Evaluating antibacterial and surface mechanical properties of chitosan modified dental resin composites

Abstract

BACKGROUND:

The antibacterial properties are beneficial and desired for dental restorative composite materials. The incorporation of various antimicrobial agents into resin composites may compromise their physical and mechanical properties hence limiting their applications.

OBJECTIVE:

The aim of the current study is to evaluate the antibacterial activity and the hardness of microhybrid and flowable resin based composites (RBCs) modified using novel antimicrobial agent chitosan (CS).

METHODS:

The antibacterial activity of microhybrid and flowable RBCs modified with 0, 0.25, 0.5 and 1% w/w chitosan (CS) against Actinomyces viscous bacteria was explored using agar diffusion test and direct contact methods. The hardness of control and experimental RBCs was determined by Vickers hardness (VH) tester.

RESULTS:

The results revealed that control and experimental flowable and microhybrid RBCs did not demonstrate growth inhibition zone in the lawn growth of Actinomyces viscous. The direct contact test revealed that colony forming unit (CFU) count of Actinomyces viscous was comparable among the experimental and control materials. The flowable RBCs containing 1% CS had significantly higher VH compared to control and other experimental flowable RBC groups. The microhybrid RBCs consisting of 0.50% CS exhibited significantly higher VH compared to experimental microhybrid RBC group containing 1% CS.

The addition of up to 1% chitosan into RBCs had no antibacterial activity against Actinomyces viscous however incorporation of CS in RBCs led to an increase in its VH, which predicts improved clinical performance of these materials.

Keywords: Antimicrobial activity, chitosan, resin based composites, Vickers hardness

1. Introduction

Globally, dental clinicians frequently use resin based dental composites (RBCs) for restoration of both anterior and posterior teeth. The increased utilization of RBCs is attributed to growing trends among patients for aesthetically pleasing fillings, questionable safety and negative impact of dental amalgam mercury on environment [1]. However, in vitro and in vivo research investigations have documented that more plaque accumulation occurs on RBCs compared to their counterparts’ namely dental amalgam and glass ionomers or tooth structures such as enamel [2, 3, 4, 5]. This increased deposition of dental plaque on modern RBCs have been attributed to four main factors; namely greater surface roughness, biodegradation, composition altering wet ability or surface free energy and lack of antibacterial activity [6, 7, 8]. Thus, during clinical life of RBCs, secondary caries that is mainly responsible for its premature failure may develop [9, 10]. The durable dental restorations are clinically appealing because they decrease the cost of restoration replacement and relieve the patient from several visits to dental clinics.

The microhybrid RBCs were selected in the present study due to its desired strength, abrasion resistance and suitable appearance. Hence, these RBCs have been widely used by clinicians for small to medium size restorations in posterior teeth. Moreover, the flowable RBCs were chosen because of its excellent handling characteristics and suitable flow into narrow pits and fissures on tooth surface [11]. Despite numerous efforts of researchers to equip RBCs with antimicrobial activity, still the scope of antibacterial properties of RBCs requires enhancement due to demand of clinicians. In order to address the challenge of secondary caries associated with RBCs and to enhance their clinical lifespan, several antimicrobial compounds have been incorporated such as chlorhexidine, quaternary ammonium, methacryloyloxydodecylpyridinium bromide (MDPB), furanone, ursolic acid, benzalkonium chloride, triclosan and zinc oxide [12, 13, 14, 15]. However, impact of these antimicrobial agents on RBCs physical characteristics have not been promising.

The RBCs hardness characteristic is another valuable predictor of its longevity. The RBCs hardness is influenced by resin and filler types, its amount; polymerization device and procedures [16, 17]. Many researchers explored the Vickers hardness (VH) of experimental and commercial RBCs due to its accuracy and ease [18, 19, 20]. Hence, we used the Vickers hardness testing for determining degree of polymerization in this study. In the literature, the Knoop hardness of experimental RBCs containing triclosan was explored and its negative impact of triclosan on hardness was observed [21]. This indicates need for new experimental RBCs modified with various antimicrobial agents which may also improve the required physical properties and have adequate clinical life span. Hence the search for new antibacterial agents is warranted which equips RBCs with desired antibacterial and physical properties. A promising option for equipping RBCs with antimicrobial properties is chitosan (CS). The number of medical and dental commercial products containing CS has been marketed. The CS is produced by alkaline deacetylation of chitin [22]. The major properties of CS include biocompatibility, safety, biodegradability, antimicrobial activity and ability to form film and gel [23, 24].

