Influence of graft thickness on tunnel technique procedures for root coverage: a pilot split-mouth randomized controlled trial

To compare the clinical outcomes of the tunnel technique (TUN) combined with either thin connective tissue graft (CTG) (TUN + thin CTG) (1 mm) or thick CTG (TUN + thick CTG) (2 mm) for the treatment of localized gingival recession (GR). Twelve patients, with 24 maxillary localized type RT1 GR were randomly assigned into two groups. Clinical, esthetic, and patient‐reported outcomes were evaluated at baseline and at 6-month follow-up. The TUN + thin CTG group showed a higher mean GR reduction (1.53 ± 0.66) (primary outcome variable) than the TUN + thick CTG group (1.44 ± 0.89), but no statistically significant difference was detected at the 6-month follow-up (p = 0.77). No statistically significant differences were found between TUN + thin CTG and TUN + thick CTG concerning complete root coverage (CRC) (58.3% versus 41.7%, respectively), gingival ticknness (GT) or Root-coverage Esthetic Score (RES) at 6 months. Regarding patient‐reported outcomes, both groups reported having experienced similar pain at 7 days (p > 0.05). No statistically significant differences were found between the groups in the analysis of the aesthetic score. Both treatments showed a similar reduction in GR. In addition, similar CRC, GT, RES, pain and self-reported esthetics were achieved in both groups. Application of thin CTG provided a similar result than thick CTG for the treatment of RT1 localized GR.


Introduction
Surgical procedures to treat of gingival recession (GR) are primarily aimed at covering exposed root surfaces and increasing the thickness and width of keratinized tissue to improve clinical parameters and patient-reported outcomes [1][2][3]. Among the most commonly used techniques to treat GR are coronally advanced flaps (CAF) [4], laterally moved flaps [5], double pedicle grafts [6] and tunneling techniques (TUN) [7], which may or may not be accompanied by subepithelial connective tissue grafts (SCTGs). Many of these surgical procedures achieve root coverage providing significant clinical attachment level gain and reduction in recession depth [3,8]. Although no single treatment has been considered superior to the others, the use of SCTG in root coverage procedures has shown to significantly increase the likelihood of reducing GR [9,10], indicating that SCTG is the "gold standard" surgical procedure in the treatment of GR [8,11].
The use of SCTG exhibits greater potential to achieve complete root coverage and offers the best long-term clinical results [12][13][14]. On the other hand, these grafts improve flap adaptation and stability to the root surface during early wound repair [15]. Moreover, they preserve the apical blood supply of the flap and have a genetic predisposition that ensures thickness and keratinization [16]. SCTGs can be obtained by connective tissue graft (CTG) harvesting procedures or by de-epithelialization of a free gingival graft [17,18], being the palate the most frequent donor area for obtaining grafts used for root coverage [17]. However, 1 3 the challenge in obtaining a CTG of the palate is the lack of thickness of the subepithelial palatal tissue, which can also vary from patient to patient [19].
Therefore, the use of thinner soft tissue grafts has gained popularity to avoid possible anatomic complications [17]. Likewise, it has been shown that CTG obtained from a deeper area is less dense and richer in fatty and glandular tissue, which makes it more prone to resorption [17,20]. A previous study [21] found that a CTG with a thickness < 2 mm showed a less painful postoperative period than grafts with a thickness ≥ 2 mm, and no significant difference in root coverage was detected. Similarly, another study [22] demonstrated that the use of even thinner grafts (1 mm) presented comparable clinical and morbidity results to those with the use of thick grafts (≥ 2 mm) at the 3-month follow-up. The use of thin grafts minimizes the risk of dehiscence of the covering flap and, consequently, exposure of the graft [21].
Nevertheless, the influence of graft thickness on the results of the TUN procedure has not yet been evaluated. Therefore, the aim of this pilot study was to compare the width of CTG (thin verus thick) for the treatment of GR using the TUN with a split-mouth design over a 6-month period.

Study design
This study was a split-mouth randomized controlled pilot trial and was performed according to the CONSORT statement (http:// www. conso rt-state ment. org/). The protocol of the study was approved by the São Leopoldo Mandic University Board (approval 30,247,720.1.0000.5374). All eligible patients signed an informed consent prior to inclusion in the study. This trial was conducted in accordance with the 2013 Declaration of Helsinki Statement on Human Experimentation.

Participants
The study participants were recruited from the clinics of the São Leopoldo Mandic University. Interested individuals were referred for initial screening by one of the investigators. Enrollment for the study opened in March 2019 and ended in July 2021. The Participants were selected according to the following inclusion and exclusion criteria:

Inclusion criteria
• Older than 18 years. • Full-mouth plaque score (FMPS) and full-mouth bleeding score (FMBS) ≤ 15% (probing of four sites per tooth of all teeth present except third molars).

