Antimicrobial Efficacy of Irreversible Hydrocolloid Impression Impregnated with Silver Nanoparticles Compared to Surface Disinfected Impressions - An In vivo Study.

BACKGROUND: Routine disinfection procedures have shown to cause incomplete disinfection and detrimental effects on dimensional properties of the impression. Hence, self-disinfecting impression materials impregnated with antimicrobial agents were developed.
PURPOSE: The purpose is to evaluate the antimicrobial efficacy of silver nanoparticles (AgNPs) impregnated in irreversible hydrocolloid (IH) impression material in in vivo conditions.
MATERIALS AND METHODS: This study comprised of four groups-IH impressions disinfected by immersion in 2% glutaraldehyde, IH impregnated with AgNPs of sizes 80-100 nm and 20-30 nm, nondisinfected impressions as control. Five impressions were made for each group and a total of 20 impression samples were made. The antimicrobial action of each sample was assessed by counting the number of colony forming units and by disc diffusion method.
RESULTS: The results were obtained and the data were statistically analyzed by Kruskal-Wallis test and tabulated. The results revealed that AgNPs of size 80-100 nm when impregnated in irreversible impression material showed effective antimicrobial action.
CONCLUSION: The anti-microbial action of 80-100 nm AgNP impregnated IH impressions is comparable to that of impressions disinfected by immersion in 2% glutaraldehyde for 10 min. Copyright:

INTRODUCTION

The contamination of irreversible hydrocolloid (IH) impressions from the oral cavity can cause cross infection to dental staff and laboratory personnel.[1234] Due to their hydrophilic nature, IH impressions were found to retain the microbes in them for a longer duration when compared to other impression materials. Several handling and disinfection protocols have been suggested for IH impressions.[567] The two main methods of disinfection of IH are spray and immersion methods. Certain drawbacks have been noticed with immersion and spray methods such as dimensional inaccuracy and incomplete disinfection, respectively.[89] To overcome these drawbacks of spray and immersion methods of disinfection, impregnated IH impression materials have been developed.[1011121314] The self-disinfectant impression materials also have an added advantage of reducing the time required for an extra chair side procedure of disinfection.

Few in vitro studies have reported that silver nanoparticles (AgNPs) impregnated IH impressions showed effective antimicrobial activity depending on the concentration and the size of the nanoparticles.[1516171819] AgNPs of size 80–100 nm have shown significant antimicrobial action than other smaller particle sizes in IH impression material. This was in contrast with earlier literature.[20] Thus, there exists a controversy in the relationship between the size of AgNPs and their antimicrobial action. Few studies also assessed the physical properties of the IH impression material when incorporated with AgNPs. It was found that the physical properties were not adversely affected when AgNPs of optimal concentration were incorporated in IH impression material.[15161718]

However, there are no in vivo studies available on the AgNPs impregnated IH impression material regarding their antibacterial action when used for making impressions in the oral cavity. Thus, the present in vivo study aims to check the antimicrobial efficacy of AgNPs of two different sizes incorporated in IH impressions and compare it to the surface disinfected impressions by immersion in 2% glutaraldehyde for 10 min.

MATERIALS AND METHODS

The ethical clearance for this study was obtained from the Institutional Human Ethics Committee (IHEC/586/2019) and informed consent was obtained from the subjects, after explaining the purpose and procedure of the study. Five healthy individuals under the age group of 24–30 years with intact dentition were selected as study subjects. The study subjects were devoid of any systemic or local complications. For this study, four groups were formed. Group I comprised of impressions disinfected by immersion in 2% glutaraldehyde, Group II and Group III comprised of IH impressions impregnated with 80–100 nm AgNps and 20–30 nm AgNps, respectively. Group IV comprised of nondisinfected control impressions.

Preparation of impregnated irreversible hydrocolloid impression material

Algitex (DPI) IH impression material was used. AgNPs of sizes 20–30 nm and 80–100 nm (Nano Research Lab, Jharkhand) and weight 2.5 wt.% were added in powder form to IH. This was subjected to ultrasonic vibration for 20 min, followed by ball milling at a speed of 200 rpm for 20 min to ensure homogeneous mixture of IH with AgNPs. Each sample was subjected to disinfection in hot air oven at 160°C for 1 h.

