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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 10  |  Issue : 2  |  Page : 47-57

A randomized, clinical split-mouth study comparing conventional techniques to lasers for second-stage implant uncovering


1 Private Practice, Houston, TX, USA
2 Department of Periodontics and Dental Hygiene, The University of Texas School of Dentistry, Houston, TX, USA

Date of Web Publication29-Dec-2016

Correspondence Address:
Andrew Rossi
2600 South, Gessner Road, Suite 304, Houston, TX 77063
USA
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2321-1385.196990

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  Abstract 

Purpose: This randomized, clinical split-mouth study compared participants' pain perception, quantified the inflammatory cytokines interleukin (IL)-1 β and IL-6, and assessed changes in the quantity of five periodontal pathogens, after implant uncovering. Materials and Methods: Twelve persons who received bilateral implants participated. After healing, implants were randomly uncovered using either a scalpel (control) or a 940 nm diode laser (experimental). The participants' pain perception was assessed by questionnaires and visual analog scale. Crevicular fluid was collected around the implants at day 1 and 7, and IL-1 β and IL-6 levels were measured by ELISA. Bacterial samples were collected around the healing abutments at day 1 and 7. Results: A statistically significant difference in change of pain score over time was found. The laser sites showed an increase in pain that continued until day 3, while the scalpel sites showed an increase in pain that declined by day 2. A global health questionnaire showed that patients overwhelmingly preferred the laser to the scalpel. IL-1 β levels were significantly lower in the laser group as compared to the control group at day 1 (387.2 pg/mL vs. 456.2 pg/mL, P = 0.02). No statistically significant differences in IL-6 or bacterial quantities were found between test and control sites. Conclusions: Even though a slightly greater amount of pain was perceived over the first 2 postoperative days, patients significantly preferred the laser to the scalpel. Less inflammatory cytokine production of IL-1 β was found after the 1 st day of healing after laser surgery. Bacterial sampling was similar between test and control groups.

Keywords: Diode laser, implant uncovering, interleukin-1 β, interleukin-6, pain perception, visual analog scale


How to cite this article:
Rossi A, Weltman R, Tribble G, Kiat-Amnuay SM. A randomized, clinical split-mouth study comparing conventional techniques to lasers for second-stage implant uncovering. J Dent Lasers 2016;10:47-57

How to cite this URL:
Rossi A, Weltman R, Tribble G, Kiat-Amnuay SM. A randomized, clinical split-mouth study comparing conventional techniques to lasers for second-stage implant uncovering. J Dent Lasers [serial online] 2016 [cited 2017 Apr 25];10:47-57. Available from: http://www.jdentlasers.org/text.asp?2016/10/2/47/196990


  Introduction Top


The original Branemark protocol for the placement of implants advocated for a two-stage approach. In this approach, implants would be placed and remain submerged subgingivally for 3-6 months, at which point the implants would be surgically uncovered and a healing abutment placed, in what was termed a second-stage implant uncovering. [1] Since the publication of this protocol, lasers have gained widespread acceptance and have found many uses in the medical and dental fields. In dentistry, lasers have been shown to be effective at cutting soft tissue with added benefits of reducing postoperative inflammation and having bactericidal activity. [2],[3] One of the more widely used lasers in dental offices is the diode laser due to its relative low cost and ease of use.

Diode lasers are commercially available in two different types based on power; either high-intensity cutting diodes or low-intensity noncutting lasers. These types are both available at different wavelengths including 810 nm, 940 nm, 980 nm, and 1064 nm. These wavelengths place diode laser energy in the near-infrared electromagnetic spectrum that is absorbed by chromophores, including heme, and melanin. The high-intensity diode laser has been utilized in dentistry for sulcular disinfection and soft-tissue surgeries, including frenectomies, excision of gingival hyperplasias, and soft-tissue tumors, vestibuloplasties, and biopsies. [4],[5] A study by Goharkhay et al. demonstrated the positive effects of the diode laser on soft tissue, including good cutting ability, enhanced hemostasis, and minimal soft-tissue damage. [6]

