Comparative evaluation of antimicrobial efficacy at the implant-abutment interface using gold and silver ion coatings - An in vitro study

Abstract

Aim:

The aim of the study is to assess the antimicrobial efficacy at Implant-Abutment Interface (IAI) coated with metal ions (silver and gold) causing peri-implantitis, with the objective of comparing its antimicrobial properties.

Study Setting and Design:

The study conducted is an in vitro experimental study.

Material and Methods:

A total of 90 specimens 45 Titanium alloy discs substitute for Implant (Ti-6Al-4V, Grade V) and 45 Stainless Steel (315L SS) disc substitute for Abutment were serially ground, polished and cleaned to be coated with gold (Au) and silver (Ag) ions by sputtering technique which was later tested for antimicrobial efficacy by means of agar disk diffusion method with gram negative microorganisms causing peri-implantitis, the measurement of inhibition zone (ZOI) encircling each sample were taken and noted.

Statistical Analysis used:

The mean zone of inhibition data for all the groups were subjected for statistical analysis by Tukey’s multiple comparison test and one way ANOVA.

Results:

The results showed that the antimicrobial efficacy of gold coated Titanium alloy-Stainless steel, (TiAu –SSAu) and Silver coated Titanium- stainless steel (TiAg-SSAg) were significantly higher with a mean ZOI value of 24.6mm (Prevotella intermedia) for gold coated and 17 mm for silver coated groups compared to Uncoated group Titanium alloy- stainless steel (Ti-SS) with a mean zone of inhibition of 10mm, also proved by Tukeys multiple comparison test with statistical significant difference in the antibacterial activity of gold coated group compared to other groups where p <0.0001 against Porphyromonas Gingivalis, Prevotella Intermedia and Aggregatibacter Actinomyces.

Conclusions:

The surface modification of titanium alloy and stainless steel with metal ion coating (Au and Ag) significantly reduces the signs of peri-implantitis and thus leading to lasting success of implants.

INTRODUCTION

Tending of implants that is been placed inside the oral cavity by prevention of any correlated infection is the major dare to the success of any dental implant.[1,2,3,4] Among the various anti-infective therapy proposed over time, the most reliable cure for peri-implantitis can be broadly categorized into surgical and nonsurgical therapy. Even so, the assurance of the long-lasting benefits of these therapies is lacking, concept of nanotechnology and the great possibilities, it can offer even in the field of dentistry is astonishing.[5,6]

Amidst the various developments of nanotechnology in the field of science and technology their potential antimicrobial effects, have been receiving considerable heed concerning the treatment of peri-implantitis. Thus, the implementation of metal ion and its oxide nanoparticles can be thought of as a desirable substitute as proposed in various recent researches for traditional antimicrobial therapies.[6,7,8,9,10,11,12,13]

The proposed null hypothesis was that the implant abutment interface (IAI) coated with metal ions Au (Gold) and Ag (Silver) will have no significant antimicrobial efficacy.

Thus, in the present study, the coating of the IAI was done by highly effective metal nanoparticles such as Au and Ag ions which have significantly reduce the bacterial growth count and present a more beneficial clinical treatment.

MATERIALS AND METHODS

The study protocol was accepted and certified by the Institutional Ethics Committee: EC/NEW/INST/2022/3131. In the present study, the fabrication of the discs followed by coating of nano ions on the disks using sputtering method followed by antibacterial efficacy testing was conducted.

Sample size determination [Table 1]

Table 1.

Sample size distribution

Based on power study of 80%, the alpha error of 5% and effect of 0.05, the sample count was computed using G. Power version 3.1.9.4 software (StatSoft Inc., Tulsa, OK, USA) and was found to be 90. The sample size distribution of 90 samples (i.e., 45 titanium disks and 45 stainless steel disks) after the coating with Au and Ag ions with sputtering method for the final step/test, i.e., the microbiological test is as shown in Table 1 [Note the codes given for the coated and uncoated disks from A to F in Table 1].