Impression making

Four IH impressions (one for each group) were made for each study subject in perforated autoclaved impression trays. Study samples were collected after 10 min from each group.

Microbiological tests

Colony forming units/ml method

The Sterile Phosphate-Buffered Saline was added on the surface of the impression. Then, sterile swab was taken from the impression surface and was directly spread on Mueller-Hinton agar (MHA) plates. The test plates were incubated for 48 h at 37°C. The colony forming unit (CFU)/ml were appreciated on the test plates after 48 h, and were counted for each sample [Figures 1–4].

Figure 1

Colony forming unit count Group I - 2% glutaraldehyde disinfected

Figure 4

Group IV - nondisinfected control group

Group II - 80–100 nm silver nanoparticles impregnated irreversible hydrocolloid impression

Group III - 20–30 nm silver nanoparticles impregnated irreversible hydrocolloid impression

Disc diffusion method

In disc diffusion method, the test pathogens (Streptococcus mutans) were spread on MHA plates. Using Bard Parker knife, disc of diameter 4 mm was cut from the surface of each sample and placed on the agar plates. Each plate consisted of four samples. The agar plates were incubated for a period of 48 h at 37°C. After 48 h time period, the zone of inhibition (ZOI) was evaluated around each test sample [Figure 5].

Figure 5

Zone of inhibition formed around Group I and II

Statistical analysis

The values of the CFU/ml assessed and the zone of inhibition evaluated were tabulated. Systat software version 12, details, Cranes software, Banglore, India (company),Chicago (Headquarter) was used to perform the statistical analysis. Kruskal–Wallis test was performed to assess the data, a difference was found among the four groups and it was statistically significant. The P < 0.001. To compare the statistical difference between the groups, Kruskal–Wallis multiple comparison test was performed.

RESULTS

As shown in Table 1, the group with 80–100 nm AgNPs showed a significant difference in the number of CFU/ml when compared to the control group and it showed comparable antimicrobial action to the standard 2% glutaraldehyde immersion disinfection group. The number of CFU/ml in 20–30 nm AgNPs incorporated in IH material was found to be lesser than the nondisinfected control group and >80–100 nm AgNPs impregnated group. Both the differences were statistically insignificant.

Table 1

Mean and standard deviation of colony forming units counted from the surface of irreversible hydrocolloid impressions

Group	Mean	SD	Minimum	Maximum	Kruskal-Wallis test	P	Kruskal-Wallis multiple comparison
I. Glutaraldehyde disinfected	0	0	0	0	17.6	<0.001
II. 80-100 nm	427.20	171.894	224	596			1>2, 3, 4
III. 20-30 nm	2646	955.797	1253	3861
IV. Control	4958	1424.287	2538	6131			2>4

SD: Standard deviation

As shown in Table 2, the 2% glutaraldehyde disinfected group and the group with 80–100 nm AgNPs impregnated in IH impression material showed the formation of zones of inhibition around their samples.

Table 2

Zone of inhibition evaluated by disc diffusion method for irreversible hydrocolloid impressions

Group	Mean	SD	Minimum	Maximum	Kruskal-Wallis test	P	Kruskal-Wallis multiple comparison
I. Glutaraldehyde disinfected	12.80	1.483	11	15	18.44	<0.001	1, 2>3, 4
II. 80-100 nm	6.40	0.348	6	7
III. 20-30 nm	0	0	0	0
IV. Control	0	0	0	0

SD: Standard deviation

DISCUSSION

In this study, it was found that AgNPs of size 80–100 nm when incorporated in IH impression material showed significant antimicrobial action when compared to the nondisinfected control group. The results of this study are in accordance with the in vitro study performed in 2016, in which AgNPs of size 80–100 nm used in two different brands of IH impression materials produced effective antimicrobial action without altering the physical properties of the material.[16] The AgNPs of size 80–100 nm exhibited superior antimicrobial action when compared to other particle sizes such as 10–20 nm, 30–50 nm, and 50–80 nm. This antimicrobial action was dose dependent.[18]