Because of these characteristics and well established safety, CS has been widely recommended for biomedical use [25, 26]. The CS based RBCs antibacterial activity against Streptococcus mutans and Lactobacilli casei has been explored previously [27, 28]. However, to the best of authors’ knowledge, there is no published study reporting the antibacterial activity of CS containing RBCs against Actinomyces species, which may comprise of up to 27% of the earliest colonizing cariogenic bacteria on human dentition [29]. Culture based investigations indicated that Actinomyces species are the prominent species during development and maturation of the dental plaque [30]. Hence, the aim of the current study is to assess the antibacterial activity of RBCs modified with novel antimicrobial chitosan (CS). The incorporation of any additive in RBCs may influence its mechanical properties making it unsuitable for restoration. Hence, surface Vickers hardness was also evaluated.

2. Materials and methods

The current study was conducted at the Medical Research Centre, Liaquat University of Medical and Health Sciences, Jamshoro, Pakistan after obtaining approval from the institutional research ethics committee (Ref#: REC/87).

2.1 Materials and sample preparation

This study was conducted using resin based composite materials; flowable (FA) RBCs (Filtek Z350 3M ESPE USA) and microhybrid (MH) RBCs (Filtek Z350 XT 3M ESPE USA). Chitosan (CS) in the form of coarse flakes/powder ( $>$ 75% degree of deacetylation; Sigma Aldrich, MO USA) was added in variable concentrations (0.25, 0.5, and 1.0% w/w) to each FA-RBCs and MH-RBCs [27, 28]. The homogeneous mixture of RBCs and CS was obtained by gentle mixing using a 50 ml glass beaker and a sterile glass rod in a dark room. The final concentration of FA-RBCs and MH-RBCs was adjusted to 0.25, 0.5, and 1.0% (w/w). The disc shaped specimens (2 mm thickness; 15 mm diameter); FA ( $n=$ 30; FA0.25, FA0.5, FA1.0; $n=$ 10 each) and MH ( $n=$ 30; MH0.25, MH0.5, MH1.0; $n=$ 10 each) were prepared using aluminium moulds according to ISO4049, 2000 [31]. The Elipar LED curing unit with 1000 mW/cm ${}^{2}$ irradiance (3M ESPE, Germany) and 10 mm diameter tip was used to cure specimens in an orbital sequence four times for 40 seconds each in overlapping shots [32]. The RBCs control group specimens of (FA0; $n=$ 10) and (MH0; $n=$ 10) were prepared without the addition of CS and using exactly the same methodology as described above. In addition, forty specimens (2 mm thickness, 5 mm diameter) for FA ( $n=$ 20) and MH ( $n=$ 20) RBCs with 0%, 0.25%, 0.50% and 1.0% (w/w) CS ( $n=$ 5) were prepared for testing the antibacterial activity via direct contact test. All specimens were inspected carefully and stored under ambient conditions until further experimentation.

2.2 Antibacterial activity test

The agar disc diffusion (ADT) and direct contact test (DCT) methods were used to evaluate the antibacterial effects of experimental RBCs. The ADT was carried out as described by Beyth et al. [33]. Briefly, an aliquot of 200 $\mu$ l of Actinomyces viscous (A.viscous) bacterium ATCC (American type culture collection) #15987Microbiologiscs USA suspension equivalent to (0.5 McFarland turbidity) was spread on petri dishes of brain heart infusion (BHI) agar. These petri dishes were allowed one hour for proper distribution and drying of bacterial suspension before the placement of experimental and control RBCs discs using sterilized tweezers. The bacterial growth inhibition zone around each RBCs specimen was measured by a metric ruler placed at bottom of the petri dish following 48 hours of incubation at 37 ${}^{\circ}$ C in a 5% CO ${}_{2}$ Incubator (Thermo Scientific, USA) [33].

