This study aims to characterize and compare the closure of open wounds in the palatal mucosa of diabetic and non-diabetic patients, evaluate clinical, patient-centered and immunological parameters as well as wound microbiome composition.
The use of autogenous grafts from the palate for the reconstruction of gingival tissues is considered the gold standard for various periodontal and peri-implant reconstructions. Given the current aging of the population, it is essential to understand the cellular mechanisms responsible for the repair response in oral tissues and how they are affected by systemic diseases, such as diabetes mellitus (DM). The objectives of the present study, conducted through a controlled clinical trial, are to characterize and compare the closure of open wounds in the palatal mucosa of diabetic and non-diabetic patients. This will be achieved through clinical analyses, patient-centered parameters, inflammatory biomarkers, and wound microbiome composition. To accomplish this, 50 patients will be divided into two groups: the Diabetic Group (D; n = 25), where diabetic patients will undergo surgery for mucogingival defect correction with the addition of a free gingival graft, and the Control Group (GC; n = 25), where normoglycemic patients will undergo surgery for mucogingival defect correction with the addition of a free gingival graft. The groups will be compared regarding clinical parameters, patient-centered measures, including remaining wound area, epithelialization, tissue thickness, immature wound area, tissue edema, early wound healing index, postoperative discomfort, quality of life, number of analgesics, and sensitivity of the operated area over a 3-month period. Furthermore, the wound biofilm will be described through microbiome analysis, and tissue, saliva, and wound exudate biomarkers will be characterized. Descriptive statistics will be expressed as mean ± standard deviation, clinical evaluations will be performed using repeated measures ANOVA, and patient-centered parameters will be assessed using the T-test. Finally, multiple linear regression and correlation tests will be employed.
Study Type
INTERVENTIONAL
Allocation
NON_RANDOMIZED
Purpose
BASIC_SCIENCE
Masking
SINGLE
Enrollment
50
With the goal to harvest the free gingival graft (FGG), an 8mm diameter scalpel will be used to ensure standardized wounds. Thereafter, a 2-mm thick FGG will be removed. The wound area will receive 4.0 silk sutures.
College of Dentistry - São José dos Campos, Sao Paulo State University
São José dos Campos, São Paulo, Brazil
RECRUITINGMauro Pedrine Santamaria and Ana Carolina Ferreira Bonafe
São José dos Campos, São Paulo, Brazil
RECRUITINGOpen Wound Area (OWA)
For this, standardized photographs will be taken. As a reference, a scale will be used to measure this area. These photographs will be exported to image software (Image J - National Institute of Health -NIH, Bethesda, USA), the area of the wound will be measured in square millimeters (Dias et al. 2015)
Time frame: baseline, 7, 14, 21 days after surgery
Tissue Thickness (TT)
An endodontic spacer (Dentsply-Maillefer Instruments S.A. - Switzerland) with a rubber cursor will be placed in contact with the area until the palatal bone is reached, without pressing the tissue. The distance between the tip of the spacer and the cursor will be measured using a digital caliper (Dias et al., 2015).
Time frame: baseline, 90 days after surgery
Epithelialization (E)
The wound will be colored with Shirley's solution and the epithelialized area will be quantified in the Image J. program. Then, with the total wound area, the % epithelialization will be calculated (Ozcelik et al., 2008).
Time frame: baseline, 7, 14, 21, 30 e 90 days after surgery
Early wound healing index (EWHI)
According to Fickl et al. 2014 any modification in wound healing will be evaluated in five different degrees: Complete wound closure with an absence of fibrin on the palate; Complete wound closure with the presence of a fibrin line on palate; Complete wound closure with the presence of a clot with fibrin on palate Incomplete wound closure with partial tissue necrosis on palate; Incomplete wound closure with total tissue necrosis on palate
Time frame: 7 and 14 days after surgery
Tissue Edema (TE)
Tissue edema will be evaluated with the score: 1 = absent; 2 = slight; 3 = moderate; or 4 = severe (Sanz-Moliner et, 2013).