Three agar plates were taken for three different microorganism namely Porphyromonas gingivalis, Prevotella intermedia, and Agregatibacter Actinomycetemcomitans as Group I, Group II and Group III respectively. In one agar plate, 3 Titanium disks was placed which was coded as A, C and E, respectively (A: Ti coated with Au, C: Ti coated with Ag, E: Uncoated Titanium). Hence, for 3 agar plates, 9 titanium disks were taken. Since the procedure (antimicrobial test) was repeated 5 times to increase the reliability of the study, i.e., n = 5, 45 titanium disks and 45 stainless steel disks were used in the overall study. The same was followed for stainless steel disks/replicas wherein a single agar plate had 3 stainless steel disks placed, coded as B, D and F, respectively (B: SS coated with Au, D: SS coated with Ag, F: uncoated stainless steel) [Figure 1 shows the schematic representation of placement of coded disks (A-F) on the agar plate groups for each microorganism].

Figure 1.

Schematic representation of coding of titanium and stainless-steel disk placement on agar plates for antimicrobial test

Steps involved in analysis of IAI antimicrobial activity:

1. Fabrication of titanium alloy discs and stainless-steel discs

2. Coating of titanium alloy and stainless-steel discs with Au and Ag ions by sputtering technique

3. Investigation of antibacterial effects

4. Statistical analysis.

Fabrication of titanium alloy and stainless-steel discs

A Titanium alloy rod (Ti-6Al-4V) and a stainless-steel rod were sliced as discs (10 mm diameter, 2 mm thickness) utilizing a lathe. A total of 90 specimens (45Ti alloy and 45 SS disks) were repetitively grounded by increasing grits of silicon carbide abrasive papers and further polished with 1 μm diamond paste to wipe off the remaining creaks. The discs were then cleaned with acetone and distilled water to remove the abrasive debris.

Coating of Ti alloy and SS discs with Au and Ag ions by sputtering technique [Figure 2a and b].

Figure 2.

(a and b) Disks coated with gold and silver ions with sputtering technique

The Ti alloy and SS discs were coated with Ag (TiAg), Au (TiAu), Ag (SSAg), and Au (SSAu) by sputtering deposition technique. The sputtering of the discs was done with SPF-332H multi-cathode sputtering system. Postdeposition, the thickness of gold ions was 114 nm and for silver ions it was 110 nm.

Investigation of antibacterial effects

The microbiological test was done using Agar disc diffusion test, three agar plates were taken for three different microorganisms namely P. gingivalis, P. intermedia, A. actinomycetemcomitans as Group I, II and III respectively [Figure 2]. The agar plates were then incubated with their respective microorganisms in the bacteriological incubator before placing the coated and uncoated discs. Following placement of discs on the agar plate [Figure 3a], the plate along with discs was incubated for 3 days and a clear zone encircling each sample was observed revealing the antibacterial activity [Figures 3b and 4]. The diameter of each zone was gauged in mm. The readings after every 24 h for 3 days for the zone of inhibition (ZOI) diameter encircling each sample were noted.

Figure 3.

(a) Placement of disks on agar plate, (b) Zone of inhibition formed around the disks for Prevotella intermedia

Figure 4.

Magnified image of a single disk showing zone of inhibition for Prevotella intermedia

Statistical analysis

The antibacterial activity of Sample specimen Groups, namely, Group A, Group B, Group C, Group D, Group E, and Group F were studied determining the ZOI produced for 3 days in a standard broth culture medium against three bacterial species, namely, P. gingivalis, P. intermedia, and Aggregatibacter actinomyces. The descriptive statistical analysis suggested the antibacterial activity in terms of ZOI. Further, the mean ZOI data after 1 day for all the Groups were subjected for Statistical analysis by Tukey’s multiple comparison test and one-way ANOVA using statistical software Graph pad Prism Version 8.0 by Dotmatics (Headquartered in Boston, Massachusetts.). The statistical tool used to verify normality was Shapiro–Wilk test.

RESULTS

The descriptive statistical analysis suggest the antibacterial activity in terms of ZOI of stated specimen groups shows maximum after 1^st day and the activity decreased after 2^nd day and 3^rd day in all the studied bacterial species. Overall results indicate the antibacterial activity was good and better against P. intermedia followed by A. actinomyces and P. gingivalis after 1^st day and same data analyzed statistically.