In the DCT method, A. viscous suspension (10 $\mu$ l) was poured in specimen based Eppendorf tube prior to one hour incubation at 37 ${}^{\circ}$ C. Then BHI broth (300 $\mu$ l) was incorporated into the tube and placed in 5% CO ${}_{2}$ Incubator (Thermo Scientific USA) for 48 hours. It was followed by serial dilutions and colony forming unit (CFU) count via Miles and Misra technique described by Tin-Oo et al. [34]. Prior to serial dilutions and CFU count measurements, the Eppendorf tube was thoroughly mixed manually to ensure uniform distribution of bacteria. The RBCs specimens prior to antibacterial activity testing were sterilized in 70% ethanol [35].

2.3 Hardness testing

Vickers hardness (VH) evaluation was performed for both experimental and control groups of each RBC. Before testing, each specimen was finished with Mylar strip and 280-grit silicon carbide paper followed by polishing with aluminium-oxide discs (3M ESPE). A digital Vickers hardness tester (Wolpert 402MVD, USA) was used to perform three indentations (load: 200 g; dwell time: 10 seconds) on the top surface of each specimen. The mean of three separate indents was calculated as the VH of the specimen.

2.4 Statistical analysis

All data were handled using the SPSS (Version 22, IBM, USA). We applied one way analysis of variance (ANOVA) and post hoc Tukey tests to assess antibacterial activity and mean VH of study groups and any differences among groups. The level of significance was set at $P\leqslant$ 0.05.

3. Results

The control and experimental microhybrid and flowable RBCs did not exhibit antibacterial activity against tested bacterial strains. The growth inhibition zone of test bacterium Actinomyces viscous is not evident around experimental microhybrid and flowable RBCs (Fig. 1A and B). The results of direct contact test revealed that Actinomyces viscous colony forming unit (CFU) count were comparable among control and experimental RBCs (Table 1). Statistically no significant difference ( $P\geqslant$ 0.05) was observed among experimental and control RBCs.

Table 1
Colony forming unit (CFU) count of Actinomyces viscous bacteria per 20 $\mu$ l after 48 hours of contact with microhybrid and flowable RBCs

Material	Mean ( $P$ value $=$ 0.6)	SD	Material	Mean ( $P$ value $=$ 0.2)	SD
Control (MH0)	21.4	1.3 $\times$ 10 ${}^{-7}$	Control (FA0)	22.2	1 $\times$ 10 ${}^{-7}$
MH0.25	21.8	1.5 $\times$ 10 ${}^{-7}$	FA0.25	20.2	2 $\times$ 10 ${}^{-7}$
MH0.5	22.2	1.3 $\times$ 10 ${}^{-7}$	FA0.5	21.4	1 $\times$ 10 ${}^{-7}$
MH1.0	21.2	1 $\times$ 10 ${}^{-7}$	FA1.0	21.6	1 $\times$ 10 ${}^{-7}$

Table 2

Mean Vickers hardness (VHN) of experimental and control flowable and microhybrid RBCs

Material	Vickers hardness number (VHN)	SD	Material	Vickers hardness number (VHN)	SD
	( $P$ value $=$ 0.002)			( $P$ value $=$ 0.015)
Control (MH0)	49 ${}^{\text{b}}$	2	Control (FA0)	82 ${}^{\text{a},\text{b}}$	4
MH0.25	48 ${}^{\text{b}}$	2	FA0.25	77 ${}^{\text{a},\text{b}}$	1
MH0.5	50 ${}^{\text{a},\text{b}}$	1	FA0.5	83 ${}^{\text{a}}$	3
MH1.0	52 ${}^{\text{a}}$	1	FA1.0	75 ${}^{\text{b}}$	5

The different alphabet letters in $P$ value column indicate statistically significant difference.

Figure 1.

No inhibition zone is evident against Actinomyces viscous around experimental microhybrid (Fig. 1A) and flowable RBCs (Fig. 1B).

