Manufacturing, Characterization and Evaluation of the Effect of Silk Fibroin Membranes, Loaded or Not With Neurotensins on Open Wounds in the Palate

Study Overview
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Brief Summary
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The aim of the present study will be to manufacture and characterize silk fibroin membranes loaded or not with neurotensin and to evaluate clinical, patient-centered and immunological parameters the effect of using these membranes on open wounds on the human palate.

Detailed Description
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Surgical procedures for the reconstruction of gingival and peri-Implant tissues with the purpose of restoring function and aesthetics are commonly used in the dental practice. The use of free gingival graft (FGG), despite being considered the gold standard, causes great morbidity and postoperative pain. Thus, in order to reduce these complications, some biomaterials have been explored to accelerate healing and bring greater comfort to the patient. The present study aims to: 1. manufacture silk fibroin membranes (SF), loaded or not with neurotensin (NT); 2. evaluate, clinically and immunologically, the effect of the membranes on open wounds at palate sites. After the manufacture of the membranes they will be characterized by visual aspect, thickness, mechanical properties, surface texture, water vapor permeability, enzymatic degradation and swelling. Posteriorly, sixty six (n = 66) patients requiring exodontia and ridge preservation for future implant placement will be randomly assigned into three groups: Control - Free Gingival Graft (n = 22): extraction and Free Gingival Graft for sealing the alveolus entrance; Group SF - Fibroin membrane (n = 22): extraction and Free Gingival Graft for sealing the alveolus entrance and placement of the fibroin membrane at the palatal wound site; Group SF + NT - Free Gingival Graft + Neurotensin-loaded Fibroin Membrane (n = 22): extraction and Free Gingival Graft for sealing the alveolus entrance and placement of neurotensin-loaded fibroin membrane at the palatal wound site. The repair of the palatine donor area will be evaluated by clinical parameters such as remaining wound area, tissue thickness, re-epithelialization and early-wound healing index at baseline, 7, 14, 21, 30, 45, and 90 days after the procedure; and patient-centered outcomes by post-operative discomfort, tissue edema and number of analgesic pills used for 14 days after the procedure. In addition, inflammatory cytokines and growth factors will be assessed using the palatal wound inflammatory exudate 3 and 7 days after surgery. For the descriptive statistics, the mean ± standard deviation will be used; Normality: Shapiro-Wilk test; Clinical trials: ANOVA for intra and inter group calculations + Tukey test for multiple comparisons when the Shapiro-Wilk p value ≥ 0.05. If p Shapiro-Wilk <0.05, will be analyzed using the Friedman test (intragroup) and Mann-Whitney tests (intergroup); Parameters centered on patients: T test.

Official Title
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Manufacturing, Characterization and Evaluation of the Effect of Silk Fibroin Membranes, Loaded or Not With Neurotensins on Open Wounds in the Palate: Randomized Clinical Study

Conditions
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Wound Healing, Palate; Wound

Intervention / Treatment
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* Procedure: Palatal Wound
* Device: Palatal Wound treated with Silk Fibroin Film
* Device: Palatal Wound treated with Neurotensin-loaded Silk Fibroin Film


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Patients at least 18 years old, healthy systemically, with good oral hygiene, assessed by means of a plaque index and gingival index less than 25% (O'Leary et al., 1972) Patients with no morphological or pathological changes in the donor palatal region; Patients who have an indication for extraction and with an indication to preserve the socket for future implant installation; The tooth included in the study, as well as the adjacent teeth, must not have loss of periodontal insertion. Exclusion Criteria: Patients with systemic problems (cardiovascular changes, blood dyscrasias, immunodeficiency, diabetes, among others), who contraindicate the surgical procedure; Patients who use medications that interfere with wound healing; Pregnant or lactating patients; Patients who present opportunistic oral lesions, mainly colonizing the palate region; Smoking patients; Patients undergoing orthodontic treatment; Patients already submitted to periodontal surgery in the area of interest in the present study; Patients with a history of allergy to any type of material used in the present study.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 65 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Parallel Assignment
Interventional Model Description: Double-blind, parallel, superiority, randomized clinical trial with a 3 months of follow-up
Masking: Double