Manufacturers of diode lasers claim that the use of lasers decreases postoperative inflammation and pain and may reduce the amount of anesthesia required during a procedure. Animal studies have indicated that the diode laser may be useful in decreasing pain sensation through alterations of specific molecular pathways, and some human studies subjectively indicate that diode lasers significantly reduce pain in soft-tissue procedures such as frenectomies and treatment of aphthous ulcers. [5],[7],[8] Surrogate markers of pain and inflammation, including levels of cytokines, chemokines, metalloproteinases, and immunoglobulins, have been used to evaluate tissue level changes in inflammation. [9] Inflammatory cytokines interleukin (IL)-1 β and IL-6 have been implicated in the pain response. [5] Wolf et al. investigated the role of IL-1 during induction and maintenance of postoperative incisional pain in a murine model. Incisions were made in the hind paws of three mouse strains impaired in IL-1 signaling and their wild-type controls. Postoperative mechanosensitivity was assessed before and up to 4 days, following plantar incisions. The wild-type mice developed significant pain responses in the incised, compared with the intact, hind paw starting 3 h after the incision and lasting up to 48 h postoperatively. The strains of mice with impaired IL-1 signaling did not display increased mechanical pain sensitivity in either the injured or noninjured hind paw. [10] Utilizing animal models, IL-6 has been upregulated in peripheral nerves, dorsal root ganglia, and spinal cord of test animals. [11] Injection of IL-6 into one hind paw of male Wistar rats evoked hyperalgesia in both hind paws, with the intensity of the pain reaching a plateau in 2-3 h and maintained for up to 6 h after local injection. [12],[13]

Diode lasers have also been shown to be very efficacious in ablating black-pigmented bacteria due to the inherent chromophores of these bacteria. [14] In proof of concept studies, diode lasers have eliminated bacteria grown on titanium discs in as little as 10 s. [15] In vivo studies utilizing diode lasers offer ample evidence that this type of laser applied within the periodontal sulcus is efficacious in reducing the numbers of bacteria cultured from plaque samples around teeth as well as from serum samples when compared to sampling from nontreated controls. [16],[17] Of great interest, there is the efficacy of the diode laser energy in the disinfection of implant surfaces in vivo. One group of investigators fabricated acrylic stents with embedded titanium tubes which were worn intraorally by participants for 10 days. Diode laser energy was applied to half of tubes with subsequent bacterial quantification. Compared to nonirradiated control sites, mean bacterial colonization on intraoral rough titanium surfaces was reduced by more than 98%. [2] Few studies have investigated the use of diode laser energy in the sustained reduction of bacteria populating implant restoration surfaces in vivo.

When a sufficient band of keratinized gingiva is found around implants, a gingival excisional procedure is performed to expose the implant platform to the oral environment. Removal of the surface gingival tissues can be accomplished either with the use of scalpel blades or with laser ablation of the tissues. Patient's comfort and acceptance are paramount to the dental practitioner. Knowledge of whether laser ablation will elicit less postoperative pain and inflammation may provide evidence for the routine use of diode laser in the dental office for second-stage implant procedures. The bactericidal effects of the diode energy on pigmented microbes may provide a bacteria-free environment around the healing implant surgical site which may also enhance healing.

The aim of this randomized clinical split-mouth study is to evaluate participants' perception of pain, to quantify the amount of the inflammatory cytokines IL-1 β and IL-6, and to assess changes in quantity of five common periodontal pathogens, (Treponema, Campylobacter, Prevotella, Porphyromonas gingivalis, and Streptococcus milleri), after second-stage implant uncovering utilizing either a scalpel or a diode laser.


  Materials and Methods Top


Data collection

Twelve participants who have received submerged bilateral implants with an overlying abundant zone of attached keratinized gingiva were invited to participate in this study. Second-stage implant uncovering surgery was scheduled at least 3 months after implant placement. The two implant sites were randomly assigned to one of two treatments: (1) Gingival excision surgery, exposing the implant platform utilizing a scalpel (control group) or (2) a procedure utilizing laser irradiation with a diode laser (iLase™, Biolase Technology ® , Irvine, California, USA) (λ = 940 ± 15 nm. power 2 ± 1 W, tip: 320 μm). The laser power utilized for each patient was adjusted to provide efficient cutting action of the absorbed laser energy. Randomization of the experimental and control sites were performed through a computer-generated randomization program, random.org/coins/. Participants were instructed to refrain from taking any nonsteroidal anti-inflammatory drug, rinsing with chlorhexidine, or taking any medication that might have interfered with cytokine or bacterial results. Participants who were wearing complete or partial dentures were instructed to refrain from wearing their prosthesis for 1 week. Participant's satisfaction and pain perception were assessed by written questionnaires and a visual analog scale (VAS) instrument, respectively. Bacterial samples were taken from around the healing abutments at day 1 and 7 postprocedure using E-swabs (Copan, Murrieta, CA, USA). The healing abutments were isolated with cotton rolls, and E-swabs were used around the occlusal and lateral surfaces of the healing abutments. The E-swabs were then placed in a test tube with 10% formaldehyde and placed in the −70°C freezer for preservation. Crevicular fluid was collected with paper points at day 1 and 7 after implant uncovering for cytokine analysis. This was accomplished by isolating the healing abutments and placing the paper points for 20 s circumferentially between the healing abutment and gingiva on the buccal surface of the healing collar. The paper points were then placed in a test tube with 10% formaldehyde and placed in a −70°C freezer for preservation.

