A New Drug Delivery System - Silk Fibroin Film Loaded or Not With Insulin on Palatal Mucosa Wound Healing

Tissue thickness of palatine masticatory mucosa will be assessed by a Cone Beam Computed Tomographic (CBCT). In order to standardize the volume measures, the FGG harvested stent, made of 1 mm thick rigid acrylic plates in vacuum plasticizer with radiopaque material, will be used by the patient at the time of the CBCT image acquisition.

Wound will be colored with Replack (Dentisply - York, Pensilvânia - USA) and quantity measured by Image J program. Then, with the total area of the wound, the epithelization % 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.

CTG will be harvested from the palatal area, the same area that received FGG protocol previously, using the same harvesting stent, during implant therapy. After CTG be harvested, a 2-mm thick will be removed from the graft (Azar et al., 2019) to perform biopsy analysis aiming to observe histological changes during healing process by previously use of silk fibroin film, charged or not with insulin, and spontaneous repair. The specimens will be immersed in 10% formalin for histological analysis which will be stained with hematoxylin-eosin and Masson's trichrome.