Comparative evaluation of the antibacterial activity of red diode laser therapy and 0.2% chlorhexidine against Aggregatibacter actinomycetemcomitans on implant healing abutments

An ex vivo study

Abstract

Aims: 

The intraoral microbiota has a high potential to undergo dysbiosis, causing inflammatory changes with respect to the tissues surrounding either a natural tooth or an implant. Thus, the longevity of implant prosthesis depends on a thorough implant decontamination protocol. Among all the techniques available for doing so, laser is garnering increasing popularity, owing to minimal bleeding, high efficiency, and faster healing. However, limited literature exists regarding the superiority of lasers over chlorhexidine (CHX), the indisputable gold standard antibacterial chemical agent. The aim of this study was to compare the percentage of bacterial reduction of Aggregatibacter actinomycetemcomitans from implant healing abutments post red diode laser therapy versus 0.2% CHX treatment.

Settings and Design: 

The current study had an ex vivo, observational, case–control design.

Materials and Methods: 

Patients reporting for the second stage of the implant surgery were taken as the source of data and the healing abutments, the clinical samples. Eleven patients were chosen with one intraoral implant serving as the test site for laser treatment and another, the control site for CHX treatment. Microbiological analysis was performed via quantitative real time polymerase chain reaction to compare the bacterial reduction percentage after each treatment.

Statistical Analysis Used: 

Repeated measures ANOVA and independent sample t test were used.

Results: 

The mean bacterial viability of the test group (laser) was 1.2%–1.6%, and 0.6%–1.4% for the control group (CHX). The former caused a mean bacterial reduction of 96.1% while the latter, 96.3%. Both the treatments caused a highly statistically significant reduction of viable bacterial counts (P = 0.001). However, when compared, there was no statistically significant difference in the bacterial reduction, when compared in between the two (P = 0.902).

Conclusion: 

Laser treatment is at par with chemical implant surface decontamination. It can help bypass the complications of CHX and revolutionize the protocols for implant surface decontamination.

INTRODUCTION

Owing to its successful and predictable outcome, there has been a profound inclination toward implant dentistry over the past few decades.[¹] Considering the crucial requirement of harmony between the various host- and implant-related factors, the basis of appraisal of the longevity of an implant has taken a gradual yet definitive shift of focus from earlier concepts such as implant surgical planning and osseointegration toward a long-term comprehensive maintenance of the implant and its associated peri-implant tissues.[²] This has led to the emergence of implant surface decontamination.

The topography of an implant surface has historically been in the spotlight as it can modulate the colonization of different microorganisms.[³] The constant salivary submergence during its entire period of function exposes the implant to a complex microbial biofilm. This biofilm may initially consist of commensals, under certain deleterious variables such as an unfavorable pH shift, systemic illnesses, or local inflammatory conditions, may undergo a dysbiotic shift to more Gram negative, anaerobic, fusiform pathogenic taxa. Various authors postulate that the peri-implant biofilm harbors a similar microbiota as that of the adjacent teeth, indicating that the residual teeth serve as the reservoir for bacterial accumulation in the biofilm surrounding the implants. Such pathobionts, combined with the host immunity response, can induce enzymatic tissue destruction and challenge the maintenance of the integrity of the attachment and functioning of the osseointegrated implant.[⁴⁵] One such putative bacterium is Aggregatibacter actinomycetemcomitans, present in high numbers at the intracoronal compartment and peri-implant sulcus of healthy implant surfaces.[⁶] This bacterium not only has a strong association with intraoral lesions like localized aggressive periodontitis but also, in rare cases, can show systemic seeding and cause brain abscess, endocarditis, etc.[⁷⁸] However, it is essential to keep this bacterial load within a critical threshold for an effective implant decontamination regimen. The literature documents various nonsurgical and surgical methods, among which, in recent times, laser therapy and chemical disinfection strategies are the most sought-after ones.[⁹¹⁰] However, there is insufficiency of literature to consider either as the preliminary conservative mainstay. Thus, the purpose of the current study was to compare the antibacterial activity of red diode laser therapy of wavelength 808 nm, with that of 0.2% chlorhexidine (CHX) treatment for the reduction of the bacterial load of A. actinomycetemcomitans, as detected from surfaces of implant healing abutments.

