Clinical outcomes of root coverage using porcine-derived collagen matrix with modified coronally advanced tunnel technique (MCAT) in multiple gingival recessions in smokers — a randomized controlled clinical trial

Root coverage (RC) is an integral part in management of gingival recessions. Unlike isolated recessions, treating multiple recessions is the most challenging scenario especially in smokers who have reported reduced RC in terms of quantity and stability. Modified coronally advanced tunnel (MCAT) technique due to its improved vascularity along with use of porcine-derived collagen matrix (PDCM) (Mucograft™) with its bilayered dense collagen matrix may be a predictable alternative to conventional gold standard subepithelial connective tissue graft (SCTG). The purpose of this study was to compare the results of RC achieved using PDCM and SCTG with the MCAT approach in the treatment of multiple gingival recessions in smokers. This is a prospective randomized controlled clinical study registered under clinical trial registry (CTRI no. CTRI/2020/03/024238) including 28 patients with 64 recession defects. Smokers (≥ 10 cigarettes/day for ≥ 5 years) fulfilling the inclusion criteria were randomized into the SCTG and PDCM groups which were treated with SCTG using MCAT and PDCM using MCAT technique, respectively. Recession depth (RD), recession width (RW), width of keratinized tissue (WKT), thickness of keratinized gingiva (TKG), and %RC were assessed at baseline, 3, and 6 months. Complete RC (CRC) at 3 and 6 months, RC aesthetic scores (RCES) at 6 months. Both groups showed significant improvement in all clinical parameters. Intergroup comparison of root coverage parameters in smokers showed better performance of the PDCM group in terms of RD, %RC, TKG, WKT, CRC (45.06%), and RCES (p < 0.001) compared to the SCTG group. Time taken for surgical procedure and visual analog pain scores were significantly lesser in the PDCM group. PDCM showed good predictable results in terms of all RC parameters compared to SCTG and thus may be used as a predictable alternative to SCTG for RC in smokers. PDCM can be used as a potential alternative to gold standard subepithelial connective tissue graft in smokers with better patient compliance.


Introduction
The loss of the marginal gingival tissues causes gingival recession, which is defined as the displacement of the gingival border apical to the cemento enamel junction (CEJ) with root surface exposure [1]. The exposed roots are unaesthetic, may cause dentinal hypersensitivity, are prone to root caries, cervical wear resulting difficulties in achieving proper plaque control, and therefore need to be covered by soft tissue wherever feasible.
The goal of periodontal plastic surgery is to achieve a stable and complete root coverage with a tissue margin attached at the CEJ, increase the dimensions of keratinized gingiva, such as thickness and width, and maintain a healthy gingival sulcus. Various surgical approaches have been evaluated in recent decades to accomplish root coverage for numerous adjacent gingival recessions with predictability and consistency. Subepithelial connective tissue graft (SCTG) is considered as a gold standard [2] as it provides the best outcomes for clinical practice because of its more significant percentages of mean and complete root coverage and considerable increase of keratinized tissue [3]. However, longer surgical time and increased patient morbidity in treating multiple adjacent gingival recessions using SCTG have become a challenge to both clinician and patient [4].
To overcome the drawbacks of patient morbidity during the harvest of SCTG, other alternatives such as allografts and guided tissue regeneration are available but have their own limitations. One such newly developed alternative is a porcine-derived collagen matrix (PDCM), i.e., Muco-graft™. Advantages of PDCM are early vascularization and good soft tissue ingrowth [5,6], excellent wound healing [7], and easy handling.
The minimally invasive mucogingival surgical approach aims to gain surgical access that minimizes tissue trauma and better stabilization of blood clots with reduced surgical morbidity. One such procedure is the modified coronally advanced tunnel technique (MCAT) proposed by Azzi and Etienne in 1998 [8], which modified Allen's design (1994). MCAT envisages providing better results and maximum root coverage and aesthetics which employs a minimally invasive approach, which has shown greater predictability due to increased vascularization by preventing vertical incisions and papillary incisions and complete graft coverage due to coronal advancement [9].
Operative technique, defect features, and patient characteristics are all factors that influence the outcome of a root covering surgery [10]. Cigarette smoking is one of the patient-related factors that has been shown to have a poor impact on the healing result following periodontal surgical treatments, such as root grafting. Miller recognized the potential negative impact of smoking on the outcome of root coverage procedures. Tobacco smoking is a known risk factor that impacts the oral environment and ecology, gingival vascularization, immunological and inflammatory responses, and periodontal connective tissue healing capacity [11]. Smokers are two to eight times most likely than non-smokers to develop periodontal disease [12]. Though there is a vast literature showing root coverage using various techniques and additives, very little is available regarding their predictability in smokers. To overcome the limitations of SCTG, PDCM can be considered as one of the treatment alternatives. Hence, this prospective randomized controlled clinical trial is intended to evaluate the root coverage obtained with PDCM and compare it with SCTG in smokers using the MCAT technique.