Inclusion criteria

The inclusion criteria were individuals who aged 18 years or older, had received bilateral submerged implants in the same dental arch, had an abundant zone of keratinized attached gingiva overlying the implants, and whose implants were osseointegrated and ready to have the second-stage uncovering procedure.

Exclusion criteria

  • Age: Younger than 18 years of age
  • Systemic diseases which would interfere with healing such as severe diabetes
  • Smoking more than ten cigarettes a day
  • Drug addiction or mentally incapacitated
  • Current systemic steroid treatment
  • Legally blind
  • Not cognizant enough to answer the questionnaires.
Subject evaluation questionnaires

Pain and treatment preference

Participants were instructed to quantify their postoperative pain level for each surgical site in the morning of 1 st through 7 th postoperative days by means of a VAS before taking any pain medication (if needed). The VAS consists of a 10 cm line anchored at one end by the label "no pain" and at the other end "worst imaginable pain." The participant marked on the line the spot for the pain intensity felt. Participants were given seven forms with two scales on each sheet (one scale for each the experimental and control sites) for the respective 7 days after surgery. All forms were returned to the primary investigator at the 1 week postoperative appointment. In addition, at the 1 week postoperative appointment, each participant was asked to indicate which implant site was easier to clean and to indicate which technique they would prefer for similar treatment in the future (global evaluation question).

Practitioner evaluation questionnaires

Gingival inflammation

Gingival inflammation was assessed by the same evaluator by means of a modified gingival index as described by Löe. [18] The practitioner evaluated qualitative changes in the gingival soft tissue in the following manner:

  • 0 = Normal gingiva
  • 1 = Mild inflammation - Slight change in color, slight edema; no bleeding on probing
  • 2 = Moderate inflammation - Redness, edema, and glazing; bleeding on probing
  • 3 = Severe inflammation - Marked redness and edema, ulceration, tendency to spontaneously bleed.
Difficulty in performance of technique

The surgeon who performed all the surgeries was instructed to quantify his level of difficulty in performance with each technique by means of a VAS, as described before, which is anchored at one end by the label "most comfortable" and at the other end "most uncomfortable." The surgeon marked on the line the spot for the comfort level based on the perceived difficulty for each procedure. Following completion of treatment, the surgeon was asked to indicate which procedure was more complicated, uncomfortable, and which treatment he would prefer for similar treatment in the future.

Time of procedure

To evaluate the time spent chairside performing each technique, the start time and end time were recorded.

Interleukin-1 β cytokine quantification

To extract the IL proteins from paper points, samples were thawed and paper points were transferred to a ×1 phosphate-buffered saline (PBS). A serial Bradford dilution was performed on eight randomly chosen samples. All diluted samples were read with the microarray spectrophotometer, set to measure a Bradford Assay (Eppendorf BioPhotometer, Hamburg, Germany). A 1:10 dilution was chosen as the ideal dilution to obtain results within the standard curve of the chosen tested cytokine. An eBioscience human IL-1 beta ELISA Ready-Set-Go! ® kit (eBioscience, San Diego, California, USA) was chosen to obtain a standard dilution curve as well as to test for the presence of cytokines in the samples. The manufacture's recommended protocol was used for all samples. After following the recommended protocol, the samples were read by a spectrophotometer at 450 nm (Tecan infinite F200PRO, Mδnnedorf, Switzerland). Standard curves were generated in triplicate and then averaged. The calculated average standard curve was graphed using Microsoft Excel 2007. From the standard curve, a slope intercept form equation was calculated by Microsoft Excel 2007 to aid in the quantification of each individual sample. The quantification of the amount of IL-1 β per sample was accomplished by inserting the value of fluorescence (quantification units), obtained from the spectrophotometer, into the equation obtained from the standard curve. The calculated values were then multiplied by 10 to account for the 1:10 dilution. The entire process was performed in triplicate and the resultant values were submitted for statistical analysis.