MATERIALS AND METHODS

The current ex vivo, observational, case–control study was carried out in the Department of Prosthodontics and Crown and Bridge, in collaboration with the Department of Periodontology and the Department of Microbiology, and was approved by the Institutional Ethical Committee (Research Protocol Number 38/2019). The selection criteria were established after purposive sampling in a way that 11 systemically healthy partially edentulous patients, having undergone prior implant placement surgery for at least two intraoral sites [Figure 1a], and now reporting to the outpatient department for the prosthetic phase, were included [Figure 1b]. Pregnant and lactating mothers; subjects under any anti-inflammatory drugs, antibiotics, and/or analgesics; subjects presenting with any other lesion not pertaining to the scope of the study; and subjects undergoing periodontal therapy at the time of recruitment were excluded.

Figure 1: Clinical sample collection from oral cavity of study subject. (a) Intraoral left lateral view of subject post second-stage surgery of implant placement at two intraoral sites. (b) Removal of the two healing abutments with the implant hex driver. (c) Collection of the two healing abutments into sterile microcentrifuge tubes prior to simple randomization for allotment to test and control groups

There were three phases to the current study, explained as follows:

Phase X: Patient recruitment phase

Patients undergoing the second-stage implant surgery for at least two sites intraorally were recruited for the study after obtaining written informed consent. Healing abutments were placed at the two sites, as per the standard treatment care decided by the clinician.

Phase A: Pretreatment sample collection

Collection of clinical samples

After 14 days of placement, the two healing abutments were removed, put into two microcentrifuge tubes, and allocated to the test/control group via simple randomization to avoid the risk of selection bias [Figure 1a-c]. After appropriate coding of both the groups, microbiological analysis was performed for the pretreatment bacterial load of A. actinomycetemcomitans from the clinical samples, without having undergone any kind of treatment. The steps for doing so have been mentioned as follows.

Preparation of clinical sample solution

Two hundred microliters of 0.9% normal saline solution was pipetted to each of the two microcentrifuge tubes and vortexed to mechanically disperse the biofilm adhered to the samples into the entire volume of the saline solution. Then, the entire volume from each microcentrifuge tube was divided into two further parts. Thus, each subject yielded four microcentrifuge tubes in the pretreatment phase, each containing 100 μL of the initial sample solutions having the biofilm collected from the subject's intraoral sites:

1. Test sample (laser, L)
1. 100 μL aliquot, for quantification of total target bacteria present in that sample, L_Dx^Ua
2. 100 μL aliquot, for quantification of viable target bacteria present in that sample, L_D√^Ua
2. Control sample (CHX)
1. 100 μL aliquot, for quantification of total target bacteria present in that sample, CHX_Dx^Ua
2. 100 μL aliquot, for quantification of viable target bacteria present in that sample, CHX_D√^Ua

Application of viability dye, propidium monoazide, PMAxx

PMAxx dye concentrate 20 mM, in H₂O (Biotium, Fremont, California, United States of America), was diluted to a stock solution of 5 mM in H₂O.

The sample solutions, coded CHX_D√^Ua and L_D√^Ua, were pipetted with 1 μL of the 25 μM dye solution. They were then covered with an opaque foil to prevent exposure to ambient light, thoroughly hand-mixed, incubated in dark for 10 min, and exposed to blue light-emitting diode (LED) light of λ = 466 nm (spectral property of dye, λ_abs= 464 nm) for 15 min.