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Time frame: 7 days
Oral Health Impact Profile (OHIP)
Will be evaluated from a questionnaire with 14 questions based on 7 domains: functional limitations, physical pain, psychological discomfort, physical disability, psychological deficiency and social deficiency. The patient should respond to the questions within 14 days after the surgical procedure, performing a postoperative diary. For each question an answer must be given, represented in numbers, being: 0- Never; 1- Almost never; 2-Occasionally; 3-Quite frequent; 4-Very common; 5-I do not know (Tonetti MS et al. 2017)
Time frame: 14 days
Number of analgesics
Number of analgesics used during 14 days after the procedure will be reported at the same postoperative diary (Tonetti et al. 2017).
Time frame: 14 days
Patient Discomfort
By a visual analogic scale (VAS) of 100 mm to assess discomfort, patients will report pain diary during the 14 days after surgery. Scale extremes will be "no pain" to "extreme." (Tonetti et al. 2017).
Time frame: 14 days
Qualitative somatosensory testing (QualST)
This analysis will evaluate somatosensorial profiles and pain conditions. For this, different stimulus will be performed on the wound and the following tests will be applied: (1) Touch stimulus will be applied with a swab by a single application for 1-2 sec in the wound; (2) Cold stimulus will be applied by a stainless steel dental spatula (kept cool in ice water, approximately 0 °C) with wound direct contact during 1-2 sec; (3) The pinprick stimulus will be performed with a periodontal probe with moderate force on the wound area for 1-2 s (Baad-Hansen et al, 2013) Patient will report hypersensitivity, hyposensitivity, or normosensitivity to touch, cold and painful stimulus.
Time frame: 7, 14 days
Immunologic Analysis
With the goal to obtain baseline data for this parameter, crevicular gingival fluid from the gingival area next to the donor area will be collected previous surgery. An absorbent paper (PerioPaper, Oraflow, Plainview, NY, EUA) will be placed at wound edges without pressure during 40s. Collects with blood contamination will be discarded. Samples will be stored into a sterilized Eppendorf containing 100 μL Phosphate Buffer Saline 0.05% Tween 2 (PBS) at - 80 C. Growth factors (VEGF and EGF), chemokines (MIP-1α, MCP-1α), and cytokines (IL1β, IL6, IL10, TNFα) levels will be determinate by the multiplex assay. Moreover, MMP-2, MMP-9, TIMP-1, TIMP-2 will be measured by the same commercial human commercial kit.
Time frame: baseline, 3, 7 days
Microbiome Analysis
The biofilm from the palatal region will be collected, and the samples will be stored in sterilized Eppendorf tubes containing 100 μL of Phosphate Buffer Saline 0.05% Tween 20 (PBS) at -80°C. Three laboratory steps will be performed for the evaluation of the palatal region's microbiome, which are as follows: (1) Bacterial DNA extraction; (2) PCR amplification of the 16S rRNA region; (3) Library preparation for sequencing - PCR targeting the V3-V4 region. Bacterial DNA extraction will be conducted using a specific kit (MasterPure Complete DNA and RNA Purification Kit - Biosearch Technologies), following the steps of cell lysis and DNA purification.
Time frame: baseline, 7, 14, 21, 30 e 90 days after surgery
Tissue Analysis
To do so, the tissue sample collected during the surgical stage will be immediately immersed in 10% formaldehyde fixative for a period of 24 hours at room temperature. After the fixation period, the sample will be washed three times with a PBS solution at room temperature and then stored in 70% ethanol at 4°C. Using multiplexed immunofluorescence imaging technology (co-detection by indexing CODEX), a tissue atlas of the repair process will be generated for both healthy and diabetic patients. Biomarkers will be analyzed to determine the inflammatory profile, cell types, cell-cell contacts, and cellular neighborhoods, following the manufacturer's instructions (Black, 2021).
Time frame: baseline
Saliva Analysis
After the collection of 5 ml of saliva, the sample will be centrifuged at 2800 g for 20 minutes at 4 ºC. The supernatant will be separated from the pellet, and to each 1 mL of saliva, 100 uL of a protease inhibitor solution (SIGMAFAST, Sigma, St. Louis, MO, USA) will be added. The following markers will be quantified through ELISA tests: (1) Histatin-1 (MBS2022124 H1, MyBioSource, San Diego, CA, USA), (2) Epidermal Growth Factor (EGF; KHG0061; Invitrogen, Waltham, MA, USA), and (3) Vascular Endothelial Growth Factor (VEGF-A; BMS277-2, Invitrogen, Waltham, MA, USA).
Time frame: baseline, 7 days