Interleukin-6 cytokine quantification

To extract the IL-6 proteins from paper points, samples were thawed and paper points were transferred to a ×1 PBS. A serial Bradford dilution was performed on eight randomly chosen samples. All diluted samples were read with the microarray spectrophotometer, set to measure a Bradford Assay (Eppendorf BioPhotometer, Hamburg, Germany). A 1:2 dilution was chosen as the ideal dilution to obtain results within the standard curve of the chosen tested cytokine. An eBioscience Human IL-6 ELISA Ready-Set-Go! ® (eBioscience, San Diego, California, USA) was chosen to obtain a standard dilution curve as well as to test for the presence of cytokines in the samples. The manufacture's recommended protocol was used for all samples. After following the recommended protocol, the samples were read by a spectrophotometer (Tecan infinite F200PRO, Männedorf, Switzerland). Two standard curves were created per test run and the cytokine values (pg/mL) were averaged to create 1 standard curve per run. The calculated standard curve was graphed using Microsoft Excel 2007. From the standard curve, a slope intercept form equation was calculated by Microsoft Excel 2007 to aid in the quantification of each individual sample. The quantification of the amount of IL-6 per sample was accomplished by inserting the value of fluorescence (quantification units), obtained from the spectrophotometer, into the equation obtained from the standard curve. The calculated values were then multiplied by 2 to account for the 1:2 dilution. The entire process was triplicated and values were submitted for statistical analysis.

Bacterial quantification

The bacteria quantified in this study were Treponema sp., Campylobacter sp., Prevotella sp., P. gingivalis, and S. milleri. It is generally accepted that periodontal disease and peri-implantitis are caused by a specific set of bacteria. The types of bacteria tend to be secondary colonizers of sites and are characteristically Gram-negative anaerobes. These bacteria were described by Quirynen et al. and Socransky as being part of the red and orange complexes of bacteria: P. gingivalis, Tannerella forsythia, Treponema denticola, Aggregatibacter actinomycetemcomitans and Prevotella intermedia, Campylobacter sp., respectively. [19] S. milleri is an unofficial name of a subset of potentially pathogenic viridians Streptococcus species. S. milleri includes S. pyogenes, Streptococcus agalactiae, Streptococcus anginosus, Streptococcus intermedius, and Streptococcus constellatus. Although commensally found in the oral cavity and the gastrointestinal tract, these bacteria have been implicated in gingivitis and have been found in periodontal and periapical abscesses. [20],[21] Recent advances in culturing techniques and 16S rDNA gene sequencing have allowed researchers to more accurately reflect the types of bacteria that are cultured from sampled sites.

To extract bacterial DNA from collected samples, the E-swabs were thawed and a placed into collection tubes for DNA isolation. The MO-BIO PowerSoil ® DNA Isolation Kit (MO-BIO, Carlsbad, California, USA) manufacturer protocol was followed for DNA isolation. The resulting DNA was purified using the Zymo DNA Clean and Concentrator-5 kit (Zymo Research, Irvine, California, USA), to remove extraneous proteins and RNA. The concentrations of the resulting DNA for all collected samples were then read using an Eppendorf Bio-Photometer (Eppendorf, Hamburg, Germany). All samples were then diluted to the lowest observed concentration (ng/μL) in preparation for real-time polymerase chain reaction (PCR) analysis.

Real-time quantitative PCR (RQ-PCR) was performed for each sample in a reaction volume of 25 μl containing 12.5 μL ×1 SYBR Green PCR Master Mix (Bio-Rad IQ SYBR Green Mix) (Life Sciences Research, Hercules, California, USA), 1 μL forward primer, 1 μL reverse primer, 1 μL of sample, and 9.5 μL water [Table 1] for primer specifications]. Reactions took place in a Bio-Rad C1000 Thermal Cycler (Life Sciences Research, Hercules, California, USA). The reaction conditions were 95°C for 5 min, followed by forty cycles of 95°C for 15 s and 60°C for 1 min. All samples were duplicated and the resulting Cq values (quantification units) were submitted for statistical analysis.
Table 1: Primers used in real-time quantitative polymerase chain reaction


