Effect of Zinc on Antibacterial Action of Bioactive Glass Coating for Dental Implant, AS Hameed, AM Abass, LJ Hamood

Tags: zinc, glass coating, bioactive glass, glass coatings, bacterial growth, inhibition, glasses, F. Goto, biochemical tests, biochemical test, antimicrobial action, brain heart infusion broth, agar plates, Laboratory diagnosis, medical bacteria, diffusion method, anaerobic bacteria, glass ionomers, glass network, Surface and coatings technology, bacterial cell wall, bacterial strains, Antibacterial activity, Caries Research, Journal of Fermentation and Bioengineering, Journal Biomedical Materials Research, glass ionomer, Bioactive glass coatings, Appleton Century Crafts
Content: Medical Journal of Babylon Vol. 12- No. 3: 612-617, 2015 http://www.medicaljb.com ISSN 2312-6760©2015 University of Babylon Original RESEARCH ARTICLE Effect of Zinc on Antibacterial Action of Bioactive Glass Coating for Dental Implant Ahmed Salah Hameed* Ahmed Muhammed Abass Luma Jasim Hamood Israa Abass Obaid College of Dentistry, University of Babylon , Hilla , IRAQ *E-mail: [email protected] Accepted 28 June , 2015 Abstract Peri-implantitis is one of the causes of implant failure. Attempts have been done to coat dental implant with bioactive glasses to enhance osseo-integration and reduce microbial attachment. However, the anti-bacterial action of these coatings was limited to certain bacterial strains . Metals such as zinc and silver have been added to these glasses to improve their anti-microbial action. In this study zinc (3 mole%)-containing and zinc-free bioactive glasses were synthesized using melt-derived route. The glass coatings on titanium discs were prepared using enameling technique. The antibacterial action of zinc-containing and zinc-free glass coatings was studied against Streptococcusmutants, Staphylococcus aureus and Porphyromonasgingivalis by disC Diffusion method. These bacterial strains were obtained by swab samples. The results showed that zinc at 3 mole% had no effect on antibacterial action of bioactive glasses and both glass coatings exhibited similar effect on bacterial strains used in this study. Since these glass coatings have similar effect on aerobic and anaerobic bacteria, these glass coatings can be used in various medical applications. Key words:Bioactive glasses, Anti-bacterial, Dental implant, Titanium.
. . . . . . Streptococcusmutants,Pyrophyromonasgingivalis, . Staphylococcusaureus, . .
Hameed et al.
Introduction
Peri-implantitisis one of the
causative factor of dental implant
failure.
Thisbacterial
infectioncauses destruction of bone
surrounding dental implant in a
mannersimilar to that of periodontitis, an
inflammatory condition affecting natural
dentition. Titanium has an excellent
biocompatible properties, but does not
exhibit antimicrobial action. One
approach to achieve better disinfection
and biocompatibility is to modify titanium
implant surfaces[1]. Surface modification
of titanium by coating or adding
antibacterial properties of metals or alloys
to reduce microbial attachment seems an
efficient way to achieve success
outcome[2]. Bioactive glasses have the
ability to dissolve in biological fluids and
release ions such as silica, sodium and
calcium. This ionic dissolution facilitates
hydroxyapatite formation and direct
bonding to bone and soft tissues[3]. In
addition, these ionic dissolution and the
rapid change in the pH of the surrounding
medium enable these glasses to exhibit
anti-bacterial action[4,5]. Hence, these
glasses can be used as coatings for
prosthetic metallic implants [6].
The antibacterial action of silica based
melt-derived bioglass® was investigated
against certain types of microorganisms
and the results were promising[7].
Though, the antiMicrobial Activity of this
glass was limited to certain types of
microorganisms. Therefore, elements such
as zinc, silver and copper have been
incorporated into glass composition to
broaden the antibacterial action of these
glasses. Many studies stated that zinc has
antibacterial action [8,9,10,11]. It was
thought that the antibacterial action of
zinc is attributed to the release of
hydrogen peroxide from its surfaces [12].
However, other reportsstated that the
inhibition of bacterial growth is related to
the change in protein structure and this
causes inhibition of specific metabolic
MJB-2015
enzymes, thus leading to inhibition of
bacterial growth [13, 14 ,15].