Exclusion criteria
• Patients with history of periodontitis, periodontal or mucogingival surgery at study sites. • Systemic diseases, smoking, pregnancy, or nursing. • Presence of carious or non-carious cervical lesions.

Sample size
The sample size was calculated using α = 0.05 and a power (1-β) of 80%, and non-inferiority sample size calculation was performed. A non-inferiority margin of 0.6 mm was established for the GR detected during follow-up. This was based on a previous clinical study reporting a mean and standard deviation of 2.1 ± 0.2 and 2.5 ± 0.2 for GR after 3-months of follow-up in groups with 1-mm CTG. and with 2-mm CTG, respectively [22]. Based on this parameter, a total of 12 sites per group was deemed necessary. However, the number of sites per group was increased by 10% considering the possibility of dropouts. Thus, 13 patients were necessary to be involved in the present study.

Randomization /Allocation concealment/ and blinding
The study was performed using a split-mouth design in which one side used TUN + thin CTG, while the contralateral side, considered as the control side, used the TUN + thick CTG. An investigator with no clinical involvement in the trial prepared the table (M.F.). Allocation concealment was obtained by means of a sealed and coded opaque envelope with the specific treatment. The envelope with the site assignment was opened after both flap elevations. Randomization for treatment site assignment was performed by computer-generated randomization. Both the examiner and patients were blinded to treatment assignment throughout the study.

Pre-surgical phase
All participants underwent periodontal examination by a calibrated examiner (V.A.T.). Before surgery, all patients received oral hygiene instructions (non-traumatic tooth brushing) and professional tooth cleaning (supragingival scaling and polishing).

Surgical procedure
All surgical procedures were performed by a single experienced operator in mucogingival surgery (D.L.C.). GR was treated by means of a modified TUN technique [23] using either thin CTG (1 mm) (test side, Fig. 1A) or thick CTG (2 mm) harvested from the palate (control side, Fig. 2A).
After local anesthesia, root planing of the exposed root surfaces was performed with manual curettes (Gracey Curettes, Hu-Friedy). Subsequently, sulcular incisions were made in the recession areas of the teeth using a 15C blade without involving the interdental papillae. A mucoperiosteal flap was then raised beyond the mucogingival junction (M.G.J.) using tunnel elevators. The tunnel was extended laterally and apically and the collagen fibers and muscle attachments were released. To allow a passive and tension-free mobilization in coronal direction of the flap, each papilla was undermined using microsurgical elevators.
Grafts were harvested from the palate using a 1.0 mm (test side) and 2.0 mm (control side) double blade technique, as previously described by Harris [24]. The grafts were positioned at the level of the cementoenamel junction (CEJ) and stabilized with single interrupted sutures, avoiding a thick blood clot formation between the teeth and grafts. Finally, the flap was coronally fixed with sling sutures.

Postoperative care
For pain, patients were instructed to take one 600 mg ibuprofen tablet at the end of the procedure, another 6 h later, and thereafter only associated with pain. Additionally, they were instructed to use a chlorhexidine mouthwash (0.12%) twice a day for two weeks. Toothbrushing was withdrawn during the first 14 days and the use of ultrasoft bristle brushes was subsequently indicated. The reevaluation was 14 days after the intervention where the sutures were removed ( Fig. 1B and 2B).

Outcomes
The following outcomes were considered: 1-Primary outcomes: Mean reduction in GR at 6 months post-treatment.

Examiner calibration
Previously, an inter-examiner agreement study was performed for the height of the RG and then a calibrated examiner (V.A.T.) performed all periodontal measurements. A set of 10 gingival recessions were evaluated twice with a two-hour interval between them. The intraclass correlation coefficient obtained was 0.87 for RG height (95% confidence interval [CI]:0.85-0.92) and 0.81 (95% CI:0.65-0.88) for GT.

Periodontal clinical measurements
All periodontal parameters were recorded before surgery and at six months postoperatively. A single calibrated and blinded examiner (V.A.T.) recorded the following variables using a periodontal probe (PCP UNC 15, Hu-Friedy): 1. GR height: distance from the free gingival margin to the CEJ (in mm). 2. GT: measured at baseline at 1 mm apical to the free gingival margin using an injection needle, perpendicular to tissue surface, and a silicon stop on the gingival surface. After removing the needle, the distance between the needle tip and the silicon stop was assessed using a digital caliper [14].
Bleeding on probing (BOP) and the Plaque Index (PI) were recorded as the presence/absence on all tooth surfaces [25] using a manual periodontal probe (PCP UNC 15, Hu-Friedy).