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To calculate the individual number of specific bacteria in each sample, a standard curve composed of several known numbers of each individual bacterial species was subjected to RQ-PCR. The Universal Primers 357F and 907R were shown to have equal efficacy with all tested bacterial species. The Cq (quantification units) values were obtained from the known concentrations of Prevotella, P. gingivalis, S. milleri, Tannerella, and Treponema and graphed using Microsoft Excel 2007. A standard curve and logarithmic scale equation were calculated for each type of bacteria so that a bacterial concentration of each sample could be obtained. The specific number of bacteria found in each sample was obtained by determining the genome sizes of each bacteria studied, found at http://www.brop.org/. The individual bacterial concentrations obtained from plugging the sample Cq values into the standard curve equation were then placed into a conversion calculator, http://molbiol.edu.ru/eng/scripts/01_07.html, along with the genome sizes, to calculate a final number of individual bacteria per species in each sample. The resulting numbers of bacteria were then submitted for statistical analysis.

Statistical analyses

To compare the categorical outcome variable, pain response, between the test and control sites over time, a generalized estimating equation (GEE) analysis was performed. Participants' preference of treatment modality was analyzed through an unpaired 2-tailed Student's t-test. Practitioner's perceived discomfort and gingival inflammation noted at treatment sites after 7 days of healing were compared within and/or between treated sites with a nonparametric 2-tailed Wilcoxon-signed rank test. To evaluate differences in the time taken per procedure, an unpaired 2-tailed Student's t-test was utilized. Inflammatory cytokine data were triplicated, and differences in cytokine levels measured on day 1 and 7, within and between treated sites, were analyzed utilizing unpaired 2-tailed Student's t-tests. A P < 0.05 was selected as the level of statistical significance.

The quantity of bacteria found in each sample was calculated utilizing the software program CLC Genomics Workbench v6, using the toolbox function "feature clustering." Statistical analysis of differences in the quantities of bacteria found around implant healing abutments 1 and 7 days after uncovering within and between treatment sites was accomplished with Student's t-tests. In addition, participants were classified according to either the reason for tooth loss, i.e., due to periodontal disease or caries, or as fully edentulous. Bacterial data were combined for test and control sites and comparisons made between groups for differences in microbial quantities and profiles with ANOVA analysis.


  Results Top


Subject's pain evaluation and treatment preference

Participants were instructed to evaluate the amount of pain felt on the right side compared to the left side, immediately postoperatively and every day for 7 days, until the final postoperative appointment. Participants were instructed to mark on a VAS the amount of pain felt at each site from 0 to 10, with 0 being no pain and 10 being the worst imaginable pain, on both the right and left sides. [Table 2] shows the mean visual analog scores for all patients over the 7-day evaluation period, and [Table 3] presents the data analysis utilizing a GEE test. The "site" variable tests for a difference in pain scores between the two sites after combining the scores for all time points. While there appears to be a trend for more pain perceived at the laser sites, no statistically significant difference was found in mean perceived pain (ignoring the time effect) between test and control sites (P = 0.063). The "day" variable tests for a difference over time after combining the scores from the two sites. While the scores noted in [Table 2] indicate an initial a trend for the scores to initially rise over the 1 st day or 2 nd day and then decline over time, there was no statistically significant effect of differences in pain perception over time (ignoring the site effect) (P = 0.086). The variable "site × day" is an interaction term which compared the test and control sites for their change in pain over time. The P < 0.001 indicates a statistically significant difference between the two sites in their change in pain score over time. The laser sites showed an increase in pain that continued until day 3, while the scalpel sites showed an increase in pain that declined by day 2.
Table 2: Mean subject perceived pain in visual analog scale (±standard deviation) scores over 7 days


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Table 3: Generalized estimating equation analysis of patient perceived pain over time


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After 7 days of healing, participants were asked through a global health questionnaire which procedure they preferred and which procedure, laser or scalpel, that they would request if the test was to be run again. Ten of the 12 participants (83%) indicated that they preferred the laser while two participants indicated that they preferred the scalpel blade. A statistically significant difference in subject preference for the laser, over the scalpel blade, was found [P = 0.0007, [Table 4].
Table 4: Participant and practitioner surveys: Comparisons of participant's treatment preference, gingival index, practitioner treatment difficulty, time length of treatment


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Gingival inflammation

For each of the test and control sites, the practitioner recorded the observed gingival inflammation 7 days after uncovering the implants utilizing a modified Loe and Silness scale as indicated above.

The experimental and control sites showed a range of inflammation from 0 to 2. The experimental laser sites displayed a mean inflammation score of 1.08, while the control sites scored a mean value of 0.92. Seven of the 12 participants exhibited the same amount of inflammation in the experimental site as in the control site. No statistically significant difference in gingival inflammation was found between experimental and control sites [P = 0.480, [Table 4].