Table 2 shows the descriptive statistical data showing ZOI values describing the antibacterial activity of P. intermedia.

Table 2.

Descriptive Statistics :Antibacterial activity of sample group specimens against Prevotella Intermedia

S.No	GROUP-A			GROUP-B			GROU- C			GROUP-D			GROUP-E			GROUP-F
	Day-1	Day-2	Day-3	Day-1	Day-2	Day-3	Day-1	Day-2	Day-3	Day-1	Day-2	Day-3	Day-1	Day-2	Day-3	Day-1	Day-2	Day-3
Zone of inhibition in mm			Zone of inhibition in mm			Zone of inhibition in mm			Zone of inhibition in mm			Zone of inhibition in mm			Zone of inhibition in mm
1	25	23	23	20	18	18	18	18	18	15	13	12	11	10	9	10	9	8
2	24	22	22	21	19	17	17	18	18	16	12	11	10	9	8	11	8	7
3	25	24	23	20	18	17	17	17	18	14	13	11	10	10	8	10	8	7
4	24	23	21	21	17	18	18	17	17	15	12	12	11	9	8	10	8	7
5	25	23	22	20	18	17	17	18	17	15	14	12	10	9	8	10	8	8
Mean	24.60	23.00	22.20	20.40	18.00	17.40	17.40	17.60	17.60	15.00	12.80	11.60	10.40	9.400	8.200	10.20	8.200	7.400
SD	0.547	0.707	0.836	0.547	0.707	0.547	0.547	0.547	0.547	0.707	0.836	0.547	0.547	0.547	0.447	0.447	0.447	0.547
SEM	0.244	0.316	0.374	0.244	0.316	0.244	0.244	0.244	0.244	0.316	0.374	0.244	0.244	0.244	0.200	0.200	0.200	0.244

GROUP - A Titanium disk coated with Gold

GROUP - B Stainless steel disk coated with Gold

GROUP - C Titanium disk coated with Silver

GROUP- D Stainless steel disk coated with Silver

GROUP E Uncoated Titanium disks

GROUP F Uncoated Stainless steel disks

F (DFn, DFd)	P
F (5, 24)=407.0	P<0.0001

The mean ZOI is summarized in Graph 1 suggesting the antibacterial activity in terms of ZOI of stated specimen groups was maximum after 1^st day and the activity decreased after 2^nd day and 3^rd day in P. intermedia and the same was conducted for P. gingivalis and A. actinomyces.

Graph 1.

Antibacterial activity of sample group specimens against Prevotella intermedia

The Tukeys multiple comparison test suggest, Group A revealed a statistically significant difference (P = 0.0075) in the antibacterial activity compared to Group B. Similarly, Group A showed a statistically significant difference in the antibacterial activity compared to Group C, Group D, Group E, and Group F (P < 0.0001). Group B revealed a statistically significant difference in the antibacterial activity compared to Group C (P = 0.0023); Group B revealed a statistically significant difference in the antibacterial activity compared to Group D, Group E and Group F, (P < 0.0001). Group C showed nonsignificant difference in the antibacterial activity compared to Group D (P = 0.0691); Group C showed a statistically significant difference (P < 0.0001) in the antibacterial activity compared to Group E and F against P. gingivalis. Group D showed a statistically significant difference (P < 0.0001) in the antibacterial activity compared to Group E and Group F; Group E showed a statistically significant difference (P = 0.0023) in the antibacterial activity compared to Group F against P. gingivalis.

The Tukeys multiple comparison test suggest, Group A showed a statistically significant difference in the antibacterial activity compared to Group B, C, D, E and Group F (P < 0.0001). Group B presented a statistically significant difference in the antibacterial activity compared to Group C, Group D, Group E, and Group F (P < 0.0001). Group C showed a statistically significant difference in the antibacterial activity compared to Group D, Group E, and Group F (P < 0.0001). Group D showed a statistically significant difference in the antibacterial activity compared to Group E, Group F (P < 0.0001). Group E showed statistically nonsignificant difference in the antibacterial activity compared to Group F (P = 0.6479) against P. intermedia.