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Sham Comparator: Control Group (CG)<br>The palatal wound area will not receive any treatment | Procedure: Palatal Wound<br>* 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.<br>|
| Experimental: Silk Fibroin Film - SF<br>The palatal wound area will receive silk fibroin film as a dressing | Procedure: Palatal Wound<br>* 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.<br>Device: Palatal Wound treated with Silk Fibroin Film<br>* 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 a silk fibroin film and 4.0 silk sutures.<br>|
| Experimental: Neurotensin-loaded Silk Fibroin Film - SF + NT<br>The palatal wound area will receive a neurotensin-loaded silk fibroin film as a drug delivery system | Procedure: Palatal Wound<br>* 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.<br>Device: Palatal Wound treated with Neurotensin-loaded Silk Fibroin Film<br>* 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 a silk fibroin film loaded with neurotensin and 4.0 silk sutures.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in the Remaining Wound Area (RWA) | 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) | baseline, 7, 14, 21, 30, 45 and 90 days after surgery | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Tissue Thickness | 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). | baseline, 45 and 90 days after the procedure | 
| Scar and Tissue Colorimetry | The presence or absence of scar or keloid in the operated area will be analyzed. In addition, the tissue color similarity of regions adjacent to the operated area and postoperative images will be analyzed through photographs. The photographs will be exported to image software (Adobe Photoshop, München, Germany), using two areas, the wound and one adjacent. The comparison (ΔE) of the areas will be made using brightness parameters (L), red-green chroma scale and blue-yellow chroma scale (b) according to the following equation (Knösel et al., 2009): ΔE =[(L.wound - L.adjacent)2 + (a.wound - a.adjacent)2 + (b.wound - b.adjacent)2] 1/ 2 | 7, 14, 21, 30, 45 and 90 postoperative days | 
| Epithelialization | 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). | baseline, 3, 7, 14 and 30 days after the procedure | 
| Early wound healing index | Any changes in wound healing will be evaluated, with five different degrees: Complete wound closure with absence of fibrin on the palate; Complete wound closure with the presence of a fibrin line on the palate; Complete wound closure with the presence of a fibrin clot on the palate; Incomplete wound closure with partial tissue necrosis on the palate; Incomplete wound closure with total tissue necrosis on the palate. | 7 and 14 days after surgery | 
| Tissue edema | It will be evaluated with the score: 1 = absent; 2 = slight; 3 = moderate; or 4 = severe (Sanz-Moliner et al. 2013). | 7 days after the surgical procedure | 
| Postoperative discomfort | Sensitivity will be measured by air spray for 5 s over the palatal region. After the air spray, patients will be required to use a 100 mm visual analog scale (VAS) to assess discomfort, where the extremes of the scale will be none and extreme to report the discomfort of this measure (Dias et al. 2015) | 7, 14, 21, 30, 45 and 90 days after the surgical procedure | 
| Number of analgesics | Patients will be given a postoperative diary in which they will note their pain intensity on a visual analog scale (VAS) 100 mm and the number of painkillers used (Tonetti et. al 2017). | until 14 days after surgery | 
| Immunologic Analysis - cytokines | The inflammatory exudate present in the palate open wound region will be collected. Samples will be obtained by placing strips of filter paper for 40 s (PerioPaper, Oraflow, New York, USA) in contact with the open wound. After the volume measurements (Periotron 8000, Oraflow, New York, USA), the strips will be placed in sterile tubes containing 300 ml of phosphate buffered saline (PBS) with 0.05% Tween-2 and stored immediately at -80 ° C. Strips contaminated with visible blood will be discarded. The following cytokines will be evaluated: Interleukin-1β (IL1β), Interleukin-4 (IL4), Interleukin-5 (IL5), Interleukin-6 (IL6), Interleukin-9 (IL9), Interleukin-10 (IL10), Interleukin-17A (IL17A), and tumor necrosis factor alpha (TNFα). | 3 and 7 days after surgery | 
| Immunologic Analysis - growth factors | The inflammatory exudate present in the palate open wound region will be collected. Samples will be obtained by placing strips of filter paper for 40 s (PerioPaper, Oraflow, New York, USA) in contact with the open wound. After the volume measurements (Periotron 8000, Oraflow, New York, USA), the strips will be placed in sterile tubes containing 300 ml of phosphate buffered saline (PBS) with 0.05% Tween-2 and stored immediately at -80 ° C. Strips contaminated with visible blood will be discarded. The following growth factors will be evaluated: Epidermal growth factor (EGF), Transforming growth factor beta (TGF-β), Platelet-derived growth factor (PDGF), Fibroblast growth factors (FGF), and Vascular endothelial growth factor (VEGF). | 3 and 7 days after surgery | 
| Immunologic Analysis - matrix metalloproteinases | The inflammatory exudate present in the palate open wound region will be collected. Samples will be obtained by placing strips of filter paper for 40 s (PerioPaper, Oraflow, New York, USA) in contact with the open wound. After the volume measurements (Periotron 8000, Oraflow, New York, USA), the strips will be placed in sterile tubes containing 300 ml of phosphate buffered saline (PBS) with 0.05% Tween-2 and stored immediately at -80 ° C. Strips contaminated with visible blood will be discarded. The following Matrix metalloproteinases will be evaluated: matrix metalloproteinase 9 (MMP-9), matrix metalloproteinase 2 (MMP-2), and Tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1/TIMP-2). | 3 and 7 days after surgery | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Periodontology, Wound healing, Silk fibroin, Randomized clinical trial, Drug Delivery System