Study design
This was a prospective randomized controlled, single blinded comparative study conducted to evaluate the outcome of root coverage using PDCM and SCTG with MCAT technique in the treatment of multiple gingival recessions in smokers.

Patient selection
The patients who satisfied all the inclusion and exclusion criteria were enrolled in the study. CONSORT guidelines were followed (Fig. 1).
Patients in age range of 18 to 60 years, current smokers who smoke on an average of ≥ 10 cigarettes per day for at least 5 years, individuals who were systemically healthy and had no contraindications to periodontal surgery, ≥ 2 adjacent Miller's Class I and Class II recessions, and teeth with no clinical signs of active periodontal disease were included. Any systemic condition that interferes with healing, pregnant or lactating women, patients treated with any medication known to affect the gingival condition, and patients who are not willing to comply with smoking cessation protocol, malpositioned teeth, active (or) untreated gingival, and periodontal disease were excluded.

Randomization, allocation concealment, and blinding
A total of 28 patients were randomly assigned to either of the study groups with 31 and 33 recession defect sites in the PDCM and SCTG groups, respectively. Allocation concealment was done by using computer generated random numbering which is sealed in opaque envelopes. The envelopes were only opened after tunnel preparation and the assignment was informed to the operator. The pre-and postoperative analysis was performed by the same examiner who was blinded about the study material used and was maintained throughout the study period. Before the start of the study, the examiner was calibrated by evaluating the clinical measurements in five patients with gingival recession at three intervals 1 day apart. A reproducibility of < 1 mm for 95% of recordings was considered acceptable. Intraexaminer correlation coefficient (k-0.931) showed good reliability.

Clinical parameters
Baseline clinical parameters were recorded at least after 4 weeks of completion of nonsurgical periodontal therapy. All the clinical parameters were performed by the same examiner who was blinded to the study groups throughout the study period. Clinical parameters, i.e., full mouth plaque score (FMPS) (Silness and Loe 1964), gingival index (GI) (Loe and Silness 1963), full mouth bleeding score (FMBS), probing pocket depth (PPD), clinical attachment level (CAL), recession depth (RD), recession width (RW), recession surface area (RSA), and width of keratinized tissue (WKT), were recorded using UNC 15 probe at baseline, 3, and 6 months postoperatively. Thickness of keratinized gingiva (TKG) was measured using endodontic spreaders (Easy Smile International Corp™, USA) with silicone stoppers at a distance of 3 mm from gingival margin placed perpendicular to the bone/root surface and evaluated using Digital Vernier Calipers (Safeseed Electronic Digital Vernier Caliper, India) with an accuracy of 0.01 mm. Percentage of root coverage (%RC), complete root coverage (CRC), and root coverage aesthetic scores (RCES) were assessed at 3 and 6 months postoperatively. VAS pain/discomfort, VAS dentinal hypersensitivity, and time taken for the procedure were recorded immediately following surgery.
Each patient was considered as a single experimental area consisting of multiple recessions. When patients presented bilateral recessions, the experimental site was selected by randomization and subsequently MCAT with SCTG was performed on one site and MCAT with PDCM was performed on the other site.