For control and experimental microhybrid and flowable RBCs mean VH values are presented in Table 2. The VH values of control and experimental flowable RBCs ranged from 47.5 to 52.2 VHN. The significantly higher VH values ( $P$ value $=$ 0.002) were exhibited by flowable RBCs based on 1% CS. The VH value for experimental and control microhybrid RBCs ranged from 75 to 82.5 VHN. The significantly higher VH values ( $P=$ 0.015) were demonstrated by microhybrid RBCs containing 0.5% CS. The incorporation of CS into RBCs enhanced its VH proving positive impact of CS. There was a statistically significant difference among the VH of control and experimental micro hybrid as well as control and experimental flowable RBCs.

4. Discussion

The present study attempted to evaluate the antibacterial activity and hardness of microhybrid and flowable RBCs modified using novel antimicrobial chitosan (CS) agent. The synthesis of antimicrobial RBCs without compromising the mechanical properties is appealing for researchers and clinicians. We used agar disc diffusion and direct contact testing against A. viscous for their antimicrobial activity and Vickers hardness to determine the hardness. Streptococcus mutans, Lactobacilli casei and Actinomyces viscous are the most common caries causing bacteria in human beings [36, 37]. These bacteria are also responsible for the secondary caries as they deposit on the outer surface of dental restorations including tooth-restoration interface. After fracture, secondary caries is considered as a major cause of restoration failure and patients frequently attend dental clinics for renewal of failed restorations. Consequently, this problem has caused concerns among clinicians as well as researchers. Therefore, to induce anticariogenic properties, investigators have incorporated fluoride releasing agents [38, 39, 40] and a variety of antimicrobial agents into RBCs; however, physical properties of RBCs appeared to be inferior after addition of such agents [41]. In recent years, use of chitosan, a biopolymer has widely been used in many commercial products due to its enhanced antimicrobial activity and safety [42, 43]. Therefore we made an effort to evaluate the antibacterial activity of RBCs containing CS coarse ground flakes and powder against one of the caries causing A. viscous bacterium.

For the evaluation of antimicrobial activity of commercially-available and experimental RBCs, mostly researchers have employed the ADT method [12, 13, 14, 15]. Following an incubation period of 48 hours, the current study observed no inhibition zone and lawn growth of A. viscous was identified in either control or experimental groups of RBCs. These findings suggested either no or low diffusion of CS from experimental RBCs in surrounding agar media. Moreover, absence of inhibition zone may be ascribed to possible copolymerization of CS biopolymer with matrix of RBCs and non-releasing behavior. Since, graft copolymerization of chitosan (CS) with methyl methacrylate, meth acrylic acid, 2-hydroxyethyl methacrylate (HEMA) and N, N-dimethyl amino ethyl methacrylate (DMAEMA) has been reported in the literature [44, 45]. In previous studies [13, 14], authors have reported no growth inhibition zone around control and experimental RBCs, which were filled with polyethylenimine (PEI) nanoparticles and MDPB antibacterial agents. It is well evident that ADT can only determine antibacterial activity of soluble agents which most likely diffuse into agar growth media and consequently leads to inhibition of bacterial growth. Thus, the insoluble antibacterial agents which exhibit bacterial growth inhibition via surface contact cannot provide meaningful data using ADT. Therefore, we also determined quantitative antibacterial activity of control and experimental RBCs by direct contact test (DCT) in accordance with the well-established literature [46].

In DCT method, caries causing bacteria are allowed to be in direct close contact with experimental and control RBCs and then are incubated under specific temperature and time. The metabolically active and vital cariogenic bacteria are essential in development of dental caries. Therefore, only living bacterial colonies were enumerated in the current research work via Miles and Misra technique following direct contact between bacteria and test material. The colony forming units (CFU) count of Actinomyces viscous were comparable among the control and experimental RBCs in the current investigation since no statistically significant difference in CFU count of control and experimental RBCs was observed. On contrary, Kim [46] has reported antibacterial effect of 2% w/w CS containing RBCs against Streptococcus mutans. The difference in results may be attributed to lower concentration of CS used in present study. More over the composition of RBCs, type of CS and antibacterial activity testing method employed in the two studies are not similar. This study attempted to determine antibacterial activity of CS, therefore, CS coarse ground flakes and powder were simply mixed manually via glass rod into the commercial RBCs without a use of solvent acetic acid to avoid antibacterial effect of solvent. Since, in this investigation ATCC pure culture of Actinomyces viscous bacteria were used in plank tonic form only, hence, we propose that antibacterial activity of RBCs against bacterial biofilm form should be evaluated. Moreover, The CS coarse ground flakes and powder was simply mixed manually in current study, which is the limitation of current study.