The Tukeys multiple comparison test suggest, Group A exhibited a statistically significant difference in the antibacterial activity compared to Group B, C, D, E, and F (P < 0.0001). Group B exhibited statistically nonsignificant difference in the antibacterial activity compared to Group C (P < 0.6675); Group B exhibited a significant difference in the antibacterial activity compared to Group D, Group E and Group F (P < 0.0001). Group C exhibited a statistically significant difference in the antibacterial activity compared to Group D, Group E and Group F (P < 0.0001). Group D showed a statistically significant difference in the antibacterial activity compared to Group E, Group F, (P < 0.0001); Group E showed statistically nonsignificant difference in the antibacterial activity compared to Group F (P = 0.6675) against A. actinomyces.

Overall, the Tukey’s multiple comparison test suggest, Group A showed a statistically significant difference in the antibacterial activity compared to other Groups where P < 0.0001 against P. gingivalis, P. intermedia and A. actinomyces.

Table 3 shows Tukey’s multiple comparisons test data for antibacterial activity of sample group specimens against P. gingivalis.

Table 3.

Tukey's multiple comparisons test data for antibacterial activity of sample group specimens against Porphyromonas Gingivalis

Tukey's multiple comparisons test	Mean Diff.	95.00% CI of diff.	Significant?	Summary	Adjusted P
GROUP-A vs. GROUP-B	1.600	0.3377 to 2.862	Yes	**	0.0075
GROUP-A vs. GROUP-C	3.400	2.138 to 4.662	Yes	****	<0.0001
GROUP-A vs. GROUP-D	4.600	3.338 to 5.862	Yes	****	<0.0001
GROUP-A vs. GROUP-E	7.400	6.138 to 8.662	Yes	****	<0.0001
GROUP-A vs. GROUP-F	9.200	7.938 to 10.46	Yes	****	<0.0001
GROUP-B vs. GROUP-C	1.800	0.5377 to 3.062	Yes	**	0.0023
GROUP-B vs. GROUP-D	3.000	1.738 to 4.262	Yes	****	<0.0001
GROUP-B vs. GROUP-E	5.800	4.538 to 7.062	Yes	****	<0.0001
GROUP-B vs. GROUP-F	7.600	6.338 to 8.862	Yes	****	<0.0001
GROUP-C vs. GROUP-D	1.200	-0.06228 to 2.462	No	Ns	0.0691
GROUP-C vs. GROUP-E	4.000	2.738 to 5.262	Yes	****	<0.0001
GROUP-C vs. GROUP-F	5.800	4.538 to 7.062	Yes	****	<0.0001
GROUP-D vs. GROUP-E	2.800	1.538 to 4.062	Yes	****	<0.0001
GROUP-D vs. GROUP-F	4.600	3.338 to 5.862	Yes	****	<0.0001
GROUP-E vs. GROUP-F	1.800	0.5377 to 3.062	Yes	**	0.0023

The analysis of variance revealed statistically significant differences among different specimen Groups (within the Groups and between the groups) against all the three studied bacterial species (P < 0.001). The results from antibacterial activity in all the studied groups found to be significant against all the three bacterial species in the order P. intermedia [Table 2] followed by A. actinomyces and P. gingivalis.

DISCUSSION

The micro aperture at IAI stands for a locale of dental Plaque Conglomeration encouraging bacterial leakage in turn upsurging inflammatory cells at IAI, leading to peri-implantitis.[14] The two parts of implants inevitably exhibit a micro aperture between the implant and the abutment.[15] Once colonized, such zones establish a bacterial reservoir subsequently contaminating the implant’s milieu and impede the peri-implant’s tissue health. These bacteria pioneered at the IAI level can be anaerobic as well as facultatively anaerobic.[16] Peri-implantitis is routinely linked with Gram-negative bacteria indistinguishable from the ones that bring about periodontal disease. The customary therapies of peri-implantitis have been unpredictable[1] and surgical therapies include resective and augmentative procedures.[5] Thus, the prohibition of bacterial infection at the IAI comes across as the key point for a healthy peri-implant site and long-term maintenance of the implant.[14]