In this study the effect of Zn-containing
bioactive
glass
coatingsonPorphyromonasgingivalis,
Strpt.mutants and S.aureuswere studied.
These bacteria were chosen due to their
relevancy in the development and
establishment
of
prei-
implantitis.Characterization, bioactivity
and cytotoxicity of the glass coatings were
investigated previously and the coatings
were able to exhibit surface apatite after 1
month immersion in simulated body
fluid[16].The prepared glasses have been
synthesized by melt-derived route and
coated on pure titanium discs by
enameling technique. The composition of
the prepared glasses is more intricate
compared to bioglass®, as they are multi-
component system and contains additional
elements such as Zn and Mg.
Materials and Methods
Glass synthesis
The zinc-containing glass and zinc-free
glass were prepared using reagent grade
chemicals (SiO2, MgO,CaCO3, Na2CO3,
K2CO3,MgF2, ZnO and P2O5) in the
appropriateproportions.
MgF2was
substituted partially for ZnO in zinc-free
glass. The composition of theseglassesare
listed in table 1. The glass batch was
melted in a 300 mL platinum­
rhodiumalloy crucible using an electric
furnace
(Hope
Valley,
LentonThermalDesigns, UK) at
temperatures between 1450 and 1460 °C
for one anda half hours. The melts were
then rapidly quenched in deionized water
to prevent crystallization and phase
separation. The glass fritproduced was
collected in a sieve and dried overnight at
120 °C. Thedried frit was then ground in a
Gyro Mill(Glen Creston, UK) for 14 min.
and sieved for 60min in a sieve shaker
(Retsch,VS1000, Germany) andseparated
into more and less than 45 mparticle size
groups.
613
Hameed et al. MJB-2015
Table1: chemical composition (mole%) of QM5MgO (Zn-containing glass) and QM5MgF2 (Zn-free glass)
Glass
SiO2 CaO MgO Na2O K2O P2O5 ZNO MgF2
QM5MgO 41.7 36.3 7.8 5.2 1.0 4.7 3.0 .........
QM5MgF2 41.7 36.3 9.93 5.2
1.0
4.7 ....... 1.0
Glass Coating
A glass coating on a disc of commercially
pure titanium (Advent researchmaterials
Ltd. U.K. purity99.6%) was synthesized
by vitreousenamelling technique, using a
porcelain
furnace
(Centurion
QuartzNEY). The precipitation method
was used in order to make bioactiveglass
to settle on a disk of commercially pure
titanium. The suspensionwas prepared by
dispersing 2.5 g of selected glass particles
(<45 m)in 50 ml ethanol by stirring
thoroughly. A disc of commercially pure
titaniumof 10 mm, which had been
previously polished with 1 mm
diamondpaper and cleaned in acetone and
ethanol in ultrasonic path, wasdropped in
glass powder suspension. This was left in
a vacuum oven at80 °C for 1 h so that the
glass powder was settled on metal
substrate,producing a uniform surface.
Confirming that the glass powder
completelydeposited, the specimen was
introduced in a pre-heated dentalfurnace
to 300 °C. Afterwards, it was heated at a
rate of 60 °C/min to740 °C followed by
30 min hold at that desired temperature.
Duringheating process, the furnace was
evacuated to 100% vacuum pressure(0.1
atm).
Samples Collection
Isolation and all biochemical tests for
diagnosis of (S.aureus,Strept.mutants, and
Pyrophyromonasgingivalis) were carried
out according to standard methods [17,
18].
1- Incubation under aerobic condition: By
swabs from caries activated in brain heart
infusion broth (B.H.I) for one hour. The
samples then cultured in blood agar and
incubated for 24 hours. The growth
appeared as mixing of microorganisms
(Streptococcus,
Staphylococcus,
Lactobacilli and fungi).