The dye was expected to cross-link with and cause permanent selective alteration of only the dead target bacterial deoxyribonucleic acid (DNA), which would aid in conducting quantitative real-time viability polymerase chain reaction (qRT-vPCR) for the pretreatment sample solutions for just the live cells.

Genomic deoxyribonucleic acid extraction and quantification by real-time polymerase chain reaction

HumqPCR real-time A. actinomycetemcomitans test kit (Lote RTq-H710-100D111220, BioIngentech Corporation, Concepcion, Chile) was procured for extraction of genomic DNA and carrying out qRT-PCR in Rotor-Gene Q PCR instrument.

The entirety of the four fractions prepared was used individually for bacterial DNA extraction before carrying out qRT-vPCR.

All the steps carried out were in conformity with the manufacturer's instructions given in the PCR test kit, and thus, the viable and total bacterial DNAs present in the pretreatment samples were quantified.

Phase B: Posttreatment sample collection

After removing the two healing abutments in the pretreatment phase of the study methodology, two new healing abutments were placed back in the oral cavity. Post 14 days, those two healing abutments were again removed, placed in two sterile microcentrifuge tubes, and aptly coded and steps were commenced for the red diode laser treatment (test site) and treatment with 0.2% CHX (control site).

Laser versus chlorhexidine treatment

Quantification parameters for carrying out laser and CHX treatments were as per Tantivitayakul et al., having had a similar study design.[¹¹]

Red diode laser (λ = 808 nm) was irradiated on each laser group sample for episodes of 30 s each, to reach a power output of 2.5 W and energy density of 703.125 J/cm² with a gap of 30 s in between each episode [Figure 2a and b].

Figure 2: Procedure for carrying out laser treatment on test sample. (a) Protocol of laser treatment followed with initial energy output of 0 J/cm² reaching a maximum of 703.125 J/cm² by the end of Phase B for samples from test site undergoing laser treatment. (b) Healing abutment from test site kept stable; dappen dish manually rotated slowly for uniform exposure of laser light to all the surfaces of the healing abutment

Each control group sample was dipped in 1 ml of 0.2% CHX digluconate solution for 30 s before swishing it gently in 0.9% normal saline solution for removal of traces of CHX [Figure 3a and b].

Figure 3: Procedure for carrying out chlorhexidine treatment on control sample. (a) Sample from control site submerged in 1 mL of 0.2% chlorhexidine digluconate. (b) After 30 seconds, sample held gently with a sterile bracket holder and washed with water lightly to remove traces of CHX. CHX: Chlorhexidine

This was followed by microbiological analysis for the posttreatment load of target bacteria from the samples.

Preparation of clinical sample solutions

Four sample solutions were prepared from the two posttreatment samples, in steps similar to the pretreatment sample solutions:

1. Test sample (laser, L)
1. 100 μL aliquot, for quantification of total target bacteria present in that sample, L_Dx^Tb
2. 100 μL aliquot, for quantification of viable target bacteria present in that sample, L_D√^Tb
2. Control sample (CHX)
1. 100 μL aliquot, for quantification of total target bacteria present in that sample, CHX_Dx^Tb
2. 100 μL aliquot, for quantification of viable target bacteria present in that sample, CHX_D√^Tb.

Thus, each patient yielded a total of eight sample solutions, four from the pretreatment phase and four from the posttreatment phase [Figure 4].

Figure 4: The final sample solutions from each patient. Phase A: L_D√ ^Ua, L_Dx ^Ua, CHX_D√ ^Ua, CHX_Dx ^Ua. Phase B: L_D√ ^Tb, L_Dx ^Tb, CHX_D√ ^Tb, CHX_Dx ^Tb. CHX: Chlorhexidine

Application of viability dye, PMAxx

One microliter of 25 μM dye solution was added to the solutions, coded CHX_D√^Ua and L_D√^Ua, followed by dark incubation and blue LED light exposure in steps similar to the pretreatment sample solutions. This would help in carrying out qRT-vPCR for the treated sample solutions for just the live cells [Figure 5].

