in 1,4-dioxane in the presence of N,N-Methylenebisacrylamideas crosslinking agent. Highly stable and uniformly distributed silver cross-linked hydrogel nanocomposite (SCHNC) was obtained via in situ reduction of silver nitrate (AgNO 3) using sodiumborohydride (NaBH4) as reducing agent. Minimum Inhibitory Concentration (MIC) of newly synthesized SCHNC was

2316 IJBPAS, November, 2014, 3(11)

Lotfipour F et al

Research Article

determined against E. faecalis by broth macro dilution method. Also in vitro experiments were carried out on 100 human single rooted maxillary incisors infected with E. faecalis. Mean MIC values for SCHNC and placebo cross-linked hydrogel (polymer without silver nanoparticles) were15ppm and 200ppm respectively. In the dental root canals in the first inoculation SCHNC lowered the E. faecalis count below 10 CFU, the hypochlorite and chlorhexidine solutions killed all the bacteria. Furthermore, the synthesized SCHNC in double inoculation with the bacteria maintained its activity in reduction of bacteria more efficiently compared to hypochlorite and chlorhexidine solutions. Placebo cross-linked hydrogel failed to show significant change from the initial bacterial count in dental samples. Findings of this study confirm the antibacterial effects of newly synthesized SCHNC against E. faecalis, and demonstrate that SCHNC can exhibit sustained antibacterial property and low toxicity. After re-inoculation of the dental root canal with the excess E. faecalis inoculum SCHNC successfully maintained its antibacterial activity compared to the hypochlorite and chlorhexidine solutions which were remarkably lost their disinfectant effects. Keywords: Enterococcus faecalis, silver-cross-linked hydrogel nanocomposites, sodium hypochlorite, chlorhexidine, antibacterial activity INTRODUCTION Studies have shown that the presence of

microorganisms present in the root canal

microorganisms is the main factor in the

systems and dentinal tubules and prevent

development of pulpal and periradicular

recontamination of the canal after the

diseases.

undergoes

treatment [2]. Cleaning and shaping of the

pathological changes due to trauma or

root canal through the use of appropriate

aggressive caries, the root canal system

intracanalirrigants reduces the bacterial load,

becomes susceptible to contamination by a

thereby increases the likelihood of successful

variety of microbes. The microorganisms not

outcomes after root canal therapy [3].

only contribute in the anatomical irregularities

Sodium hypochlorite (NaOCl) is a commonly

of the root canal system but also invade the

used root canal irrigant. In addition toits

dentinal tubules and can re-infect a poorly

bactericidal and proteolytic properties, NaOCl

treated root canal system [1]. The main

solution offers some advantages including its

objectives of endodontic therapy are to

ability

remove the infected tissue, eliminate the

increase the permeability of the dentin, and

When

tooth

pulp

to

dissolve

organic

substances,


Lotfipour F et al

Research Article

dissolve necrotictissue [4-7]. However, the

Bacillus subtilis, Klebsiella mobilis, and

use of this irrigant is limited due to its

Klebsiella pneumonia [16- 17].

undesirable taste and smell. Moreover, it has

Different forms of silver nanomaterials have

been proven to have cytotoxic effect on

been already reported including metallic

periradicular tissues in the case of extrusion

silver nanoparticles, silver chloride particles,

[8, 9]. Recently with the major advances in

silver-impregnated

nanotechnology, novel agents such as silver

activated carbon materials, dendrimer–silver

nanoparticles with advanced antimicrobial

complexes and composites, polymersilver

activityhave been developed [10]. These

nanoparticle

nanoparticles

nanoparticles coated polymers [16-17].

possess

improved

zeolite

composites,

and

and

silver

characteristics include the high surface area to

Several

volume ratio, greater solubility and chemical

influence the antimicrobial activity of silver

reactivity

nanoparticle like particle size, shape, surface

and

accordingly

bactericidal

factors have

powders

been reported to

activities [11]. The antimicrobial property of

chemistry,

silver

environmental factors such as pH, ionic

nanoparticles

concentration

and

depends release

on rate.

its The

coating agents,

as well as,

strength, and the presence macromolecules.

antimicrobial activity of silver is attributed to

Stability of silver nanoparticleshas a critical

the interaction of silver ions with specific

role in their properties sincefast oxidation and

thiol

also formation of silver aggregates tends to

groups

that

contain

sulphur

and

hydrogen and are found in different structural

decrease their antimicrobial activity.

compounds of bacterial enzymes and proteins

For biomedical applications, it is necessary to

[12-15]

stabilize

thereby

causing

less

bacterial

silver

nanoparticles.