In this study, AgNPs of size 20–30 nm impregnated in IH impression material did not show any significant antimicrobial action compared to the control group. The results of this study are in accordance with the in vitro study conducted in 2018, in which AgNPs of different concentrations and sizes such as 10–20 nm, 30–50 nm, and 50–80 nm did not show any antimicrobial action against Escherichia coli, Staphylococcus Aureus, and Candida albicans.[18]

AgNPs of size 80–100 nm impregnated in IH impression material produced effective antimicrobial action and was comparable to the glutaraldehyde disinfected group. Disinfection by immersion in 2% glutaraldehyde has shown to cause complete eradication of microbes.[2122] However, this method has shown to cause detrimental effect on dimensional properties of the material, thus affecting the quality of resultant gypsum casts. It also has certain toxic effects due to the presence of the aldehyde group.[2324] The physical properties were found to be not altered in the in vitro studies on IH impregnated with AgNPs in optimum concentrations.[1517]

In a study, conducted in 2010, it was found that AgNPs of size 20–30 nm is the most effective particle size among other sizes without significant cytotoxicity suggesting their use as an effective antimicrobial agent,[20] which is in contrast with our study. The reason for the absence of antimicrobial action in the 20–30 nm group in the present study, could be due to the addition of AgNPs in powder form. When the particle size reduces, the aggregation of particles increases and reduces the surface area available for anti-microbial action.[25] The aggregation of the AgNPs of smaller size can be clearly seen in the in vitro study conducted in 2018.[18] Consequently, there is less aggregation in 80–100 nm due to increased particle size which makes increased amounts of AgNPs made available for antimicrobial action.

In many studies on this topic, disc diffusion method is used to assess the antimicrobial action. Agar diffusion method is reported to be the most efficient and qualitative test that shows the resistance of microbial growth against the particular antimicrobial agent used in the test. However, this test does not have the ability to distinguish between bacteriostatic and bactericidal effect.[26] S. mutans was chosen for disc diffusion method in this study, as it most commonly present oral microflora along with S. aureus. In CFU count, the number of viable bacterial cells retained in the impression after 10 min of impression making were counted.[27] The reduction in colonies from the control specified the bactericidal action of the sample, which could not be appreciated in disc diffusion method. There existed an inverse correlation between the two tests. The lesser the CFU count, the larger is the ZOI and which is equal to the antimicrobial efficacy of the particular group.

In this study, AgNPs of concentration 2.5 wt. % was used in both the particle sizes 20–30 nm and 80–100 nm. This was in accordance with the in vitro study performed in 2019, where it was concluded that 2.5 wt.% of AgNPs is an optimal concentration without affecting the physical properties of the material.[19] The mechanism of antibacterial action of AgNPs is not clearly known, though several authors have come with various theories. Formation of pits in cell wall of bacteria followed by disturbance in the membrane permeability and then leading to cell death, is one of the widely accepted concepts.[28]

Further studies are needed to assess the antimicrobial action of AgNPs impregnated in IH impression material in different concentrations. The colloidal form of AgNPs can reduce the problem of agglomeration and further studies are needed to prove its efficacy.

CONCLUSION

Thus, from the findings of the present in vivo study, it can be concluded that:

AgNPs of size 80–100 nm impregnated in IH impression material showed an effective antimicrobial action by both the CFU and zone of inhibition methods

AgNPs of size 20–30 nm impregnated in IH impression material did not produce any antimicrobial action

When compared with the impressions disinfected by immersion 2% glutaraldehyde for 10 min, the 80–100 nm AgNPs impregnated impressions produced comparable antimicrobial action

IH impression material impregnated with AgNPs of size 80–100 nm can be used as an effective alternative to impressions disinfected with 2% glutaraldehyde by immersion method.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.