Evaluation of Local Doxycycline in Smokers With Chronic Periodontitis
Condition: Chronic Periodontitis
MeSH terms: Periodontitis; Chronic Periodontitis
Study type: Interventional
Phase: Phase 2

Summary: 
      Summary The aim of this study is to evaluate through clinical, microbiological and
      immunological parameters using PLGA microspheres (lactic-co-glycolic acid polyacid)
      containing doxycycline in periodontal therapy in smokers with chronic periodontitis. For this
      study, 40 patients will be selected, with an average age between 30 and 59 years old of both
      genders (male and female), who spontaneously seek the clinic graduate of the Piracicaba
      Dental School -FOP / UNICAMP, and exhibiting at least four bleeding sites, including 2 sites
      with a probing depth (PD) between 5 and 6 mm and two sites PS ≥7 mm in uniradiculares teeth.
      Patients should have a minimum of 20 teeth. Will be excluded from relevant systemic disease
      carriers or antibiotics used in the 6 months preceding the study. All patients will receive
      oral hygiene instruction and will undergo a pre-treatment, which will be held supragingival
      sweeps and removal of plaque retentive factors. Patients will be divided into 2 groups (DB +
      DOX: Debridement + Doxycycline, test group and DB: only debridement, control group) of 20
      patients who will receive the following treatments: periodontal debridement ultrasound for 45
      minutes associated with the local administration of 1 mg microspheres containing doxycycline,
      or just mechanical debridement, for each selected site. Will be assessed the following
      clinical parameters: gingival index (GI) Plaque Index (PI), Bleeding on Probing (BOP),
      Position of the gingival margin (PGM), Probing Depth (PD) and Relative Clinical Attachment
      Level (RCAL). The microbiological evaluation will be made by means of polymerase chain
      reaction - real time ( "real time" - PCR) to detect bacteria: Porphyromonas gingivalis,
      Tannerella forsythia, Treponema denticola, Aggregatibacter actinomycetemcomitans and
      Prevotella intermedia. The enzyme immunoassay (LUMINEXMAGPIX) allow the detection of cytokine
      levels of GM-CSF, INF-γ, IL-10, IL-12, IL-13, IL-1β, IL-4, IL-6, IL-8 and TNF-α. The analysis
      of the proteolytic enzyme activity is accomplished by using zymography to assess MMP-8 and
      MMP-9. The described parameters will be evaluated at baseline, 45 and 90 days after the
      initial visit. The results are compared statistically by analysis of variance with repeated
      measures with 5% significance level.
    

Detailed description: 
      VI- Methods

        1. Sample Selection

           After submission and approval of this project to the Research Ethics Committee (CEP) of
           the Piracicaba Dental School - UNICAMP, will be selected among the patients who seek
           spontaneously treatment in clinics Graduate FOP-UNICAMP (where also will be performed
           the clinical phase of the study), 40 patients with an average age between 30 and 59
           years, of both genders (male or female) according to the following inclusion criteria:

             -  Patients smokers, but overall good health

             -  patients initially diagnosed with moderate or severe chronic periodontitis
                (according to the American Academy of Periodontology- AAP, 1999) verified the
                presence of radiographic periodontal pockets and bone loss;

             -  Patients who have at least four bleeding sites with probing depth (PD)> 5 mm in
                uniradiculares teeth.

             -  Presence of a minimum of 20 teeth;

             -  formal consent to participate in research, after explaining the risks and benefits
                for individual not involved in it. (Resolution No. 196 of October 1996 and the
                Dental Code of Professional Ethics (C.F.O.) 179/93).

           The exclusion criteria are:

             -  periapical or pulp change Presence

             -  Presence of systemic changes or use of medications (6 months prior to the study)
                that may influence response to periodontal treatment (eg, phenytoin, cyclosporine,
                calcium channel antagonists, and anti-inflammatory steroids and non-steroidal);

             -  Presence hypersensitivity to tetracyclines and their derivatives;

             -  Teeth with involvement furcation in the case of premolars.

             -  Pregnancy and lactation

        2. Study design

           The study will be clinical, randomized, controlled, double-blind, parallel design, and
           the treatments are randomly distributed among the patients. Selected patients will be
           divided into 2 groups, whose teeth receive the treatments, as follows:

           DB group (20 patients), - mechanical debridement in one session and application of
           placebo microspheres in four periodontal pockts greater than 5 mm DB + DOX group (20
           patients), - mechanical debridement in one session associated with the application of 1
           mg of microspheres loaded with doxycycline per periodontal pockt, into four sites equal
           to or greater than 5 mm.

           The PLGA microspheres, used in this study as a controlled drug delivery system, will be
           produced in the Biomaterials Laboratory of Medicine of the Catholic University of São
           Paulo, according to Moura, 2015. The microspheres are loaded with doxycycline
           hydrochloride ( EMS Sigma Pharma-Generics, Hortolandia, Brazil).

