Evaluation of the Effect of Smoking on Palatal Donor Site Tissue Profile in the Success of Connective Tissue Grafts
Evaluation of the Effect of Smoking on the Palatal Donor Site Tissue Profile in the Success of Connective Tissue Grafts
This single-center prospective clinical study investigates how smoking affects palatal donor site tissue biology and, in turn, the success of connective tissue grafts used in root coverage surgery. A total of 24 systemically healthy patients with Cairo Type 1 (recession type 1 [RT1]) gingival recession will be enrolled: 12 smokers and 12 non-smokers. All participants will receive a coronally advanced flap combined with a subepithelial connective tissue graft (CAF + CTG) harvested from the palatal donor site.
Both donor and recipient sites will be assessed at baseline and postoperatively (2 weeks, 1 month, and 3 months) using ultra-high frequency ultrasonography (UHFUS) to measure tissue thickness, echogenicity, structural integrity, and vascularization. Residual connective tissue fragments obtained during routine graft preparation will undergo histological, histochemical, and immunohistochemical analysis (including COL1A1, COL1A2, COL3A1, α-SMA, CD31, Factor XIIIa, and IL-33). Smoking status will be biochemically confirmed via salivary cotinine enzyme-linked immunosorbent assay (ELISA). Clinical outcomes (recession depth, clinical attachment level, keratinized tissue height, percentage of root coverage) and patient-reported outcome measures (PROMs) will be compared between groups.
Primary Hypothesis: Smoking negatively affects palatal donor site biology, impairing the clinical and ultrasonographic healing outcomes of CAF + CTG procedures.
Study Overview
Status
Intervention / Treatment
Detailed Description
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.
Study Type
Enrollment (Estimated)
Phase
- Not Applicable
Contacts and Locations
Study Contact
- Name: Sivge Kurgan, PhD
- Phone Number: +905325495235
- Email: sivgeakgun@gmail.com
Study Contact Backup
- Name: Husna OKTOPCU KARAKOYUN, DDS
- Phone Number: +905397835782
- Email: husnaansuh34@outlook.com
Study Locations
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Yenimahalle
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Ankara, Yenimahalle, Turkey (Türkiye), 06560
- Ankara University Faculty of Dentistry
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Contact:
- Husna OKTOPCU KARAKOYUN, DDS
- Phone Number: +905397835782
- Email: husnaansuh34@outlook.com
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Contact:
- Elif Unsal, PhD
- Phone Number: +905337627542
- Email: unsal.e@gmail.com
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Participation Criteria
Eligibility Criteria
Ages Eligible for Study
- Adult
- Older Adult
Accepts Healthy Volunteers
Description
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).
Study Plan
How is the study designed?
Design Details
- Primary Purpose: Treatment
- Allocation: Non-Randomized
- Interventional Model: Parallel Assignment
- Masking: Double
Arms and Interventions
Participant Group / Arm |
Intervention / Treatment |
|---|---|
|
Active Comparator: Group 1 - Non-smokers
Systemically and periodontally healthy non-smoking patients requiring root coverage treatment due to esthetic concerns or root hypersensitivity, treated with a coronally advanced flap combined with a subepithelial connective tissue graft (CAF + CTG) harvested from the palatal donor site (n = 12).
|
All defects are treated by a single experienced periodontist using microsurgical All defects are treated by a single experienced periodontist following the technique of Cairo et al. (2012).
After local anesthesia, root surfaces are debrided, conditioned with 24% EDTA for 2 minutes, and rinsed.
Vertical releasing and intrasulcular incisions are made; a full-thickness flap is raised to the mucogingival junction, then split-thickness dissection allows coronal advancement.
A subepithelial connective tissue graft (0.5-1 mm) is harvested from the palate, adapted to the root surface, and secured with sutures, and the flap is positioned 1-2 mm coronal to the cemento-enamel junction.
|
|
Experimental: Group 2 - Smokers
Systemically and periodontally healthy smoking patients requiring root coverage treatment due to esthetic concerns or root hypersensitivity, treated with the same coronally advanced flap combined with a subepithelial connective tissue graft (CAF + CTG) harvested from the palatal donor site (n = 12).