Surgical procedure
Local infiltration with 2% lignocaine with adrenaline at a concentration of 1:2,00,000 was used during the surgical procedure. In cases where the graft was harvested, a greater palatine nerve block was used to anesthetize the donor palatal location. In the SCTG group, intrasulcular incisions were given and mucoperiosteal flaps were raised using sharp tunnel elevators, Tunnelling Knife I/II. A full thickness mucoperiosteal dissection is extended beyond the Fig. 1 Consort flowchart of the study mucogingival junction (MGJ) and under each papilla. A split thickness flap was elevated apical to the mucogingival junction so that the flap can be moved in a coronal direction without tension. Muscle fibers and any remaining collagen bundles on the inner aspect of the flap alveolar mucosa are cut using Gracey curettes with extreme care in order to avoid perforation of the flap and to obtain a passive coronal positioning of the flap and the papilla. Tunnelling Knife I is used for initial tunnel preparation and Tunnelling Knife II is used to remove the attachment till the mucogingival junction. The tunnel was extended one or two teeth beyond the area of interest to mobilize gingival margins (Fig. 2a). A sterile saline solution was used to irrigate the region. Because the trapdoor technique is a more common and predictable method for harvesting an adequate amount of connective tissue, it was used to create a second surgical site on the palate, and incisions were made between the canine and maxillary first molar regions. The harvesting of the graft distal to first molar was avoided to prevent complications caused due to damage to the greater palatine vessels. A connective tissue graft of sufficient size and thickness of 2 mm was obtained. The donor area was closed with polyglactin sutures (Fig. 2b). The graft was drawn into a tunnel (Fig. 2c) and graft stabilization was done using horizontal stabilizing sutures and mattress followed by coronal advancement of the flap using vertical mattress suture placed into interdental gingiva by using polyglactin (Fig. 2d). The suture is tied so that the knot is positioned at the midpoint of each tooth. Flowable composite resin was used to seal the suture knots, preventing apical relapse of the gingival margin during the early stages of recovery. Non-eugenol periodontal dressings were used to protect the operated site because they are less irritating and provide a tightly adapted adhesive barrier to saliva and other irritants.
In the PDCM group, MCAT procedure was done (Fig. 3a). PDCM was trimmed to defect size and the graft was positioned in a way that the compact structure of PDCM faces outwards and the spongy structure towards the bone and/or periosteum (Fig. 3b). The graft was drawn into tunnel (Fig. 3c) and graft stabilization was done using horizontal stabilizing sutures and mattress followed by coronal advancement of flap using vertical mattress suture suspended on the midbuccal aspect using polyglactin sutures and knots were secured by placing composite resin over the knot. Only midbuccal suspensory sutures were preferred as recession defects do not require displacement of the interdental papilla. A non-eugenol periodontal dressing was used to protect the surgical site (Fig. 3d).

Post-surgical care
Smoking cessation was recommended to the patients. If not, at least advised to quit 2 weeks before and after surgery. Analgesics were given to all of the participants after surgery (combination of ibuprofen 40 mg, paracetamol 325 mg thrice daily for 3 days). Following the procedure, the patient was given the usual postoperative instructions. For 4 weeks, the patients were told not to brush their teeth at the surgery site and to rinse their mouths with 0.2% chlorhexidine gluconate mouthwash once a day for 6 weeks. After 2 weeks, sutures were removed and in the operated area, the patients were recommended to begin gentle brushing with soft bristled toothbrush.

Statistical analysis
With a statistical power of 0.8, an alpha of 0.05, and an effect size of 0.22 and 0.59, the required sample size was calculated to be 56 defect/tooth sites using G*Power software (version 3.1.9.4). All the data collected at baseline, 3 months, and 6 months was subjected to statistical analysis using unpaired t test for intergroup comparison and ANOVA for intragroup comparison. Descriptive statistics were expressed as mean ± standard deviation. p-value < 0.05 was considered statistically significant for all the analysis. The statistical software, namely IBM SPSS 20.0 (IBM SPSS, IBM Corp., Armonk, NY, USA), were used for analysis of the data and Microsoft Word and Excel have been used to generate tables.

