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

Comparison of Palatal Wound Healing in Diabetic and Non-diabetic Patients: Controlled Clinical Trial and in Vitro Study

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.

Study Overview

• Status: Recruiting
• Conditions: Diabetes; Wound Heal; Palate; Wound
• Intervention / Treatment: Procedure: Palatal Wound

Detailed Description

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
• Enrollment (Estimated): 50
• Phase: Not Applicable

Contacts and Locations

Study Locations

• Brazil
  • SP
    • Sao Jose dos Campos, SP, Brazil, 12245-310
      • Recruiting
      • College of Dentistry - São José dos Campos, Sao Paulo State University
      • Contact: Mauro P Santamaria, PhD; Phone Number: 55 12 3947 9055; Email: mauro.santamaria@unesp.br
  • Sao Paulo
    • São José Dos Campos, Sao Paulo, Brazil, 12245000
      • Recruiting
      • Mauro Pedrine Santamaria and Ana Carolina Ferreira Bonafe
      • Contact: Ana Carolina F Bonafe, MS; Phone Number: +55 12 991711401; Email: ana.bonafe@unesp.br

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: Yes

Description

Inclusion Criteria:

• Patients with at least 18 years, systemically healthy, with good oral hygiene, assessed by plaque index and gingival index less than 25% (Ainamo, Bay, 1975);
• Patients with no morphological or pathological conditions on the palatine donor area;
• Patients who present indication for extraction and ridge preservation;
• The tooth included in the study, as well as, the adjacent teeth do not present loss of periodontal insertion;
• Patients who agreed to and sign the formal consent to participate in the study after receiving an explanation of risks and benefits from an individual who was not a member of the present study (Resolution no. 118 - May, 2012, and Ethics and Code of Professional Conduct in Dentistry - 118/12).
• Patients diagnosed with type 2 diabetes for more than 5 years who are using oral hypoglycemic agents or insulin supplementation, with HbA1c levels ranging from ≥ 6.1% to 8.5%.
• Non-diabetic patients with HbA1c levels below 6.1%.

Exclusion Criteria:

• Patients with systemic problems (cardiovascular, blood dyscrasias, immunodeficiency, and diabetes, among others) that will contraindicate the surgical procedure;
• Patients taking medications known to interfere with the wound healing process or that contraindicate the surgical procedure;
• Smokers patients;
• Pregnant or lactating patients;
• Patients who had had periodontal surgery on the study area;
• Patients who presents opportunistic oral lesions, mainly colonized the palate region;
• Use of dental prosthesis with palatal cover;
• Thin palatal mucosa (~2.0mm).

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Basic Science
• Allocation: Non-Randomized
• Interventional Model: Parallel Assignment
• Masking: Single

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Active Comparator: Diabetic (D); Palatal wound healing in diabetic patients	Procedure: Palatal Wound; 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.
Active Comparator: Control Group (CG); Palatal wound healing in normoglycemic patients	Procedure: Palatal Wound; 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.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Open 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)	baseline, 7, 14, 21 days after surgery

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Tissue Edema (TE)	Tissue edema will be evaluated with the score: 1 = absent; 2 = slight; 3 = moderate; or 4 = severe (Sanz-Moliner et, 2013).	7 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).	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).	14 days
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).	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).	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	7 and 14 days after surgery
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)	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.	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.	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.	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).	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).	baseline, 7 days

Collaborators and Investigators

• Sponsor: Universidade Estadual Paulista Júlio de Mesquita Filho

Publications and helpful links

General Publications

• Kamalathevan P, Ooi PS, Loo YL. Silk-Based Biomaterials in Cutaneous Wound Healing: A Systematic Review. Adv Skin Wound Care. 2018 Dec;31(12):565-573. doi: 10.1097/01.ASW.0000546233.35130.a9.
• Dias SB, Fonseca MV, Dos Santos NC, Mathias IF, Martinho FC, Junior MS, Jardini MA, Santamaria MP. Effect of GaAIAs low-level laser therapy on the healing of human palate mucosa after connective tissue graft harvesting: randomized clinical trial. Lasers Med Sci. 2015 Aug;30(6):1695-702. doi: 10.1007/s10103-014-1685-2. Epub 2014 Nov 6.
• Fickl S, Fischer KR, Jockel-Schneider Y, Stappert CF, Schlagenhauf U, Kebschull M. Early wound healing and patient morbidity after single-incision vs. trap-door graft harvesting from the palate--a clinical study. Clin Oral Investig. 2014 Dec;18(9):2213-9. doi: 10.1007/s00784-014-1204-7. Epub 2014 Feb 23.
• Black S, Phillips D, Hickey JW, Kennedy-Darling J, Venkataraaman VG, Samusik N, Goltsev Y, Schurch CM, Nolan GP. CODEX multiplexed tissue imaging with DNA-conjugated antibodies. Nat Protoc. 2021 Aug;16(8):3802-3835. doi: 10.1038/s41596-021-00556-8. Epub 2021 Jul 2.

Study record dates

Study Major Dates

• Study Start (Actual): August 1, 2023
• Primary Completion (Estimated): August 1, 2026
• Study Completion (Estimated): January 6, 2027

Study Registration Dates

• First Submitted: August 2, 2024
• First Submitted That Met QC Criteria: August 2, 2024
• First Posted (Actual): August 6, 2024

Study Record Updates

• Last Update Posted (Actual): August 6, 2024
• Last Update Submitted That Met QC Criteria: August 2, 2024
• Last Verified: January 1, 2024

More Information

Terms related to this study

• Keywords: Diabetes; Wound healing; Controlled Clinical Trial; Periodontology
• Additional Relevant MeSH Terms: Wounds and Injuries
• Other Study ID Numbers: UEPJMF 14

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES
• IPD Plan Description: Data will be available at the end of the study after a direct request
• IPD Sharing Supporting Information Type: STUDY_PROTOCOL; ICF

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No