Effectiveness of Three Different Mouthrinses in Dental Plaque Control and Early Wound Healing
Condition: Periodontitis; Wound Healing
MeSH terms: Periodontitis; Dental Plaque; Wounds and Injuries
Study type: Interventional

Summary: 
      Aim: This study compared the effectiveness of three different mouthrinses (alcohol and
      non-alcohol chlorhexidine, alkyl dimethyl glycine / alkyl dimethyl amine oxide - C31G) in
      plaque control and early wound healing, postoperatively.

      Materials and Methods: In this, randomized, double-blind, controlled clinical trial 42
      patients were allocated to three groups assigned to two weeks rinsing after periodontal
      surgery with C31G (group A), alcohol-free chlorhexidine 0.12% (group B) or alcohol-based
      chlorhexidine 0.12% (group C). At weeks 1 and 2, plaque and early wound healing indices were
      recorded. At day 14, total bacterial counts were estimated utilizing real - time qPCR.
      Statistics included linear and generalized linear mixed models.
    

Detailed description: 
      Materials and Methods

      Patients' enrollment and all clinical procedures were conducted at the Department of
      Periodontology, School of Dentistry, National and Kapodistrian University of Athens, Greece.

      Clinical Procedures:

      All surgical interventions were conducted by first and third year postgraduate residents.
      Supragingival debridement and polishing of the operated area was performed immediately before
      surgery. In cases of residual periodontal pocket elimination / reduction surgeries,
      intrasulcular incisions followed by papilla preservation incisions at interdental areas were
      applied. In crown lengthening cases, resective gingival incisions were applied in presence of
      sufficient width of keratinized tissues (≥ 3mm). Following flap elevation, root surfaces were
      thoroughly debrided with instruments and ultrasonic devices. Ostectomy / osteoplasty with
      carbide burs was applied, as deemed appropriate. Buccal and palatal / lingual flaps were
      fully adapted to each other and sutured together with monofilament slowly resorbable suture.
      Periodontal dressings were not used. No systemic antibiotics were prescribed. Ibuprofen
      400mg, three times daily for 4 days, was suggested immediately after surgical intervention.
      Smokers were advised to cease smoking for the first postsurgical week. Sutures were removed
      at 7th postoperative day. During that period no accidental loss of sutures was recorded.

      Randomization and allocation concealment:

      Subjects were randomly assigned to one of three groups using a computerized assignment
      procedure (Random Sequence Generator, www.random.org) generated by the examiner A.G. Each
      subject was given an identical number between 1 and 42. Participants received bottles with
      one of the three solutions under investigation. Neither patients nor clinicians (surgeons and
      examiner A.G.) were informed about the identity of mouthrinse administered in each case
      (double - blind design). Masked bottles were given to participants by a third independent
      person who was not a dentist (member of Postgraduate Clinic's assistance staff).

      Post - surgical maintenance protocol:

      During the first 14 postoperative days, participants were instructed to rinse with 15ml of
      the administered solution for one minute, twice daily.

      During the same period, subjects refrained from any mechanical plaque removal at the operated
      areas.

      Intra-examiner reproducibility:

      Prior to the beginning of the study, the examiner (A.G.) who performed the measurements was
      calibrated in order to establish intra - examiner reproducibility. Calibrations were
      performed with two subjects not included in the study. During post-operative maintenance
      using 0.12% chlorhexidine, at the 7th postsurgical day, early wound healing index (EHI) and
      plaque index (PI) were recorded on two separate occasions during the same day, however at
      least 8 hours apart. Intra-class correlation analysis was used to calculate intra-examiner
      agreement for repeated measurements. The calibration was accepted if both measurements were
      similar with a deviation of +/- 10% (intra-class correlation coefficient > 0.9).