In the current study, the significantly higher VH values were exhibited by flowable RBCs based on 1% CS and microhybrid RBCs containing 0.5% CS, which indicate no negative effect of addition of CS on degree of polymerization of experimental RBCs. Moreover, increased hardness of experimental RBCs may be accredited to probable copolymerization of CS biopolymer with matrix of RBCs and non-releasing behavior. Since, graft copolymerization of chitosan (CS) with methyl methacrylate, meth acrylic acid, 2 hydroxy ethyl methacrylate (HEMA) and N, N-dimethyl amino ethyl methacrylate (DMAEMA) has been reported in the literature [44, 45]. On the contrary, addition of triclosan negatively influenced the hardness of RBCs in a previous study [21]. This could be linked to triclosan influence on translucence and the dispersion of light and consequently its degree of polymerization [47, 48]. The results of present study revealed that flowable RBCs containing 0.50% and 1% CS had higher VH values compared to control group. Whereas RBCs containing 0.25% CS exhibited slightly decreased VH compared to control specimens. Hence, addition of 0.50 and 1% CS into flowable RBCs improved the VH. The VH values of flowable RBCs are significantly lower than those for human enamel and dentin. Therefore, these materials cannot be used in high stress areas such as posterior teeth [49]. However, to obtain superior adaptation to tooth cavity and reduced micro leakage flowable RBCs can be used in conjunction with conventional RBCs so as to improve the performance of latter [50]. In current investigation, VH values of experimental flowable RBCs were found similar to control or significantly improved. These findings may be attributed to chemical interaction of CS with resin matrix and better curing capacity of CS modified flowable RBCs. The micro hybrid RBCs containing 0.25% and 1% CS exhibited lower VH values in comparison to control specimens. However, micro hybrid RBCs consisting of 0.50% CS exhibited VH values similar to control group. Due to lack of homogeneity in the VH results of micro hybrid RBCs, the direct association between the percentage of CS in these RBCs and its impact on surface VH could not be established. Authors of this study do not know what really differs among the experimental microhybrid RBCs that lead to inconsistent results. However, it is probable that the nature of interaction of CS with organic and inorganic component of the RBCs play a decisive role. Although every effort was made to remain consistent during specimen preparation and VH test. The assumption that CS would be a determinant factor in RBCs hardness value was not confirmed. There are no convincing evidences regarding the VH of CS based RBCs. It is recommended that further studies should be conducted to see the interaction of CS with constituents of RBCs so as to aid understanding.

5. Conclusions

The antibacterial activity testing suggested that there were no growth inhibition zones against Actinomyces viscous bacteria around the experimental and control resin composite materials. The addition of up to 1% chitosan into RBCs led to an increase in its Vickers hardness value.

Footnotes

Conflict of interest

The authors have no conflict of interest to report.

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Evaluating antibacterial and surface mechanical properties of chitosan modified dental resin composites

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

1. Introduction

2. Materials and methods

2.1 Materials and sample preparation

2.2 Antibacterial activity test

2.3 Hardness testing

2.4 Statistical analysis

3. Results

Table 1 Colony forming unit (CFU) count of Actinomyces viscous bacteria per 20 μ l after 48 hours of contact with microhybrid and flowable RBCs

5. Conclusions

Footnotes

Conflict of interest

References

Table 1
Colony forming unit (CFU) count of Actinomyces viscous bacteria per 20 $\mu$ l after 48 hours of contact with microhybrid and flowable RBCs