Practitioner perceived difficulty in performance of techniques

The clinician who performed the second-stage procedures was instructed to mark on a VAS the difficulty experienced performing the procedure at each site, from 0 to 10, with 0 indicating no difficulty and 10 indicating the worst imaginable difficulty. In every participant, the clinician indicated that the experimental procedure (laser gingival excision) was more difficult to perform than excision of the gingival tissues with a scalpel (control procedure). The mean VAS score for the experimental sites was calculated as 2.25 visual analog units, while the score for the control sites was 0.25 visual analog units. The experimental procedure demonstrated a significantly greater amount of difficulty for the practitioner to perform when compared with the control procedure [P < 0.01, [Table 4].

Time taken for uncovering

The clinician recorded the length of time (in minutes) taken to complete the experimental and control techniques. Uncovering the experimental sites with a diode laser took significantly more time (mean = 6.5 min) than uncovering the control sites through scalpel excision (mean = 1.4 min) [P < 0.01, [Table 4].

Interleukin-1 β levels

Crevicular fluids absorbed on paper points from the sulci surrounding the healing abutments of the experimental and control sites were analyzed for the presence of the inflammatory cytokine IL-1 β at day 1 and day 7 posttherapy. ELISA testing was performed, results triplicated, and averaged for analysis. The control site samples at day 1 measured a mean IL-1 β level of 456 pg/ml, while the experimental sites samples at day 1 measured a mean IL-1 β level of 387 pg/ml. By day 7, the observed mean value of IL-1 β had decreased to 329 pg/ml for the control sites and 256 pg/ml for the experimental sites [Table 5].
Table 5: Mean interleukin-1â concentrations pg/ml (±standard deviation) measured at the control sites and laser sites on day 1 and day 7


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Statistical analysis between test and control sites found a significant difference in levels of IL-1 β only on day 1 with the scalpel sites producing more cytokine that the laser sites (P = 0.02). While levels of IL-1 β decreased over the healing period, the change in IL-1 β levels at test and control sites, from day 1 to day 7, did not reach statistical significance [Table 5].

Interleukin-6 levels

Crevicular fluids absorbed on paper points from the sulci surrounding the healing abutments of the experimental and control sites were analyzed for the presence of the inflammatory cytokine IL-6 at day 1 and day 7 posttherapy. ELISA testing was performed, results triplicated, and averaged for analysis. The control site samples at day 1 measured a mean IL-6 level of 25 pg/mL, while the experimental sites samples at day 1 measured a mean IL-6 level of 22 pg/ml. By day 7, the observed mean level of IL-6 had decreased to 19 pg/ml for the control sites and 15 pg/ml for the experimental sites [Table 6].
Table 6: Average interleukin-6 concentrations pg/ml (±standard deviation) measured at the control sites and laser sites on day 1 and day 7


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Statistical analysis between test and control sites did not find statistically significant differences in levels of IL-6 on day 1 or day 7. Levels of IL-6 decreased over the healing period; however, only the change, from day 1 to day 7, in IL-6 levels at the laser control sites reached statistical significance (P < 0.01) [Table 6].

Bacterial data

Standard curves of known concentrations of the tested bacteria including Prevotella, P. gingivalis, S. milleri, Tannerella, and Treponema were created. Samples of bacteria were collected circumferentially around the healing abutments, at day 1 and day 7. RQ-PCR was performed on all of the samples, testing for the presence and quantity of the five bacterial types in each sample, using the standard curves.

The quantities of each type of bacteria cultured from the swab samples in the experimental groups were compared to those in the control groups. This comparison was represented by heat mapping. A heat map is a graphical representation of the quantities of bacteria, in a color-coded format, with bright red bars indicating the greatest number of bacteria and blue indicating few or no bacteria [Figure 1]. Comparisons of the quantities of each tested bacteria found no statistically significant differences between the samples obtained from the experimental and control healing abutments (P > 0.05).
Figure 1: Heat map of the number of bacteria sampled at the test sites (L) versus the control sites (C) on day 1 and day 7

