Evaluation of the Effect of Smoking on Palatal Donor Site Tissue Profile in the Success of Connective Tissue Grafts

Gingival recession, particularly in the esthetic zone, exposes root surfaces and leads to root hypersensitivity, esthetic impairment, and reduced periodontal stability. The coronally advanced flap combined with a subepithelial connective tissue graft (CAF + CTG) is considered the gold standard for root coverage, offering high rates of long-term root coverage and soft tissue volume gain. Smoking is a well-recognized patient-level factor that adversely affects wound healing through its impact on vascularization, fibroblast activity, and collagen metabolism, potentially compromising both the biological integrity of the donor graft and its integration at the recipient site. However, the relationship between these clinical differences and the underlying biology of the palatal donor site remains poorly understood. This single-center, prospective, controlled clinical study aims to examine the relationship between palatal donor site biology and connective tissue graft success using an integrated clinical, ultrasonographic, histological, and molecular approach, with the central hypothesis that smoking negatively affects donor site biology and thereby impairs the clinical and ultrasonographic healing outcomes of CAF + CTG procedures. A total of 24 systemically healthy patients presenting with Cairo Type 1 (recession type 1 [RT1] / Miller Class I-II) gingival recession and requiring root coverage for esthetic concerns or root hypersensitivity will be enrolled and allocated into two groups of 12: smokers (≥20 cigarettes/day for at least 5 years, biochemically confirmed by salivary cotinine) and non-smokers. The sample size was determined via G*Power (α = 0.05, effect size = 0.4, power = 0.95), yielding a minimum of 10 subjects per group, increased to 12 to account for potential dropout, and all patients will be followed for 3 months under standardized imaging and biological sampling protocols.

Every participant will receive the same standardized surgical protocol: a coronally advanced flap with a subepithelial connective tissue graft harvested from the palatal donor site between the second premolar and second molar using an extraoral de-epithelialization approach, with root surfaces conditioned using 24% ethylenediaminetetraacetic acid (EDTA), following a previously described technique. Ultra-high frequency ultrasonography (LOGIQ P10 XDClear with an L8-18i-RS high-frequency linear probe) will be performed at both the palatal donor site and the recipient site at baseline and at 2 weeks, 1 month, and 3 months postoperatively, with custom three-dimensional (3D)-printed surgical stents standardizing probe positioning for reproducibility. The ultrasonographic evaluation includes Doppler and B-Flow color quantification of vascularization and blood flow, linear measurements of epithelial thickness, lamina propria thickness, and total soft tissue thickness at the donor site along with marginal mucosal, flap, and graft thickness at the recipient site, and echo intensity analysis of mean gray value, tissue homogeneity, and structural integrity performed with ImageJ.

Residual connective tissue fragments naturally trimmed during routine graft preparation, which require no additional surgical intervention or patient morbidity, will be collected for laboratory analysis. These samples will undergo morphological evaluation with hematoxylin-eosin, histochemical assessment of extracellular matrix organization with Masson's Trichrome and collagen typing with Picrosirius Red under polarized light to distinguish Type I from Type III collagen, and immunohistochemical evaluation of collagen type I alpha 1 (COL1A1), collagen type I alpha 2 (COL1A2), and collagen type III alpha 1 (COL3A1) for collagen matrix, alpha-smooth muscle actin (α-SMA) for fibroblast and myofibroblast activity, Factor XIIIa for extracellular matrix stabilization and graft integration, cluster of differentiation 31 (CD31) for microvascular density, and interleukin-33 (IL-33) for inflammatory signaling.

Blinded clinical measurements including recession depth, recession width, pocket depth, clinical attachment level, keratinized tissue height, bleeding on probing, plaque index, and percentage of root coverage will be recorded at baseline, 1 month, and 3 months. Patient-reported outcome measures will capture postoperative pain, donor site discomfort, functional recovery, esthetic satisfaction, and analgesic consumption, and salivary samples for cotinine enzyme-linked immunosorbent assay (ELISA) will be collected before surgery and at 1 month to biochemically verify smoking status. Through this multidimensional design, the study will characterize how palatal donor site biology shapes connective tissue graft success and will scientifically define the role of smoking in these processes, contributing to the development of personalized, biology-based treatment approaches in periodontal plastic surgery.

Inclusion criteria

• Aged 18 years or older.
• Systemically healthy, with no systemic disease that could directly affect status or wound healing.
• Presence of an isolated facial recession on non-molar maxillary teeth:

greater than 2 mm, less than 5 mm (up to a maximum of 5 mm).

• Recession classified as Cairo Type 1 (RT1 = Miller Class I and II), meaning gingival recession without interproximal attachment loss.
• No previous soft tissue surgery at the surgical site.
• Good oral hygiene (full-mouth bleeding on probing below 10%).
• No soft tissue graft previously harvested from the palatal donor site.
• Able to comply with all study-related procedures, including attendance at all follow-up visits.

Exclusion criteria

• Untreated periodontitis or peri-implantitis.
• Any systemic disease that could potentially impair wound healing, such as diabetes or connective tissue disorders.
• Pregnancy or breastfeeding.
• Previous or concurrent use of medications affecting mucosal healing (e.g., steroids or high-dose anti-inflammatory drugs).
• Coagulation or bleeding disorders.
• Cleft palate.
• Active orthodontic treatment with fixed or removable appliances.
• A history of malignancy, radiotherapy, or chemotherapy for malignancy within the past 5 years.
• Antibiotic therapy within the past 6 months.
• Previous connective tissue graft harvested from the palatal donor site.
• Any other surgical procedure at the palatal donor or recipient site (e.g., apical resection or crown lengthening).