      Microbiological assessment:

      Supragingival dental plaque pooled samples were collected at the 14th postoperative day from
      both interproximal tooth surfaces respectively to the interdental papilla with the highest
      EHI value, using curettes. Pooled samples were individually collected in 150μl TE buffer
      solution. Samples were stored at -80 until processing, which was performed at the Laboratory
      of Cell and Matrix Pathobiology at the Institute of Biosciences and Applications, National
      Center for Scientific Research (N.C.S.R.), Athens, Greece. Bacterial genomic DNA was isolated
      and purified using the PureLink® Genomic DNA Μini Kit, following the Gram (+) bacterial cell
      lysis manufacturer's protocol. Purified DNA concentrations were determined
      spectrophotometrically (NanoDrop 1000). Specimens were analyzed using quantitative real time
      PCR (qPCR) in order to evaluate the total bacterial count in each pooled sample. qPCR was
      conducted with application of a set of universal primers (forward primer: 5'
      TCCTACGGGAGGCAGCAGT 3' and reverse primer: 5' GGACTACCAGGGTATCTAATCCTGTT 3', with an amplicon
      size of 466 base pairs), reported to detect DNA sequences of gene encoding for 16S ribosomal
      subunit of at least 50 different bacterial species (aerobic and anaerobic Gram positive and
      Gram negative) and SYBR Green Ι fluorescent probe. Serial dilutions of DNA extract of pure
      bacterial cultures of Escherichia coli were performed in order to generate a standard curve
      for the qPCR. Reactions were performed for 40 cycles at 94 °C for 30 sec, 58 °C for 45 sec
      and 72 °C for 10 min in a Light Cycler 96 qPCR Device. Results were analyzed using the Light
      Cycler 96 1.1 Software. Total bacterial count of each sample is expressed as total bacterial
      DNA mass (ng) and bacterial copy numbers (x106).

      Sample size calculation:

      Sample size was estimated through one-way ANOVA based calculations, assuming that the minimum
      clinically significant difference is one unit in median EHI values at 14 days between at
      least one pair of groups. 14 patients for each group were needed for 80% power achievement
      and for median differences detection in under evaluation parameters among the groups.

      Statistical analysis:

      Demographic and clinical characteristics of the sample are presented by group in tables
      containing absolute and relative (%) frequencies for categorical variables and medians and
      interquartile ranges (Median - IQR) for quantitative variables. Between groups differences in
      these characteristics were assessed through Fisher's exact tests for categorical variables
      and Kruskal-Wallis tests for quantitative ones. The distributions of the indices of interest
      (Early wound healing index - EHI, Plaque index - PI, Plaque area index - PA%) were summarized
      through medians and IQRs.

      Indices' data were analyzed through linear or generalized linear mixed models taking into
      account the potential correlations between multiple measurements taken on the same
      individual. In particular, for the analysis of EHI and PI, ordinal logistic regression models
      were used. For the PA% index, which was a proportion ranging from 0% to 100%, a linear model
      was used after a logit transformation of the dependent variable. Model selection, including
      checks for potential interaction effects, for the final multivariable models was based on
      likelihood ratio tests.

      Total microbial count differences among groups or in relation to other sample characteristics
      were analyzed using linear regression model after log10 transformation. Results are presented
      as relative differences %.

      P-values less than 0.05 were considered statistically significant. All analyses were
      performed using the statistical package Stata 13.1 (Stata Corp., USA).
    

Intervention name(s): Alcohol-based Chlorhexidine Gluconate 0.12%; Alcohol-free Chlorhexidine Gluconate 0.12%; C31G
Intervention type(s): Drug; Drug; Drug
Intervention description(s): Rinsing with 15ml for one minute twice daily for 14 days; Rinsing with 15ml for one minute twice daily for 14 days; Rinsing with 15ml for one minute twice daily for 14 days
Intervention MeSH terms: Ethanol; Chlorhexidine; Chlorhexidine gluconate
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)
Primary purpose: Supportive Care
Keywords: chlorhexidine; alcohol; C31G; Mouthrinses; Early postsurgical wound healing