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Since no differences were found in the presence and amount of bacteria at the experimental and control sites, bacterial data for the experimental and control sites were combined and the participants were subdivided into three groups based on the way in which each individual lost his/her teeth (necessitating the placement of implants). Four participants (participants #2, 4, 5, and 11) lost their teeth due to periodontal disease. Two participants (participants #8 and 10) lost their teeth due to caries. Six participants (participants #1, 3, 6, 7, 9, and 12) presented as edentulous before implant placement. ANOVA analysis found no significant differences in the bacterial quantities between the three subcategories of tooth loss history for all measured species except Tannerella [P = 0.034, [Figure 2]. Tukey's honestly significant difference post hoc test showed the amounts of Tannerella found around healing abutments in the edentulous and periodontitis groups were similar.
Figure 2: Heat map of the number of bacteria sampled from test and control sites stratified according to tooth loss patterns: caries, periodontal disease, edentulism

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  Discussion Top


The current study examined the differences seen between uncovering implants with a traditional scalpel blade or a 940 nm diode laser on a patient level, an immunologic level, and a bacterial level. One of the most noteworthy observations of this study was the fact that participants reported feeling a significantly greater amount of pain, in the first 3 days postoperatively, at the sites which received laser application. After 3 days, the VAS scales indicated that the pain subsided and there was no appreciable difference in pain or discomfort between the test and control sites by day 7. Despite the significant difference in pain perception, when asked through the global health questionnaire which treatment was preferable, the overwhelming majority of patients, 83%, opted for the laser if the procedure was to be performed again. This result does not seem to be related to the participant's clinical experience, as it took a significantly longer amount of time to uncover the implants with the laser and participants felt a significantly greater amount of pain during initial healing with the laser. The participant's preference for the diode laser appears to be a mental preference for a different type of procedure that does not involve a scalpel blade, has good hemostasis, and appears to be "futuristic," as explained by some of the participants. Studies show that given an alternative treatment, participants tend to prefer procedures in which there is no perceived "cutting" of the skin. [22] In this study, patients may have preferred the diode laser due to their preconceived notions that lasers are a futuristic, safe, and effective option for a variety of procedures despite not actually knowing the research behind their particular operation.

The results revealed immunologic trends consistent with the hypothesis that the use of a diode laser will decrease the amount of inflammatory cytokines produced at application sites, with the amount of IL-1 β measured 1 day after laser application yielding significantly lower levels at experimental laser sites as compared to the control scalpel treated sites. The levels of IL-6 were lower in the laser-treated sites than the control sites; however, statistical significance was not reached. Over the 7-day evaluation period, levels of both cytokines were lowered in all treated sites. Studies have shown that after injury to the gingiva, inflammation is an immediate response which peaks day 3 postoperative before subsiding. [23] Histological studies in animals have also shown that the application of diode lasers after a scalpel incision decreases the inflammatory phase. [24] In addition, animal studies that evaluated the effects of low-level diode energy application on IL-1 β and IL-6 levels demonstrated a significant reduction of the cytokines compared to the controls. [25],[26],[27] Although the literature lacks studies demonstrating the effects of a high-intensity diode laser on IL-1 β and IL-6 levels, there is evidence that scatters from the high-intensity diode creates a low-intensity photo-stimulatory effect on adjacent tissues.

Interestingly, the immunologic trends did not correlate with the clinical outcomes of patient perceived pain or gingival inflammation. Local production of IL-1 β and IL-6 by nascent cells populating gingival tissues of wounded sites has been implicated with hyperalgesia, recruitment of inflammatory cells, and release of other inflammatory cytokines. [11] Injection of IL-6 into one hind paw of male Wistar rats evoked hyperalgesia in both hind paws with the intensity of the pain reaching a plateau in 2-3 h and maintained for up to 6 h after local injection. Cunha et al. injected IL-6 into the hind paws of male Wistar rats and found that the hyperalgesia response reached maximum intensity after 2-3 h and was maintained for a least 6 h after injection. [13] Our participants reported greater pain perception at the laser-treated sites over the first several days after second-stage implant uncovering surgery as compared to the scalpel-treated sites. The reported gingival indices for laser and scalpel sites, which scales clinically observed signs of gingival inflammation, were not significantly different. Since the pain levels and gingival inflammation were considered "mild" for both treatment groups, cytokine levels may not have played a significant role in these treatment outcomes.