Various

resistance than other antibiotics. Silver-

methods for stabilizing and capping of

containing nanomaterials have been widely

nanoparticles have been reported [18- 21].

used in biomedical products due to the broad-

Coating of silver nanoparticle, embedding

spectrum antimicrobial properties. Already,

silver nanoparticles in different polymeric

silver nanoparticles have been shown to be

composites and surface modification with

effective against bacteria such as Escherichia

functional polymers, have been used to

coli, Staphylococcus aureus, Staphylococcus

increase nanoparticles stability.

epidermis,

We have previously reported the antibacterial

Leuconostocme

senteroides,

activity of silver nanoparticles embedded in 2318 IJBPAS, November, 2014, 3(11)

Lotfipour F et al smart

Research Article

poly (N-isopropylacrylamide)-based

hydrogel

networks

of

N-isopropyl acrylamide (NIPAAM) (Fluka,

cross-linked

poly(N-

Deisenhofen, Germany) was purified by

isopropylacrylamide-methacrylic

acid-

recrystallization in hexane and dried under a

[P(NIPAAm-

vacuum at 25°C. Vinyl pyrrolidone (VP)

MAA-HEM)] have been obtained by cross-

(Merck,Hohenbrunn, Germany) was freed

linking- free radical polymerization method.

from

Highly stable and uniformly distributed silver

distillation with continuous bubbling argon.

nanoparticles have been obtained within the

Methacrylic acid (MAA) (Fluka) was used as

hydrogel networks via in situ reduction of

supplied. Benzoyl peroxide (BPO), silver

silver nitrate using sodium borohydride

nitrate (AgNO3), and sodiumborohydride

(NaBH4) as reducing agent. The antibacterial

(NaBH4) were from Merck Chemical Co.

activity

N,N-Methylenebisacrylamide(NNMBAAm)

thermosensitive

hydroxyethyl

of

[22].

methacrylate)

these

A

series

Materials

hydrogel–silver

the

stabilizer

In the present paper we have reported the

without further purification. Muller-Hinton

synthesis of a novel silver nanocomposite

Broth medium and Agar-Agar from Merck

prepared from silver nanoparticles embedded

(Germany), Todd Hewitt Broth, Bile Esculin

in

N-

Agar and Soybean Casein Digest Medium

polymeric

(tryptone Soya Broth) from Hi Media

poly(N-

Laboratories (India) were used. Enterococcus

acid-vinyl

faecalis (PTCC 1237) was purchased from

thermosensitive

isopropylacrylamide-based hydrogel.

Cross-linked

isopropylacrylamide-methacrylic pyrrolidone)

used

vacuum

(Sigma-Aldrich

cross-linked

was

twice

nanocomposites has been studied.

a

Co.)

by

directly

[P(NIPAAm-MAA-VP)]

Persian Type Culture Collection (Iran).

copolymer was synthesized and used as

Preparation and Characterization of

hydrogel

Cross-Linked P (NIPAM-MAA-VP)

networke.

The

antibacterial

activities of silver-free hydrogel and hybrid

Hydrogel

silver-polymer nanocomposite were studied in

Poly

dental root canals infected with Enterococcus

acid-vinypyrrolidone) (Poly (NIPAM-MAA-

faecalis and compare with two commonly

VP) copolymer was synthesized by free

used irrigants; 2.5% sodium hypochlorite and

radical copolymerization of monomers in 1,4-

2% chlorhexidine gluconate.

dioxane under N2 atmosphere according to the

MATERIALS AND METHODS

method described previously [24]. The ratio

(N-isopropylacrylamide-methacrylic


Lotfipour F et al of

NIPAM:

Research Article MAA:VP

85:5:10.

25C for 1 day. The dried specimens were

Monomers were dissolved in 1,4-dioxane to

examined by a LEO 906 transmission electron

form a 5 wt% solution containing PBO (0.1

microscopy (TEM) operated at 80kV.

wt% weight ratio of total monomers) and

Preparation of Tooth Samples

NNMBAAm (1 wt%) as a cross-linking

The study was conducted on 100 human

agent. The polymerization was carried out at

single-rooted upper anterior teeth with fully

70 C for 10 h under N2 atmosphere. The

developed apices and straight roots devoid of

resulting copolymer was precipitated in

any abnormalities, which were extracted due

excess cold n-hexane. The crude polymer was

to periodontal reasons. The teeth were stored

purified by dissolving in tetrahydrofurane

in a phosphate-buffered saline solution until

(THF) and reprecipitation in diethyl ether to

used. Root surfaces were cleaned using an

remove the reactant residues. The copolymer

ultrasonic device and teeth appearing as

was finally dried by pumping under reduced

cracked or calcified on radiographs were

pressure. Chemical structure of copolymers

excluded from the study. Canal lengths of the

was determined by FT-IR (Shimadzu 8400,

selected teeth were measured by introducing a

Kyoto, Japan) and 1H-NMR (Bruker AC 80,

K-flexofile#20

Rheinsteten, Germany) spectroscopies.