Figure 5: Exposure of sample solutions to blue LED light after application of viability dye, PMAxx. LED: Light-emitting diode

Genomic deoxyribonucleic acid extraction and quantification by real-time polymerase chain reaction

Extraction and quantification of the viable and total bacterial DNAs present in the treated samples was done similar to the steps followed for the pretreatment samples.

RESULTS

After verification of the validity of the experimental setup, qRT-vPCR was carried out for all the coded clinical samples. Raw data were acquired by the Rotor-Gene Q Analysis Software for quantification of the template bacterial DNA present in all the samples which was then analyzed using IBM Corp. Released 2013. IBM SPSS Statistics for Windows, Version 22.0. (Armonk, NY: IBM Corp.).

This mean total reduction of 322296.13 copies/μL was found to be highly statistically significant (F = 42.812, P = 0.001) [Table 1]

Table 1: Mean viable bacterial counts before and after treatment of laser and chlorhexidine groups with descriptive statistics and results of repeated measures analysis of variance

Before treatment, the mean total viable bacterial count (VBC) was 325967.93 copies/μL which had been reduced to 3671.80 copies/μL, irrespective of the groups. This mean total reduction of 322296.13 copies/μL was found to be highly statistically significant (F = 42.812, P = 0.001).

However, when analyzed across the laser and CHX groups, the reduction in the mean total VBC in the laser group was 325361.28 copies/μL and in the CHX group was 319230.97 copies/μL, which were statistically insignificant (F = 0.004; P = 0.951) [Graph 1].

Figure

The mean total reduction in the BVR of 96.23 was found to be highly statistically significant (F = 22580.893, P = 0.001) [Table 2]

Table 2: Mean bacterial viability ratio before and after treatment of laser and chlorhexidine groups with descriptive statistics and results of repeated measures analysis of variance

Before treatment, the mean total bacterial viability ratio (BVR) was 97.38 which had been reduced to 1.15, irrespective of the groups. The mean total reduction in the BVR of 96.23 was found to be highly statistically significant (F = 22580.893, P = 0.001).

However, when analyzed across the laser and CHX groups, the mean reduction of the BVR in the laser group was 96.14, and in the CHX group was 96.3, which were statistically insignificant (F = 0.015; P = 0.902) [Graph 2].

Figure

Independent sample t test revealed that the ratio differences between the two groups were statistically insignificant (t = 0.124; P = 0.902) [Graph 3] [Table 3]

Table 3: Mean bacterial percentage reduction and bacterial viability ratio of laser and chlorhexidine groups and the results of independent sample t-test

Figure

The mean bacterial percentage difference between the before treatment and after treatment groups was 96.14 for the laser group, whereas it was 96.30 for the CHX group. Independent sample t-test revealed that the ratio differences between the two groups were statistically insignificant (t = 0.124; P = 0.902) [Graph 3].

The ratio between the viable and the total counts after treatment for the laser group was 1.32, whereas it was 0.99 for the CHX group. The independent sample t-test revealed a significant mean difference in the BVR values of after treatment values with the t = 3.458, significance level being 0.002 [Graph 4].

Figure

DISCUSSION

Pokrowiecki et al. state that the lack of periodontal ligament, the origination from postoperative scar tissue, reduced vascularity, and an increased sulcus depth make peri-implant tissues more susceptible to bacterial penetration as compared to periodontal tissues.[⁴¹²] Hence, it may be equated that these fundamental differences and the lack of complete resolution of any inflammatory condition, warrants a sustained and thorough implant maintenance regimen.

