Clinical and Histological Evaluation of Subepithelial Connective Tissue Graft Maturation at Different Healing Timepoints, With and Without Topical Application of a Polynucleotide- and Hyaluronic Acid-Based Gel

Clinical and Histological Evaluation of Subepithelial Connective Tissue Graft Maturation at Different Healing Timepoints, With and Without Topical Application of a Polynucleotide- and Hyaluronic Acid-Based Gel: A Randomized, Blinded, Controlled Clinical Trial With a Split-Mouth Design

This study evaluates whether a 9-week healing interval is sufficient for subepithelial connective tissue graft (SCTG) maturation after palatal harvesting, and whether topical application of a polynucleotide and hyaluronic acid gel (Regenfast®) improves tissue quality. This is a randomized, blinded, controlled clinical trial with a split-mouth design involving 15 participants. Each participant undergoes bilateral SCTG harvesting from the palate in two surgical sessions 9 weeks apart. In the first session, one randomized side receives a single topical application of Regenfast® after graft removal; the contralateral side serves as control. Standardized tissue fragments (3×3 mm, 1.5 mm thickness) are collected from each graft for histological (HE, Picrosirius Red) and immunohistochemical analyses. Comparisons are made between grafts collected at baseline and after 9 weeks, and between sides treated with or without the gel.

Surgical procedures and sample collections will be performed at Instituto Kahn Odontologia (Rio de Janeiro, Brazil). Laboratory analyses will be conducted at the Histology Laboratory of the School of Dentistry of Ribeirao Preto, University of Sao Paulo (FORP-USP).

SCTG removal will be performed using the double incision technique (Harris, 2003) with a standardized 1.5 mm guide scalpel, under local anesthesia (2% lidocaine, 1:100,000 epinephrine). From each graft, a standardized fragment (3×3 mm, 1.5 mm thickness) will be collected from the distal portion and immediately fixed in 10% buffered formalin for 24-48 hours, then transferred to 70% alcohol.

Laboratory analyses will include: (1) Hematoxylin-eosin (HE) for general morphological assessment; (2) Picrosirius Red under polarized light for quantification of collagen density and proportion of type I and type III collagen fibers; (3) Immunohistochemistry for vascularization assessment, measuring microvessel density (n/mm²) and vascular area (%).

Statistical analysis will use paired Student's t-test or Wilcoxon test for paired samples, depending on data distribution (Shapiro-Wilk test), with significance level of 5% (p<0.05).

Inclusion Criteria:

• Age between 18 and 60 years
• Good general systemic health
• Presence of multiple bilateral gingival recessions with indication for root coverage

Exclusion Criteria:

• Smoking of any intensity
• Pregnancy or lactation
• Oral lesions or alterations in the palatal mucosa
• Previous history of graft harvesting from the donor area
• Continuous use of medications that interfere with the inflammatory response, such as corticosteroids or immunosuppressants
• Autoimmune diseases, coagulation disorders, or other systemic conditions affecting wound healing