Of late the concept of nanotechnology has proffered enormous possibilities in varied grounds of science and technology.[6] Morphological and physicochemical features of the nano metals have shown beyond doubt to employ an effect on their antimicrobial activities.[6] Previous studies have shown nanoparticles on implant surfaces to have a potent antibacterial effect but a study on the antimicrobial effect of metal ions such as Au and Ag on the IAI was not been conducted.[6,8,9,11]

In the last tenner, a myriad of studies has been recorded about the potentiality of titanium surface modifications and coatings to cut back microbial adherence, impede the formation of biofilms, and provide effectual bactericidal effect protecting implanted biomaterials.[4,17,18]

This study was conducted in two parts comprising, first coating of implant replicas and abutment replicas with Ag as well as Au ions using sputtering deposition method followed by latter part of the study consisting of testing the efficacy of the coating by microbiological study.

An Agar disc diffusion test was done to examine the antimicrobial efficacy of the disc samples, specifically for Gram-negative organisms namely P. gingivalis, P. intermedia, and A. actinomyces which are the most common microorganism known to cause peri-implantitis.

In the present study, the coating of the disks was done by sputtering technique.[19,20,21,22,23] In this sputtering technique means the material (e.g., Au and Ag ions in the present study) to be deposited is bombarded with positive inert ions with kinetic energy outstripping the heat of sublimation of the target material (IAI in the present study) results in dislodging of target atoms and their ejection into the gas phase succeeded by deposition of the substrate (like gold or silver being deposited on IAI) using SPF-332H multi-cathode sputtering system. When coating by sputtering, the bond formed between the deposited material and the substrate is primarily a physical bond at the atomic level due to the high energy involved in the process, essentially creating a very strong adherence amidst the film and the substrate through close packing of atoms at the interface.

Previous studies have evaluated the antibacterial efficacy of metal coated titanium implant surfaces by various methods such as polymerase chain reaction (PCR),[14,15,16] broth culture test. While reviewing a study by Mishra et al., it was proved that the disc-diffusion method is cost-effective as opposed to other tests, easy to conduct, and no special materials and techniques required in contrast to the e-test susceptibility method.[25]

In the present study, an agar disc diffusion test was done to test the antimicrobial efficacy of the replicas.[26] Carinci et al. used the PCR analysis in their study in which an antimicrobial coating of polysiloxane was put into effect with chlorhexidine digluconate (PXT). In a time, span of 48 h only 0.3% of the bacteria that contaminated the exterior of the implant-abutment junction penetrated the internal chamber and was later completely inactivated as shown by the PCR analysis.[14]

Kheur et al. veneered silver onto Ti abutments (Grade 5 Ti discs) using direct current sputtering deposition and tested for its antimicrobicity.[27] An excellent antibacterial effect of TiAg was seen on Pseudomonas aeruginosa and Streptococcus mutans at a very low conc. (Ag content 1.2 and 2.1 µg/mm² and at a higher amount of Ag (6.1mug/mm²) on Staphylococcus aureus and Candida albicans. His study proved a lasting antibacterial effect, which was revealed by the dispense of silver for 22 days in simulated body fluid for Ag-coated Titanium abutments.[27]

Hameed et al.,[26] in their study, compared the antibacterial action of Ti, TiCu, TiHA, and Ti Cu/HA against P. gingivalis, the results indicated that the surface alteration of Ti-6Al - 7Nb alloy with Cu/HA may prosper as a coating for local infection control.

The result of the present study revealed that Au and Ag released from TiAu, TiAg, and SSAu and SSAg had strong antibacterial effects as compared to the antibacterial efficacy of uncoated titanium and stainless-steel replicas against most common microorganisms that cause peri-implantitis, i.e., P. gingivalis, P. Intermedia, A. actinomyces.