        3. Research Plan Initial examination: Selection of 40 patients according to pre-established
           criteria.

      Initial therapy: All patients will be educated about the causes and consequences of
      periodontal disease as well as on the importance of prevention techniques, (sulcular brushing
      and flossing) for periodontal therapy. In this phase, there will be supragingival periodontal
      debridement of all mouth and removal of plaque retentive factors.

      Treatment: All patients will be treated by subgingival debridement of mouth all by the same
      operator and will be reassessed in 3 months by the study investigator. Randomization will be
      performed through a list generated by computer.

      Reviews of treatment: Patients will be instructed in oral hygiene and will be held the
      professional control of supragingival biofilm in follow-up visits. To standardize the
      location of the collection of clinical data, guiding probing devices will be made - "stents"
      with acrylic sheets 1mm thick in a vacuum plasticizing, to survey the sites with PS≥ 7 mm.

      Clinical: The clinical parameters are:

        1. Gingival Index - IG (Löe and Silness, 1963): Inflammatory signals are collected through
           the following scores: 0 (absence of gingival inflammation); 1 (mild inflammation - small
           change in color, texture, absence of bleeding on probing); 2 (moderate inflammation -
           moderate brightness, redness, swelling and hypertrophy, bleeding on probing); 3 (severe
           inflammation - marked redness and hypertrophy; spontaneous bleeding, and tendency to
           ulceration)

        2. Plaque Index - IP (Ainamo & Bay, 1975): Evaluation of the presence / absence of plaque
           in binominal standard. Number of faces engaged with plate divided by the number present
           in all faces the patient's teeth 0- no visible plaque; 1- presence of visible plaque

        3. Bleeding on probing - SS (Mühlemann & Son, 1971): Standard dichotomous. 0-absence of
           bleeding; 1- presence of bleeding.

        4. position gingival margin - PMG: distance of demarcation in the "stent" to the free
           gingival margin.

      e Insertion Relative Clinical Level - NICR: distance of demarcation in the "stent" to the
      base, clinically detectable, the periodontal pocket.

      f- Depth Survey - PS (NICR - PMG): distance from the gingival margin to the base, clinically
      detectable, the periodontal pocket.

      All clinical parameters are obtained using a periodontal probe of the North Carolina type at
      baseline, 45 and 90 days after.

      Description of the methods that affect the research subjects:

      Once selected the study volunteers, they receive all the guidelines relating to the Consent
      and Informed research following the recommendations of Resolution 466/12 of the CNS. Any
      discomfort that may be caused by periodontal debridement treatment by ultrasound are not
      different from those caused by treatment coronorradicular scraping, consisting of moderate
      pain in the treated area and the possibility of dentine hypersensitivity due to exposure of
      dentinal tubules on the root surface. To minimize possible discomfort for these volunteers
      will be prescribed aftertreatment analgesics and mouthwash fluoride solution in all cases,
      regardless of whether or not feature dentinal hypersensitivity. Local administration of
      antibiotics (doxycycline) poses no risk to volunteers, since the administered dose will cause
      only local effects, according to the studies consulted for the preparation of this project.
      Moreover, even if undesirably the administered antibiotic does not achieve the expected
      results, it can be said that this will not result in patient exposure to some kind of risk,
      since the dose to be given is insufficient to cause increased discomfort. Still, if the
      conventional treatment with or without the local use of the antibiotic does not yield results
      in some patients, they will be benefited by the methods of oral hygiene education and
      clarification on oral health, which contribute to improving the quality of life of patients .

      Microbiological evaluation:

      It will be done by polymerase chain reaction (PCR) of the "real time". This test will allow
      the quantitative detection of the following bacteria: Porphyromonas gingivalis, Tannerella
      forsythia, Treponema denticola, Aggregatibacter actinomycetemcomitans and Prevotella
      intermedia, held in the Discipline of Periodontology Laboratories, located in FOP / UNICAMP.

        1. collection of subgingival The area for the collection of material, will be properly
           insulated and dry with sterile cotton rolls. The portion of supragingival bacterial
           biofilm is removed to then obtain samples of subgingival plaque, by placing strips of
           paper (Periopaper - (OraFlow Inc., New York, USA)) inside bags moderate (5-6 mm ) and
           deep pockets (≥ 7 mm) of each patient, for 45 - 60 seconds. The volume of gingival
           crevicular fluid collected is measured with the Periotron 8000 (OraFlow Inc., New York,
           USA) and the paper strips are then placed in microcentrifuge tubes (Eppendorf), coded
           for each of the patients, and 400 or 200l phosphate buffered saline (PBS) and 0.05%
           Tween-20.