Switzerland) into the canal until the tip was

Preparation

visible at the apical foramen.

of

Silver

was

cross-Linked

(DentsplyMaillefer,

Hydrogel Nanocomposites (SCHNC)

Tooth crowns were cut-off at CEJ using

Precisely measured dry hydrogel was steeped

diamond disks(D&Z, Diamond, Germany)

in water for two days and the swollen

such that the length of the roots were 12mm

hydrogels were transferred to a beaker

and then, using a Maillefer, Dentsply,

containing aqueous solution of 50 ml of

Switzerland k-flexo file #20the canal working

AgNO3 (30 ppm) and stirred at 25 ˚C for 72

length was set at 1 mm short of the apical

hours for equilibration. These silver salt

foramen. Next, root canals were prepared

absorbed hydrogels were then added to 50 ml

using #3 and #4 Gates-Glidden drills and

of aqueous solution NaBH4 (60 ppm) and

rotary files size 40 (10%) and size 35 (8%) of

stirred lightly for 4 hours to reduce the silver

the RaCe system (FKG Dentaire, La-Chaux-

ions into silver nanoparticles.The resulted

de-Fonds, Switzerland) and according to the

silvercross-linked hydrogel nanocomposite

crown-down technique. Physiological serum

(SCHNC) was dried under vacuum oven at

was used for canal irrigation and the smear 2320

IJBPAS, November, 2014, 3(11)

Lotfipour F et al

Research Article

layer was removed using 5.25% sodium

Determination of the Antibacterial Activity

hypochlorite (Taj Corp, Tehran, IRI)

of SCHNC Against Enterococcus faecalis

(3

minutes) and 17% EDTA (Pulpdent Corp,

In order to evaluate the in vitro antibacterial

MA, USA). The teeth were divided into 10

activity of newly synthesized SCHNC, firstly

groups, each group including 10 samples that

the MIC value was determined by broth

were classified into A and B subgroups.All

macrodilution technique according to CLSI

teeth were sterilized by autoclaving for 20

[25]. In brief, serially diluted concentrations

minutes at 121C and 15 psi. To check the

of SCHNC were prepared in sterile water

sterility of the samples, they were incubated

ranging from 60 to 0.93 ppm. Muller-Hinton

for 24 hours at 37C in brain-heart infusion

Broth medium containing the inoculum in the

broth (Merck, Darmstadt, Germany).

final concentration of 106CFU/mlwas added

Inoculum Preparation

to the serially diluted concentrations of

Enterococcus

faecalis(PTCC

1237)

SCHNC. After 24 hours of incubation at 37

wasobtained in lyophilized form Persian Type

C, the tubes were checked for any evidence

Culture Collection (Iran), which was activated

of bacterial growth. The MIC value was

by incubation in Todd Hewitt Broth for 24

defined as the lowest concentration of

hours at 37°C. 50 µl from the grown bacteria

SCHNC with no sign of bacterial growth. The

was transferred and spread on the surface of

tubes

bile esculin sodium azide agar medium and

(polymer without silver nanoparticles) were

incubated overnight at 37°C. The black

also included in the test as negative control

colonies confirmed the identity of entroccucus

group.

species. A Single colony from the plate was

In the next step, the teeth canals were

transferred into 4 ml fluid tryptone Soya

inoculated, under aseptic conditions, with the

Broth and incubated over night at 37°C and

bacterial inoculum and left in the room

200 rpm in shaking incubator. The cells were

temperature for 4 hours. Then the samples

harvested by centrifugation at 3000 rpm for

were rinsed three times with 5 mL of 0.9%

15 min. Subsequently, they were washed

normal

twice and re-suspended in Ringer solution to

bacteria. Accordingly, the samples in groups a

provide bacterial concentrations between 107–

and b (1 to 5) were filled with 250µl of

8

10 CFU/ml [25].

withplacebo

saline

to

cross-linked

eliminate

hydrogel

un-attached

SCHNC (30 ppm), 2.5% sodium hypochlorite,


Lotfipour F et al

Research Article

2% chlorhexidine gluconate, 30 ppm placebo

pair-wise comparisons. The level of statistical

cross-linked hydrogel and 0.9% normal saline

significance was set at p