Esthetic evaluation by clinician
The RES was used to evaluate esthetics after six months of follow-up, as previously reported by Cairo et al. 2009 [1]. The final RES value ranged from 0 to 10 (10 being the best esthetic score). Standardized photographs of the treated RG zones at baseline and at six months post-intervention were evaluated by two different examiners, masked, and calibrated (k > 0.85).

Questionnaires
The esthetic results were assessed by the patients with the following question 6 months after surgery-Which operated side do you like the most? Patient discomfort (postoperative pain) in the palatal and buccal areas was assessed using a Visual Analog Scale (VAS) (from 0 to 100) at 7 days after surgery. The VAS questionnaire used a horizontal line measuring 100 mm, and subjects were asked to mark a line to indicate their level of pain perception between the extreme limits of the line (worst on the left and least on the right) [14].

Statistical analysis
The statistical analyses were performed using IBM® SPSS 22.0 (SPSS Inc., Chicago, USA). Descriptive statistics were used for summarizing data using mean ± standard deviation and median (interquartile range) for quantitative variables. Frequencies and percentages were used for qualitative variables. The primary outcome variable was the mean root coverage. Secondary variables included the mean reduction in GR, CR., GT, the mean difference in GT, postoperative pain, esthetics and the RES.
The site of GR was defined as the unit of analysis. The Shapiro-Wilk test assessed data distribution for all outcomes, and an asymmetrical distribution was detected for all variables, except for a mean reduction in GR and mean difference in the GT. Therefore, comparisons between groups were performed by the Mann-Whitney or t-test for independent samples (continuous variables) or the chi-square test (categorial variables). A p-value < 0.05 was established for statistical significance. Figure 3 shows the flowchart of the study design. In total, 150 subjects were assessed for eligibility, One patient did not follow the post-operative protocols, starting the mechanical control of biofilm in the same day of the oral surgery, and it was excluded. Therefore, 12 participants with 24 localized GR were included in the study (TUN + thin CTG group n = 12 sites; TUN + thick CTG. group, n = 12 sites). All study participants completed the 6-month follow-up. No adverse events were reported, and no non-compliance was detected.

Clinical findings and esthetic evaluation
The TUN + thin CTG and TUN + thick CTG groups did not differ in terms GR height and GT at baseline (p > 0.05) ( Table 1). FMPS and FMBS were maintained at ≤ 20% (data not shown) throughout the study. All study sites showed no visible plaque or BOP during the study period. The mean GR at baseline was 1.93 ± 0.64 and 2.02 ± 0.89 mm for the TUN + thin CTG and TUN + thick CTG groups, respectively. GR and GT showed significant improvement at 6-month follow-up compared to baseline values in both groups (p < 0.05). At the 6-months followup, the GR values were 0.40 ± 0.61 and 0.58 ± 0.61 for the TUN + thin CTG and TUN + thick CTG groups, respectively (p > 0.05). Professional evaluation using the RES scale during the follow-up period showed not statistically difference between groups (p > 0.05). The frequency of teeth exhibiting CRC showed no statistically significant differences between the groups at 6 months (p > 0.05). CRC was achieved in 58.3% (7 teeth) in the TUN + thin CTG group and 41.7% (5 teeth) in the TUN + thick CTG group at 6 months after surgery ( Table 2). In addition, the mean GT at 6 months showed no statistically significant difference between groups (1.89 ± 0.39 and 1.95 ± 0.49) (p > 0.05) ( Table 1). Table 2 presents the mean changes

Patient-reported outcomes
The results of esthetic satisfaction among the patients were similar in both groups at 6 months with no significant differences between groups (p > 0.05) ( Table 3). Likewise, no significant differences were detected between groups with respect to pain (p > 0.05) ( Table 3).