The constant exposure to the oral cavity brings an implant in contact with a bacterial biofilm composed of a community of commensals that generally help the host in maintaining homeostasis.[⁵] Certain concomitant factors pertaining to the patient systemic history (uncontrolled diabetes mellitus, head and neck radiotherapy), local parameters (dental plaque, calculus, and overhanging restorations), any drug induced pH alteration (antacids and antihypertensives), deleterious habit history like smoking or alcohol consumption, etc.; may cause a dysbacteriosis to a more pathological, Gram negative, anaerobic taxa, leading to the breakdown of the surrounding peri- implant tissues.[¹³]

Various authors have affirmed the persistent presence of A. actinomycetemcomitans in and around healthy and diseased peri-implant sites, which, unlike the red and orange complex microbes, may not be a polymicrobial conglomerate, but can still pose a latent bacterial challenge to the health of implant and its associated tissues.[¹⁴¹⁵¹⁶] It resists and dodges host defense and interferes with tissue repair mechanisms. The combined infectious pattern of multiple serotypes may exhibit a difference in the individual antibiotic susceptibility rates.[¹⁷¹⁸] Moreover, Di Murro et al. correlated compromised systemic health and the prevalence of peri-implantitis and noted pronounced higher bacterial loads of A. actinomycetemcomitans in patients with systemic comorbidities as compared to healthy patients.[¹⁹] Thus, regular surface decontamination from this spp. is imperative toward the overall success of the maintenance of the implant.

Conventional nonsurgical techniques for doing so include mechanical methods such as implantoplasty, air-powder abrasives, chemical disinfectants (bis-biguanides, essential oils etc.), antibiotics and lasers.

Literature states that implantoplasty has adverse effects such as embedment of debris within surrounding tissues and postoperative marginal recession. Air-powder abrasives, despite being an excellent noninvasive technique, have the possibility of persistent attachment of the powder particles to the implant surface and subcutaneous emphysema. Metallic-tipped scalers can cause extensive surface texture alteration that can serve as the niche for further bacterial accumulation. On the other hand, nonmetallic scalers have a questionable surface decontamination and re-osseointegration.[²⁰²¹²²]

CHX is considered the gold standard antibacterial chemical disinfectant and has been reported to cause a significant decrease of aerobic and anaerobic bacteria.[²³] This was substantiated by Kadkhoda et al. after having checked the antibacterial effect of 0.2% CHX against A. actinomycetemcomitans, isolated from peri-implantitis sites.[¹⁰] However, it is said to have certain rising complications both at a macro-local level, such as staining of restorations and tissues, mucosal ulcerations, taste alteration, an elevation of supragingival calculus, and parotid swellings, and at the microcellular level, such as detrimental effects on cellular proliferation, growth, enzymatic activity, polymorphonuclear leukocyte disruption, and apoptotic and necrotic cell deaths.[^23242526]

Lasers, owing to their thermal energy based degradation have a pronounced fungicidal and bactericidal effect. They also promote rapid wound healing and repair, have a low possibility of developing bacterial resistance, can showcase selective targeting of microflora and cause minimal tissue damage without any surface texture alteration.[^927282930] This was in concordance with Aimetti et al., who concluded that lasers brought about a decrease in clinical parameters such as bleeding on probing, plaque index, and probing pocket depth that was comparable to the conventional mechanical debridement alone.[³¹]

With respect to the results derived from the current study, both kinds of treatments, laser and CHX, brought about a major bacterial reduction of viable A. actinomycetemcomitans [Tables 1 and 2]. This mean total reduction of 322296.13 copies/μL was found to be highly statistically significant (F = 42.812, P = 0.001). However, when analyzed across the laser and CHX groups, the reduction in the mean total VBC in the laser group was 325361.28 copies/μL and in the CHX group was 319230.97 copies/μL, which were statistically insignificant (F = 0.004; P = 0.951) [Graph 1].

Laser treatment resulted in the postoperative bacterial viability of 1.2%–1.6% and CHX resulted in the postoperative bacterial viability of 0.6%–1.4%. Conversely, laser treatment caused a mean bacterial reduction of 96.1%, while CHX caused a mean bacterial reduction of 96.3% of A. actinomycetemcomitans [Table 3]. Independent sample t-test revealed that the ratio differences between the two groups were statistically insignificant (t = 0.124; P = 0.902) [Graph 3].