Microbiologic assays in this study revealed no statistically significant difference between the quantities of the five microbes measured from around the healing abutments at the experimental and control sites. It is likely that the healing abutments were contaminated with saliva soon after abutment attachment. The energy emitted from diode laser does not penetrate deep into the soft tissues and dissipates once absorbed into the target tissue. As such, the laser energy utilized in this study would have been absorbed into the gingival tissues in performance of the excision of gingival tissues coronal to the implant platform. Reapplication of the laser to the peri-implant soft tissues after abutment placement was not part of the methodology of this study and therefore would have no inhibitory action on subsequent bacterial contamination of the healing abutments after the laser uncovering procedure. In addition, due to the methodology of this study, the bacteria identified and quantified around the healing abutments may be more reflective of the population of bacteria indigenous to the oral cavity, i.e., those bacteria harboring in the crevices in the tongue, cheek, and mucous membranes. When no differences were found in the microbial counts between test and control sites, the data were combined and then stratified on a subject-level based on the modality in which teeth were lost: Caries, periodontal disease, and edentulism. Data analysis revealed that there were significantly fewer Treponema bacteria in the participants who lost their teeth due to caries as compared to those who were previously edentulous or those who lost their teeth due to periodontal disease. Participants who were edentulous seemed to harbor similar amounts of periodontal pathogens as participants who had been treated for periodontal disease. Previous studies have evaluated the makeup of bacteria that colonize around implant restorations in edentulous individuals. The majority of studies have shown the presence of many periodontopathogenic bacterial species, especially in peri-implant sulci, including Prevotella, and Tannerella, but edentulous individuals typically show severely reduced levels of Porphyromonas as compared to dentate individuals. [28],[29] It is possible that despite a lack of teeth or exposed implants, the tested periodontal pathogens could have found safe harbor in other areas of the mouth. If oral hygiene is not meticulous, Prevotella, Tannerella, Treponema, Streptococcus, and Porphyromonas species may reside in the dorsum of the tongue, oral vestibule, or buccal mucosa, providing an avenue for bacterial contamination of the exposed healing abutments. [30],[31]

There were study design limitations that may have influenced the results of this study. For one, the practitioner performing all of the gingival excisions was a senior periodontal resident and was proficient with the use of a scalpel blade. There was a natural learning curve to using the diode laser and thus may result in the diode laser taking a longer period as well as being perceived as more difficult to use, when compared to a technique that the practitioner is proficient at performing. It is possible that in the hands of a novice in both techniques, there would be no differences in time taken or difficulty of performing gingival excisions when using a scalpel compared to a laser. Another limitation of the study involves the variable power of the laser. The study protocol allowed for the adjustment of the power of the laser, up to 2W, depending on the perceived cutting ability of the laser on each participant's tissue. No effort was made to measure the actual laser power being emitted from the laser tips as there may exist levels of variability in power being emitted.

Another limitation of the study involves the collection of both the crevicular fluid using paper points and collection of bacteria using E-swabs. Many of the implants were located immediately adjacent to the submandibular gland ducts. While isolation of the healing abutments was attempted, the fluid absorbed into the paper points may have been a combination of saliva and crevicular fluid (along the same line of reasoning, saliva contamination of the implant abutments may have contributed to the microbiology outcomes as noted above). Furthermore, the amount of crevicular fluid was not measured, making the calculation of concentrations of the inflammatory cytokines harder to standardize.

Sampling of bacteria was performed with E-swabs around the healing abutments in a circumferential manner, taking care to sample only bacteria present on the healing abutment and not from the gingival tissue. In this process, it is likely that supragingival plaque was collected around the healing abutments and would therefore contain fewer anaerobic bacteria, which made up the majority of the bacteria being analyzed. This may help explain some quantitative differences seen in the relative amounts of streptococci versus the anaerobic bacteria.


  Conclusions Top


Within the limits of the present study, the following conclusions may be made:

  • The diode laser excisional technique was preferred by participants over the scalpel blade tissue excision even though pain perception was reported more intense and longer lasting with the laser
  • The practitioner rated the laser as being more difficult to use due to an increased amount of time taken with the laser
  • IL-1 β-levels were significantly lower at laser sites 1 day after second-stage implant uncovering but were not correlated with reduced pain or inflammation at these sites (as compared to controls)
  • There are no differences in the amounts of the five periodontal pathogens: Prevotella, P. gingivalis, S. milleri, Tannerella, and Treponema between the diode laser sites and the control sites.
Acknowledgments

We would like to thank Dr. Ali Obeidi for the initial design of this study; to Todd Rigney, Doan Dao, Jennifer Kerr PhD, and Lansara Jaruthien for their assistance in Dr. Gena Tribble's Laboratory; to Mr. Stanley Cron for statistical analysis; and Dr. Barros and Dr. Wang for their expertise in laser technology and laboratory techniques, respectively.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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