Discussion
The primary aim of this study was to compare the tickness (thick versus thin) of CTG plus TUN for the treatment of localized RT1 GR over a 6-month follow-up using a split-mouth design. To our knowledge, this is the first clinical study designed to evaluate the influence of graft thickness on the results of the TUN procedure with a split-mouth design. Overall, there were no significant difference between groups in all the outcomes assessed. CTG, in conjunction with CAF, is considered the gold standard treatment for localized or multiple GRs [26,27]. However, palatal graft harvesting has been associated with a high level of pain [28]. Efforts have been made to decrease the morbidity of the palatal wound by developing different extraction techniques, as well as reducing the size and thickness of the graft [29,30]. Previous studies have evaluated the impact of the graft thickness on surgical and patient-related outcomes performing CAF [21,22,31].
The literature on whether graft thickness could influence the root coverage procedure so far is controversial, in the present study, a split-mouth design was used to eliminate the effects of host response [22]. Previous studies showed no difference in reduced GR, CRC, or increased keratinized tissue height on comparing different CTG thicknesses (≤ 2 mm and ≥ 2 mm) when performing CAF technique [21]. In the same line, Moissa et al. (2019) found no difference clinical and morbidity with grafts of 1 mm versus 2 mm using the modified Ratzke envelope flap [22]. Our study employed another technique (TUN technique). To our knowledge, this is the first clinical study using grafts of 1 mm and 2 mm with this technique to treatment of localized GR. The TUN technique is another widely used approach to treat GRs [7,32], demonstrating an average root coverage and similar esthetic results in comparison to CAF [28,33,34]. One systematic review with meta-analysis evaluated the efficacy of TUN technique in the treatment of localized and multiple GRs showed that the mean of root coverage of TUN for localized GRs was 82.75 ± 19.7%, while the mean of root coverage of TUN for multiple GRs was 87.87 ± 16.45%. The authors concluded that TUN technique is efficacy to treat localized and multiple GRs [35]. In our study, no differences were found between the two groups concerning a reduction in GR, CRC% at 6 months (58.3% versus 41.7%, respectively) or the frequency of teeth exhibiting CRC. These results are comparable to those reported in a recent systematic review that investigated the predictability of the TUN technique [35].
Previous studies have reported that a thicker CTG may result in increased postoperative donor site morbidity. Zucchelli et al. [21] found that a thinner CTG (< 2 mm) showed less postoperative pain than a CTG ≥ 2 mm. However, another study with a 3-month follow-up showed that the use of very thin grafts (1 mm) presented comparable clinical and morbidity results to those with the use of thick grafts (2 mm) [22]. This difference could be explained by the harvesting techniques of CTG. In the study of Zuchelli et al. [21] the CTGs were obtained from extraoral de-epithelialization, while another study [22] used the parallel blade technique described by Harris [24]. In our study, we used the same technique to harvest CTG and found no significant difference in terms of postoperative pain.
This study also found no difference in GT gain in concordance with another study that evaluated thick (2 mm) versus thin (1 mm) CTG for the treatment of GR [22]. In the present study, the baseline GT of 1.0 ± 0.1 mm in the 1 mm group was increased by 1.0 ± 0.1 to 1.9 ± 0.1 mm while in the 2 mm group, GT was increased by 1.2 ± 0.1 mm, from 1.0 ± 0.1 to 2.2 ± 0.1 mm (P < 0.05) [22].
Likewise, in the present study there were also no significant differences when comparing esthetic satisfaction of the patients in both groups. This result is different from that obtained by Zucchelli et al., Which may be due to the fact that color matching and keloide formation is directly related to graft exposure. In the present study, no patient had gingival graft exposure during the healing phase. However, the study of Zuchelli presented early (2 weeks) shrinkage of the covering flap with graft exposure in 8 patients with CTG (> 2 mm) and 2 patients with CTG (< 2 mm) (p < 0.05).
There are certain limitations to the present study. Our sample size was relatively small. Power calculation was performed, but only for the primary outcome (root coverage). Another limitation could be the exact thickness of the grafts. In this study, we used doble blade technique, as previously described by Harris to harvested the gingival grafts [24], but we do not measure the exact thickness of the graft at the time of surgery. Conversely, a highly experienced operator performed the harvesting of all grafts. This is aligned with the literature, which compared the impact of connective tissue graft thickness in root coverage procedure used the same technique to harvested the grafts [22].
The results of the present study showed no significant differences between the two treatment groups in terms of root coverage and patient-related outcomes, but a superior trend was observed with the 1 mm thick grafts. Although our findings provide new insight for performing future clinical trials, the limitations of this pilot study do not allow for broad conclusions and the results must be taken with caution. Comparative studies with a larger sample size are awarranted for definitive conclusions. It would also be beneficial to compare the results over 12 months of follow-up.

Conclusions
The present study is the first to compare thin versus thick CTG thickness with the TUN technique for the treatment of single GR using a split-mouth design.Within the limits of this pilot study, it can be concluded that in patients with type RT1 GR, CTG thickness in conjunction with a tunneling procedure is not a significant factor in the parameters measured at 6 months post-treatment.
Authors' contributions All the authors have made substantial contributions to the conception and design of the study. VAT was involved in data collection. MF, DC and FWMGM were involved in data interpretation. JMM, AVB, VAT, MAMM and DC were involved in drafting the manuscript. All the authors critically revised and approved the final version of the manuscript to be published.

Declarations
Ethical approval The study was conducted in compliance with the principles outlined in the experimentation involving human subjects in the Declaration of Helsinki of the World Medical Association (2013). The study protocol was reviewed and approved by the Research Ethics