Participants are classified as smokers if they report smoking ≥20 cigarettes per day (one pack per day) for at least 5 years, confirmed biochemically by salivary cotinine analysis.
|
All defects are treated by a single experienced periodontist using microsurgical All defects are treated by a single experienced periodontist following the technique of Cairo et al. (2012).
After local anesthesia, root surfaces are debrided, conditioned with 24% EDTA for 2 minutes, and rinsed.
Vertical releasing and intrasulcular incisions are made; a full-thickness flap is raised to the mucogingival junction, then split-thickness dissection allows coronal advancement.
A subepithelial connective tissue graft (0.5-1 mm) is harvested from the palate, adapted to the root surface, and secured with sutures, and the flap is positioned 1-2 mm coronal to the cemento-enamel junction.
|
What is the study measuring?
Primary Outcome Measures
Outcome Measure |
Measure Description |
Time Frame |
|---|---|---|
|
Percentage of Root Coverage
Time Frame: Time Frame: Baseline to 3 months postoperatively
|
The percentage of root coverage achieved at the treated site, calculated as [(preoperative recession depth - postoperative recession depth) / preoperative recession depth] × 100.
Recession depth is measured with a calibrated UNC-15 periodontal probe as the distance from the cemento-enamel junction to the gingival margin.
This is the principal clinical measure of treatment success and allows direct comparison of graft effectiveness between smokers and non-smokers.
|
Time Frame: Baseline to 3 months postoperatively
|
Secondary Outcome Measures
Outcome Measure |
Measure Description |
Time Frame |
|---|---|---|
|
Ultrasonographic Soft Tissue Thickness at the Donor and Recipient Sites
Time Frame: Baseline, 2 weeks, 1 month, and 3 months postoperatively
|
Quantitative change in soft tissue thickness measured non-invasively with ultra-high frequency ultrasonography (UHFUS), including epithelial thickness, lamina propria thickness, and total soft tissue thickness at the palatal donor site, and marginal mucosal, flap, and graft thickness at the recipient site.
Measurements are standardized using custom 3D-printed surgical stents.
This outcome captures tissue healing dynamics and volumetric stability over time.
|
Baseline, 2 weeks, 1 month, and 3 months postoperatively
|
|
Donor Site Tissue Vascularization
Time Frame: Baseline, 2 weeks, 1 month, and 3 months postoperatively
|
Quantitative assessment of blood flow and microvascularization at the palatal donor site using Color Doppler / B-Flow ultrasonography, analyzed with integrated Color Quantification software within a standardized region of interest.
Vascularization is a key indicator of donor site healing capacity and is expected to be reduced in smokers.
|
Baseline, 2 weeks, 1 month, and 3 months postoperatively
|
|
Donor Site Histological and Immunohistochemical Tissue Profile
Time Frame: At the time of surgery (single time point)
|
Characterization of the palatal donor tissue obtained from residual connective tissue fragments trimmed during routine graft preparation.
This includes collagen organization and Type I/Type III collagen ratio (Picrosirius Red under polarized light), extracellular matrix organization (Masson's Trichrome), microvascular density (CD31), fibroblast/myofibroblast activity (α-SMA), matrix stabilization (Factor XIIIa), collagen expression (COL1A1, COL1A2, COL3A1), and inflammatory signaling (IL-33).
This outcome defines how donor site biology differs between smokers and non-smokers.
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At the time of surgery (single time point)
|
Collaborators and Investigators
Sponsor
Publications and helpful links
Study record dates
Study Major Dates
Study Start (Estimated)
Primary Completion (Estimated)
Study Completion (Estimated)
Study Registration Dates
First Submitted
First Submitted That Met QC Criteria
First Posted (Actual)
Study Record Updates
Last Update Posted (Actual)
Last Update Submitted That Met QC Criteria
Last Verified
More Information
Terms related to this study
Other Study ID Numbers
- İ01-36-26
- TDH-2026-4827 (Other Grant/Funding Number: Ankara University Scientific Research Project Unit)
Plan for Individual participant data (IPD)
Plan to Share Individual Participant Data (IPD)?
Drug and device information, study documents
Studies a U.S. FDA-regulated drug product
Studies a U.S. FDA-regulated device product
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