Comparison of Palatal Wound Healing in Diabetic and Non-diabetic Patients

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).

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).

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

Tissue edema will be evaluated with the score: 1 = absent; 2 = slight; 3 = moderate; or 4 = severe (Sanz-Moliner et, 2013).

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)

Number of analgesics used during 14 days after the procedure will be reported at the same postoperative diary (Tonetti et al. 2017).

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).

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.

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.

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.

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).

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).