Effect of Probiotics on Oral Wound Healing - Pilot Study
Condition: Wound
MeSH terms: Wounds and Injuries
Study type: Interventional

Summary: 
      This research will study the healing of standardized wounds created in the oral mucosa of
      volunteer participants during daily intake of tablets and topical application of oil
      containing the probiotic bacterium Lactobacillus reuteri. Our null hypothesis is that the
      consumption and topical application of probiotic supplements containing L. reuteri does not
      improve clinical healing of oral wounds in healthy participants when compared to controls.
    

Detailed description: 
      Intervention: after informed consent and baseline collection of clinical data (age,sex), the
      participants will be randomly assigned to one of the order of the treatments. As a run-in
      period, they will be given a supply of probiotic or placebo tablets enough for seven days.
      The participants are instructed to take two tablets daily (one in the morning and one in the
      evening, days 0-7) and are asked to refrain from any other foods or supplements containing
      probiotics. On day eight, the participant will attend to the clinics and a punch biopsy will
      be performed. Depending on the allocation group, probiotic or placebo oil will be supplied.
      The participant will be instructed to apply the oil locally on the wound with a plastic
      microbrush for 9 days every evening, and strongly reinforced to continue with the assigned
      tablets twice daily. The participant will attend the clinics on days two, five and eight
      post-wounding for follow-up photographies of wound healing. The first intervention period
      terminates with a wash-out period of four weeks, and then the second run-in an intervention
      period start following the same procedures.

      The probiotic tablets and the probiotic oil (at the recommended dose) contain 2x10^8 CFU of
      Lactobacillus reuteri. Both the placebo tablets and oil are identical in size and composition
      but without the addition of the probiotic strains. The tablets and oil are packed in
      identical pots/bottles with color coding. The code will be kept from the investigators until
      the analysis are finished. Both the tablets and oil are provided by BIOGAIA AB.

      Clinical procedures and sampling

      All clinical procedures and sample collections will be conducted by a qualified and
      authorized dentist from the Institute of Odontology, Section for Cariology, Endodontics,
      Pediatric Dentistry and Clinical Genetics, Faculty of Health and Medical Sciences, University
      of Copenhagen. Collected samples (saliva, wound exudate and bacterial smear) will be stored
      in a freezer at -80 °C until all samples from all patients are gathered -for a maximum of six
      months. Running of all collected samples at the same time allows laboratory analyses to be
      carried out efficiently and avoids errors.

      Punch biopsy: on day eight, a standardized circular wound of 3.5 mm diameter and
      approximately 1.5 mm deep will be created in the mucosa of the hard palate with aid of a
      biopsy punch. The biopsy is performed under aseptic conditions and local anesthesia using
      lidocaine 2% combined with epinephrine for local hemostasis. A scalpel is used to remove the
      surface epithelium and underlying superficial connective tissue, which will be discarded
      according to biosafety regulations from the Institute of Dentistry, University of Copenhagen.
      After wounding, the wound site will be photographed (blind-coded) with an intraoral camera,
      placing a standard-sized 6 mm diameter template around the wound as reference. The
      participant will attend to the dental clinics on days two, five and eight post-wounding to
      have a photograph of the wound taken by Ph.D student Gina Castiblanco. The wound-size on
      photographs will be measured by two raters and will be expressed as the ratio of the wound
      surface area relative to the standard label size with aid of software (Canvas 7, ACD Systems
      of America). The endpoint will be the percentage of initial wound size, measured at days two,
      five and eight post-wounding.

      Saliva sampling: six samples of unstimulated whole saliva (UWS) fro m each subject will be
      collected (one at baseline, one right after the run-in period and one at the end of the
      intervention period). For USW collection, the participant will be sitting in a comfortable
      position with the head slightly bent. The participant is asked to keep the eyes open and make
      as few moves as possible, including swallowing. Then, he/she is asked to rinse the mouth in
      water and spit it out again. Thereafter, he/she is instructed to let saliva flow out of the
      mouth down a designated plastic cup for ten minutes. Aliquots of saliva (1.5 ml) will
      immediately be placed on ice and stored in a freezer at -80 °C until further analysis.

      Collection of wound exudate: eight samples of wound exudate will be collected in total. The
      wound site will be dried with cotton pellets and periopaper strips (ProFlow, Amityville, NY,
      USA) will be gently pressed on the wound for 20 s. In case of blood contamination, the strip
      is discharged and the sampling is repeated after five min. The volume of exudate is recorded
      using a Peritron 8000 (Proflow) and expressed in µL. One strip holds approximately 0.1-0.4 µL
      of exudate. The strips are then transferred to plastic tubes and stored at -80 until further
      analysis.

      Bacterial smear collection: four bacterial smears will be sampled. A sterile swab will be
      passed through the wound site, placed in a plastic tube and stored freezed at -80 °C until
      further analysis. The smears will be subjected to conventional microbial cultivation to
      detects the presence/absence and counts of bacterial species commonly associated with delayed
      wound healing (β-hemolytic streptococci and S. aureus).

      Perception of pain and discomfort: a one-page logbook will be handed to the participants at
      the beginning of each run-in period so they can register the intake of tablets and their own
      perception of pain and discomfort according to a VAS-scale. The participant is also
      encouraged to register factors that could possibly influence the outcome (physical activity
      and number of alcoholic drinks). The participant will be asked to bring the filled logbook in
      the end of the intervention period.

      Perceived stress and general health: in the end of the intervention period, the participant
      will be asked to fill the validated General Heath Questionnaire GHQ-12 (10) and the validated
      Perceived Stress Scale (11). These factors (self-perception of stress and general health) are
      known to be possible cofounders of wound healing outcomes.

      Compliance: at the beginning of each intervention period, the participants will be provided
      with tablets enough for 16 days and oil for topical use enough for nine days application.
      Compliance will be checked through the participant´s completion of a logbook in which the
      daily intake of tablets and oil application is registered. For the last appointment, the
      participants will be asked to bring the remaining tablets and oil for counting/estimating.

      Data analysis: all data will be processed with the open source statistics software "R" and
      controlled for normal distribution. Multivariate and univariate analysis of variance (MANOVAs
      and ANOVAs) will be used to assess within-subject change over time. A p-value less than 0.05
      will be considered statistically significant. The obtained results will be published in an
      internationally recognized journal. If the results cannot be published in a journal, they
      will be available at the website of the University of the Odontology Institute. We make
      emphasis in that positive, negative and inconclusive results will be published.

      Data management: all collected data will be treated in compliance with the Act on Processing
      of Personal Data and "The Health Law". At inclusion the participants will be assigned a
      unique number and a separate protocol without personal identification number. This study will
      not be reported to "Datatilsynet" as it is a private scientific health project
    

Intervention name(s): L. reuteri DSM 17938/ATCC PTA; Placebo
Intervention type(s): Dietary Supplement; Dietary Supplement
Intervention description(s): Ingestion of active lozenge twice daily for 16 days and topical application of active oil once daily for 8 days; Ingestion of inactive lozenge twice daily for 16 days application of inactive oil once daily for 8 days
Allocation: Randomized
Intervention model: Crossover Assignment
Intervention model description: Cross-over pilot trial
Masking: Double (Participant, Care Provider)
Primary purpose: Supportive Care
Keywords: oral wound healing; probiotics