        2. DNA Purification The bacterial strains obtained from the American Type Culture
           Collection (ATCC) P. intermedia (Pi) (ATCC 25611), P. gingivalis (Pg) (ATCC 33277), A.
           actinomycetemcomitans (Aa) (ATCC 29522) from a culture pure T. denticola (Td) and
           T.forsythia (TF) will be used as positive controls and standards. The bacterial DNA is
           purified using DNA isolation kit PowerSoil® (MO BIO Laboratories, Inc.). The DNA
           concentration is adjusted to 15 ng / ul in water and then an additional six dilutions in
           10-fold serial be made. Three replicate samples of each dilution (5 ul / sample) are
           then assayed as described below for each specific DNA. The sensitivity of each assay was
           determined as the lowest dilution of DNA that produced a statistically significantly
           lower (p <0.001) higher than the control without template using a t-test.

        3. real PCR team DESIGN OF PRIMERS: The "primers" specific to intermediate P., P.
           gingivalis, A. actinomycetemcomitans, T. and T. forsythensis denticola will be used. All
           "primers" will be checked for their specificity by verifying the Melting curve (obtained
           after running the LightCycler) and running gel for product verification.

      OPTIMIZATION OF REACTIONS: The use of SYBR Green I requires getting specific PCR products, on
      that basis the effectiveness of responses to "primer" will be optimized before the start of
      the actual reactions. Concentrations ranging from 2 to 5 mM MgCl 2 and 0,2 to 0,5 uM of each
      "primer" will be used to determine the reaction conditions which have the best efficiency.
      These conditions suggested by the manufacturer of the equipment.

      REACTIONS OF REAL-TIME PCR (RT-PCR): The RT-PCR reactions are performed with the LightCycler
      system (Roche DiagnosticsGmbH, Mannheim, Germany) using the kit "FastStart DNA Master SYBR
      Green I (Roche DiagnosticsCo.) ". The profile of the reactions will be determined following
      the equipment manufacturer's recommendations. For each of the "racing" Aqua will be used as
      negative control, and the product of the reaction is quantified using the manufacturer's own
      program (Software LightCyclerRelativeQuantification - DiagnosticsGmbH Roche).

      immunoenzymatic evaluation of gingival crevicular fluid For a comprehensive analysis of the
      immune-inflammatory markers present in the gingival crevicular fluid collected from
      periodontal sites, will use the Luminex / Magpix system for the identification and
      quantification of these inflammatory markers: IL-1β, IL-4, IL-6, IL 8, IL-10, IL-17, IL-23,
      TGF-β, TNF-alfa, IFN-alfa and GM-CSF. For this analysis, are added to beads labeled sample to
      monoclonal antibodies specific for each marker to be quantified. After the addition of the
      beads, the sample is hybridized placed on plates and incubated for 2 hours at 37 ° C. The
      plate is then washed and a secondary antibody joined to a substrate (biotin) is added. This
      assembly is then incubated for 1 hour until, after washing, fluorescent marker (Streptavidin
      / R-phycoerythrin). The plates will then be analyzed by a Luminex 100 instrument program
      (MiraiBio, Alameda, CA, USA). This identifies and quantifies the fluorescence of each bead,
      corresponding to the inflammatory marker. This fluorescence is then transformed, by means of
      standard curves (third-orderpolynomialequation) values are processed in pg / ml.

      Analysis of proteolytic enzyme activity - zymography Will evaluate the presence and activity
      of two proteolytic enzymes - MMP 9 (gelatinase - which degrades mainly collagen type IV) and
      MMP 8 (collagenase - major degradation of collagen type I, II, III) after three months of the
      completion of debridement mechanical subgingival. For this purpose it will be used zymography
      technique. This technique is based on digestion of association between the substrate and
      verification of the molecular weight of the protease. It is used to identify proteolytic
      activity in enzymes separated (bands) in a polyacrylamide gel under nonreducing conditions.
      Gels are stained with Coomassie blue and the enzyme activity is demonstrated by the absence
      of coloration (white streaks) in areas where the substrate was degraded.

      Gel zymography:

      The gel zymography resembles gel electrophoresis also being mounted on a plate system. This
      gel is composed of two parts: Starting from the gel and gel separation. The two are divided
      as to have different position and composition.
    

Intervention name(s): Doxycycline; Mechanical debridement; Placebo
Intervention type(s): Drug; Procedure; Drug
Intervention description(s): Application of 1 mg of doxyxycline microspheres at 4 sites (two means and two deep periodontal pockets) after mechanical debridement.; Mechanical debridement (full mouth) of 45 minutes associated with the application of 1 mg of microspheres placebo in selected periodontal pockets.; The application of 1 mg of microspheres placebo
Other intervention name(s): PLGA microspheres; Full mouth debridement; Full mouth debridement
Intervention MeSH terms: Doxycycline
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)
Primary purpose: Treatment
Keywords: Local Drug Delivery; Antibiotics; Doxycycline; Smokers; Chronic Periodontitis