Laboratory diagnosis was carried out by gram stain and biochemical test (catalase, coagulase tests) and antibiotic sensitivity test on Muller Hinton agar with 5% blood. 2- Incubation under anaerobic condition: The subgingival plaque samples were inoculated into 2 ml of Brucella broth supplemented with 0.4-µl/ml vitamin K and 5µg/ml hemin. After that, they were diluted and plated onto trypticase soy agar supplemented with 10% defibrinated horse blood,5mg/ml hemin and 0.4 µl/ml vitamin K. The plates were incubated and duplicated in anaerobic atmosphere for 710 days or in air plus 10% CO2 for 2-4 days and staining. The anaerobic bacteria were identified by API20 and rapid ID32A, biochemical tests. black pigmented,anaerobic ,gram negative rods is considered Porphyromonasgingivalis. Antimicrobial Activity Antibacterial action of zinc-containing and zinc-free glass coatings was investigatedin vitro using disc diffusion method. Bacterial strains (S.aureus, Strept.mutants, and P.gingivalis) were obtained by swab samples and used to analyze the antimicrobial action. The antimicrobial tests were carried out on the previously prepared solidified and sterilized Mueller-Hinton agar plates. The coated samples were sterilized by dipping in 100% ethanol for 1 minutes.After dryness, they were placed in the agar plates and incubated for 48 hours at 30 C . After the incubation period, the inhibitory zone was measured in millimeter (mm) using a transparent ruler. Uncoated titanium disc was used as a control. Results and Discussion The antimicrobial efficacy of Zncontaining and Zn-free glass coatings
614
Hameed et al. were investigated against bacterial cultures of S. aureus, Strept. mutantsand P.gingivalis by disc diffusion method. The two bioactive glasses produced almost equal zones of inhibition ranged between 35-45mm as shown in table 2 and figure 1. However, Zn-containing coating showed slightly wider inhibition zone (50 mm) against P.gingivalis compared to Znfree glass coating (45 mm). These results indicated that zinc at 3 mole% has little, if any, effect on antibacterial action of bioactive glasses. Zinc at 3 mole% was used in order to prevent cytotoxicity and not to interfere with glass solubility and bioactivity.Ainaand her-coworkers[19] studied the effect of incorporating zinc at 5-20 wt% into bioglass® composition and concluded that high zinc content in bioglass retards glass dissolution and enhances cytotoxicity. The results of present study are consistent with the ionic dissolution studies of the glass coating containing zinc, as the
MJB-2015 concentration of the released zinc in the biological fluid was not significant; because this ion has the ability toenter the glass network and form Si-O-Zn bonds and is therefore not released in the medium[16].Hence, zinc was unable to potentiate the antibacterial action of the Zn-containing glass. In general, the antibacterial action of Zncontaining and Zn-freeglass coatings could be ascribed to the high concentration of certain ions such as calcium and silica. It is thought that calcium stimulates auto-agglutination of bacteria on the glass surface [4]; whereas silica inhibits bacterial growth by promoting calcium-phosphate layer formation [20],which could interfere with the integrity of bacterial cell wall. Furthermore, the antibacterial action of these glasses could be assigned to the ionic dissolution of these glasses which increases the alkalinity of the physiological medium[5,21].
Table2:Sensitivity of bacterial strains to QM5MgO and QM5MgF2 glasses on Mueller-Hinton agar plates
Glass coatings
Bacterial strains
QM5MgO QM5MgF2 QM5MgO QM5MgF2 QM5MgO QM5MgF2
Staphylococcus aureus Streptococcusmutans Porphyromonasgingivalis
Inhibition zone (mm) 35 45 45 45 50 45
615
Hameed et al. MJB-2015
A B
C
Figure 1:Effect of (A) Zn-containing (B) Zn-free bioactive glass coatings against S. aureus (C) is uncoated (control) titanium disc. The coated surfaces facing bacterial growth.
This study showed that the investigated glasses (with and without Zn) have antibacterial action against aerobic and anaerobic bacterial strains found in the oral cavity.For this reason, these glass coatings can be used to coat the body and the neck of dental implant to prevent the establishment of peri-implantitis.In addition, this feature renders these glasses are useful for other clinical applications such as coating of surgical sutures and pin fixation used in oral and maxillofacial surgery. The uncoated titanium discs did not show zone of inhibition. This indicates that titanium has no antimicrobial effect on the bacterial strains used in the present study. Conclusion Zinc at 3 mole% has no effect on antibacterial action of bioactive glass. The glass containing zinc and zinc free glasses exhibit similar inhibitory action on growth and multiplication of S. ureus, Strept.mutants andP.gingivalis.These glasses can be used in a variety of medical and dental applications. References 1. Groessner-Schreiber B., M. Hannig, A. Dьck, M. Griepentrog, D.F. Wenderoth.Do different implant surfaces exposed in the oral cavity of humans show different biofilm compositions and
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