Antiplaque Effect of Essential Oils and 0.2% Chlorhexidine on an in Situ Model of Oral Biofilm Growth.
Condition: Oral Biofilm; Mouthwash; Periodontitis
MeSH terms: Periodontitis
Study type: Interventional
Phase: Phase 4

Summary: 
      The accumulation and maturation of oral biofilm in the gingival margin is widely recognised
      to be the primary aetiological factor in the development of chronic gingivitis. Based on this
      association, the current treatment of gingivitis is focused on biofilm disruption, which will
      normally include mechanical processes, both professionally and at home. However, for
      patients, it is not easy to achieve a proper level of plaque control. The efficient plaque
      control techniques are very time consuming and require a special motivation and skills for
      their optimum use. It was at this point where mouthwashes become important, due to the fact
      that they include diverse types of antimicrobial agents to complement the results of
      mechanical oral hygiene measures.

      Chlorhexidine is considered the "gold standard" of oral antiseptics; nevertheless it has not
      been recommended for long periods of time due to its well-known secondary effects. All of
      these inconveniences have limited its acceptability among dental professionals and users; in
      contrast, however, are the exceptional antiseptic properties, promoting the interest of
      researchers in other alternative antiplaque agents. Mouthwashes containing essential oils in
      their formulation have received a lot of attention. Their antiplaque activity has been
      demonstrated in numerous clinical studies, in which they were used in conjunction with
      mechanical oral hygiene measures.

      In order to achieve a better understanding of the clinical effects that antimicrobial agents
      produce in the interior of the biofilm, it is necessary to apply a methodology in which the
      biofilm grows directly in the interior of the oral cavity but its three dimensional structure
      is not distorted by manipulation.

      The aim of this study was to evaluate the in situ antiplaque effect of 2 antimicrobial agents
      (essential oils formulation and 0.2% chlorhexidine) in the short term with a posterior
      analysis on "non-destructured" biofilm with Confocal Laser Scanning Microscope combined with
      fluorescence staining.
    

Detailed description: 
      The volunteers wore two individualised splint which was capable of holding 6 glass disks (6
      mm in diameter, 1 mm thickness); this was polished at 800 grit. . These splints are formed of
      2 sheets, an internal 1 mm soft vinyl sheet where the disks are attached and an external 1 mm
      rigid one made of polyethylene terephtalate that is fenestrated.

      • Mouthwash protocol During the 96 hour (4 days) duration of each course of study, each
      volunteer wore the splints with the glass disks, withdrawing them from the oral cavity only
      during meals (they were stored in an opaque container in humid conditions) and to perform
      oral hygiene procedures, using only the mechanical removal of bacterial plaque with water,
      without the use of any toothpaste or mouthwash.

      Using the permitted mechanical oral hygiene measures (without the splints), the volunteers
      performed the following protocols based on the manufacturers' instructions, with the splints
      in the oral cavity, during the 4 days in the morning (8.30) after breakfast and at night
      (22.00) after dinner:

      A) 20 ml rinses for 30 seconds with essential oils/2 times daily. ----14 days---- B) 10 mL
      rinses for 30 seconds with 0.2% chlorhexidine/2 times daily. ----14 days---- C) 20 mL rinses
      for 30 seconds with sterile water (negative control). Using an internet-based balanced
      randomisation system, indicating the mouthwash that each subject would use first, second and
      third, all volunteers performed the 3 rinsing cycles, with a rest period of 14 days between
      each test.

      • Collection of the samples of Plaque like-biofilm Sample collection was done individually at
      8 am in the morning, so that the samples of each volunteer were analysed on different days.
      It was determined that a minimum of 10 hours should have elapsed since the last mouthwash on
      the previous night.

      As the glass disks were removed from the splint, they were immediately immersed in 100 µL of
      fluorescence solution LIVE/DEAD® BacLight™ and kept in a dark chamber at room temperature for
      15 minutes. Microscopic observation was performed by a single investigator who was unaware of
      the study design, using a Leica TCS SP2 laser scanning spectral confocal microscope (Leica
      Microsystems Heidelberg GmbH, Mannheim, Germany) with an HCX APOL 63x/0.9 water-immersion
      lens.

      • Processing of the samples of Plaque like-Biofilm Four selected fields or XYZ series in the
      central part of each disk were evaluated. These fields were considered representative of the
      whole sample after the observer's general examination. Fluorescence emission was determined
      in a series of XY images in which each image corresponded to each of the Z positions (depth).
      The optical sections were scanned in 1 µm sections from the surface of the biofilm to its
      base, measuring the maximum thickness of the field and subsequently the mean thickness of the
      biofilm of the corresponding sample. The maximum thickness of biofilm field was defined as
      the distance between the substrate (in perpendicular) and the peaks of the highest cell
      clusters. The maximum biofilm thickness of each field was divided into 3 zones or equivalent
      layers: outer layer (layer 1), middle layer (layer 2) and inner layer (layer 3).

      The capture of the data was done with the same settings in all cases. The spatial scan mode
      (XYZ) and the 1024x1024 pixels scan format resolution were used. The Argon-ion and DPSS laser
      were used at a 13% and 78% of maximum intensity, respectively. The values for the pinhole,
      zoom and scan speed were 121.58 microns, 1 and 400Hz, respectively. The only values that were
      different depending on the sample were the offset (range between -1% to 1%) and PMT gain
      which was different for channel red and green, being in general terms, higher for green than
      for red (test and positive control), due to the fact that there was more presence of green
      than red signal, being for the negative control the opposite. These values were always
      adjusted to get a good quality capture without background noise, avoiding excessive
      saturation of the brightest pixels of the image. As the technician was blind to the
      experiment, they were advised to make the adjustments always consistent with what was seeing
      by the objective of the microscope, obtaining an image which was the closest as possible to
      reality.

      Quantification of bacterial vitality in the series of XY images was determined using
      cytofluorographic analysis (Leica Confocal Software). In this analysis, the images of each
      fluorochrome were defined as "channels" (SYTO 9 occupies the green channel and propidium
      iodide the red channel). Square capture masks were used to measure the area occupied (µm2) by
      the pixels in each channel, determining the total area occupied by the biofilm and the
      corresponding percentage of vitality. The intensity ranges that were considered as positive
      signal were between 100 and 255. Determination of the mean percentage of bacterial vitality
      in each field required sections with a minimum area of biofilm of 250 µm2, and the mean
      percentage of bacterial vitality of the biofilm was calculated for the corresponding sample
      and for each biofilm layer.

      For quantification of the percentage of surface substrate covered by the biofilm (covering
      grade), the cytofluorogram itself was used. From the maximum projection (superposition of all
      planes captured) of each of the analysed fields, the percentage of covering grade was
      obtained by calculating the sum of the bacterial mass (vital and non-vital) in regard to the
      total surface of the field (% positive within total area).
    

Intervention name(s): Essential oils; 0.2% chlorhexidine; Sterile water
Intervention type(s): Drug; Drug; Drug
Other intervention name(s): Listerine; Oraldine Perio
Intervention MeSH terms: Chlorhexidine; Listerine
Intervention model: Crossover Assignment
Masking: Double (Investigator, Outcomes Assessor)
Primary purpose: Prevention
Keywords: Biofilms; Chlorhexidine; Microscopy, confocal; Dental plaque; Mouthwashes