The ratio between the viable and the total counts after treatment for the laser group was 1.32, whereas it was 0.99 for the CHX group. Independent sample t-test revealed a significant mean difference in the BVR values of after treatment values (t = 3.458, P = 0.002) [Graph 4].

The above three statistical results implied that even though CHX was more potent in reducing the total VBC, laser therapy was also equally effective.

This study validated the results of an in vitro study conducted by Tantivitayakul et al. having used red diode laser and 0.2% CHX digluconate solution against A. actinomycetemcomitans cultured on natural teeth in vitro. Red diode laser had shown 93.425% of bacterial reduction, and CHX, 99.994%.[¹¹]

The clinical implications and recommendations from this study include the mandatory need for regular systemic and oral health checkups for a constant vigil over clinical and radiographic parameters of the implant and the prosthesis. For in-office debridement of the implant site, both the lasers and CHX can be provided to the patient. Keeping in mind the clinical scenario and the financial aspect involved, the final call can be made.

Misch states that the first decontamination appointment should be succeeded by a follow-up visit after about 1 month. This can be followed by regular 3-month recalls. In case of a good intraoral health and condition, a 3–6-month recall system can be established. Patient education and motivation about a stringent home care regimen is also equally essential. This can include a combination of tooth brushing aids, auxiliary hygiene devices, and antibacterial mouthwashes.[³²]

The strengths of the current study included the conservative target approach for implant maintenance. All the steps were in compliance with good clinical practices. Culture-independent techniques increased the speed and sensitivity of microbiological analysis and the viability dye gave the accurate quantification of the viable bacteria on the samples before and after laser and CHX treatments.

However, further research can be done with larger sample sizes, in vivo study designs, other causative microorganisms of peri-implantitis, and further in-depth research regarding the effect of lasers on different surface designs and characteristics of an implant fixture and prosthesis.

CONCLUSION

1. Contemporary times call for the need of early detection and conservative management of peri-implant diseases. Out of all the documented conservative treatment protocols for implant detoxification, laser and CHX were two treatments that have been under the radar for recognition as the relatively superior one among the two
2. The results of the current study showed that both diode laser treatment and CHX disinfection treatment gave a similarly high degree of antibacterial efficacy against A. actinomycetemcomitans, present on implant healing abutments. However, when compared in between the two, laser treatment gave a postoperative BVR of A. actinomycetemcomitans that was marginally higher as compared to the one observed after CHX treatment
3. Consequently, laser and CHX treatments gave similar high percentages of overall bacterial reduction of A. actinomycetemcomitans present on implant healing abutments. However, on drawing a comparative evaluation, it was seen that CHX brought about a bacterial reduction that was marginally higher as compared to the one seen after laser treatment
4. Laser therapy is garnering increasing popularity among all fields on dentistry, owing to minimal bleeding, high efficiency, and faster healing. Hence, it can also be considered to be an efficient treatment modality for implant surface decontamination and implant maintenance that is at par with other conventional nonsurgical mechanical and chemical options for carrying out similar procedures.

Financial support and sponsorship

This study was financially supported by the Indian Council of Medical Research (ICMR) Annual Thesis Financial Assistance for MDS students (Ref No. 3/2/June-2020/PG-Thesis-HRD (48D).

Conflicts of interest

There are no conflicts of interest.

Acknowledgment

We sincerely acknowledge and express our heartfelt gratitude toward the Indian Council of Medical Research (ICMR) for having provided us with the financial aid for the current study in the form of the annual ICMR PG Thesis Research Grant for MDS students, June 2020 (Ref No. 3/2/June-2020/PG-Thesis-HRD (48D). The amount has been duly used for the smooth conduct of our study.