Tukey’s multiple comparison test suggests, Group A (i.e., Titanium coated with gold ions) revealed a statistically significant difference (P = 0.0075) in the antibacterial activity in contrast to Group B. Similarly, Group A also exhibited a statistically significant difference in the antibacterial activity compared to Group C to Group F (P < 0.0001) for P. gingivalis.

Furthermore, the study showed similar results against P. intermedia and A. actinomyces also, which was in accordance with the work of Hameed et al.[26]

In a study conducted by Kheur et al., the viable count diminished radically after 6 h in all Ag coated abutments in the case of S. mutans and P. aeruginosa.[27]

In the current study, the antibacterial effect of TiAg (silver-coated implant replicas) and SSAg (silver coated abutment replicas) was maximum in 24 h in the case of P. gingivalis, P. intermedia, and A. actinomyces. The antimicrobial performance of Ag ions released from nanoparticles is accredited to their integration to the bacterial cytoplasmic membrane that is negatively charged inactivating cellular enzymes, which disturbs the membrane permeability causing lysis and eventually cell death.[24,27,28,29]

In a study conducted by Cui et al., the bactericidal property of Au nanoparticles was assessed.[7] The minimum bactericidal concentration (MBC) of Au nanoparticles showed a range of 10–16µg/ml and the minimum inhibitory concentration (MIC) of 4mug/ml. The MBC/MIC ratio was <4 suggesting that Ag nanoparticles having bactericidal properties against Escherichia coli. In the present study, the mean ZOI of Au-coated implant replicas was highest (i.e., 25 mm in 24 h) among all other groups against P. intermedia. The ZOI gave similar results against P. gingivalis and A. actinomyces suggesting the high antibacterial efficacy of Au nanoparticles. This antibacterial pursuit in regard to Au ultrafine particles can be accredited to (1) Affinity of Au to the bacteria with subsequent membrane potential alteration and depletion of the quantity of ATP and (2) Hampering of tRNA linked to the ribosome.[7,30,31]

Tiwari et al. had also put to test the microbial activities (bacterial and fungal) of the Au ultrafine particles along with 5-fluorouracil against Micrococcus luteus, S. aureus, P. aeruginosa, Aspergillus niger, Aspergillus fumigates, and E. coli.[11] It was indisputably shown that Au nanoparticles attributing to their easier introjection into the Gram-negative bacteria showing more exertion than on Gram-positive ones, also Au nanoparticles are reliable and safe to mammalian cells as compared to other nano metals due to the reactive oxygen species-independent mechanism of their antimicrobial action. Besides, the peak impact of these nanoparticles for practicality earmarks them as antimicrobial agents.[9]

The null hypothesis of the study that the IAI coated with metal ions (Gold and Silver) will have no significant antimicrobial efficacy was rejected.

The research hypothesis is accepted since the IAI coated with metal ion lay significant antimicrobial efficacy.

Within the limitations of this study, though the research was done on the antimicrobial efficacy of IAI modified with Au and Ag ions coating with a specific group of the organism i.e., P. gingivalis, P. intermedia, and A. actinomyces, it can be conducted on various other complex microorganisms such as Fusobacterium nucleatum, Bacteroides, and other Gram-negative bacteria that cause peri-implantitis and ultimately failure of the implants. Clinical relevance can be further investigated with coated IAI.

Clinical implication

The clinical significance of coating IAI with metal ions is that it can prevent the formation of biofilms that causes aggregation of bacteria thus preventing peri-implantitis. The coating done by the sputtering technique is economical when done on a large scale. Coating of the IAI also reduces the need for various surgical and nonsurgical procedures that are required for the maintenance of implants during or after placement of implants in the oral cavity.

CONCLUSIONS

Based on the findings of this in vitro study, the following conclusions were drawn:

1. A statistically significant antimicrobial efficacy was noted with Au and Ag ions coated IAI as compared to uncoated interface against microbes causing peri-implantitis

2. Au coated samples have shown the most significant difference in the antibacterial activity compared to Ag and uncoated groups where P < 0.0001 against P. gingivalis, P. intermedia, and A. actinomyces.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.