Results
A total of 64 recession defects in current smokers (≥ 10 cigarettes daily) with either Class I or Class II recessions were included in the study. All the patients were followed up for the entire study period. No cases of post-surgical bleeding or paresthesia or surgical complications were encountered. Satisfactory healing was noticed in both the recipient area and donor area.
The mean age in the SCTG group was 37.39 ± 6.403 and in the PDCM group was 37.81 ± 6.911 years. Mean number of cigarettes in the SCTG group was 6.72 ± 2.63 and in the PDCM group was 6.87 ± 2.77 (Table 1). Mean pack years in the SCTG group was 2.98 ± 1.36 and in the PDCM group was 2.92 ± 1.38 (Table 1). The PDCM group included 25.8% incisors, 16.1% canines, 45.2% premolar, and 12.9% molars. In the SCTG group, incisors were 6.1%, premolars were 75.8%, and molars were 18.2% (Table 1).
On intragroup comparison, the mean plaque index (PI), GI, and sulcus bleeding index (SBI) scores reduced in both the groups significantly from baseline to re-evaluation. Intergroup comparison showed no significant difference in PI and GI scores at any time interval. However, statistically significant difference in SBI scores was detected at 3 months (p = 0.035*) and 6 months (p = 0.001) with the PDCM group showing lesser bleeding tendency (Table 2).
On intragroup comparison in both groups, significant improvements were observed in the mean periodontal probing depth (PPD) and CAL values (p < 0.001*) during the study period. Intergroup comparison showed no statistical significant difference in PPD at baseline (p = 0.575), 3 months (p = 0.686), and 6 months (p = 0.56) ( Table 3).  However, a statistical significant difference was observed in mean CAL at 3 months (p = 0.007) and 6 months (p = 0.018) with test group showing greater improvement in CAL (Table 3). Both the PDCM and SCTG groups showed significant improvements in RD, RW, RSA, WKT, and TKG (p < 0.001) ( Table 4) (Fig. 4).
The PDCM and SCTG groups showed a mean gain in RD of 2 mm and 1.52 mm at 6 months re-evaluation. Similarly, a greater reduction in RW of 1.9 mm was observed in the PDCM group as compared to the SCTG group showing 1.63 mm of RW reduction. At 6 months, a mean gain of 1.29 mm and 0.58 mm of WKT were observed in the PDCM and SCTG groups, respectively. Also, greater gain in TKG of 0.78 mm was observed in the PDCM group as compared to the SCTG group (Tables 3 and 4).
A significant difference in the mean %RC was observed at 6 months (p < 0.001) with the PDCM group showing greater %RC, i.e., 79.03 ± 20.16, as compared to the SCTG group with 56.81 ± 23.97%RC (Table 5).
At 3 months interval, 6 out of 33 recession defects in both the SCTG and PDCM groups achieved CRC. However, at 6 months, 3 recession defects in the SCTG group (9.1%) and 14 recession defects (45.16%) in the PDCM group achieved CRC with significantly better performance in the PDCM group (p < 0.001) ( Table 5). The mean RCES was 8.19 ± 1.70 in the PDCM group and 6.36 ± 1.81 in the SCTG group which was statistically significant (p < 0.001) ( Table 5).
The mean pain scores immediately after surgery were 6.81 ± 0.50 and 2.81 ± 0.54 in the SCTG and PDCM groups, respectively. On intergroup comparison, significant difference was noticed in the pain scores immediately and 1 week after surgery (p < 0.001) ( Table 6). No significant difference in the dentinal hypersensitivity scores was observed before surgery (p = 0.132), immediately after surgery (p = 0.12), 3 months (p = 0.18), and 6 months (p = 0.81) between the groups ( Table 6). The mean time for surgery was 26.23 ± 1.87 min in the PDCM group and 48.85 ± 1.57 min in the SCTG group with a significant difference (p < 0.001) ( Table 6).

Discussion
Several treatments of recession-type defects are based on periodontal plastic surgery principles. Over the years, various techniques have been tried to obtain root coverage. The anatomy of the defect site, such as the size of the recession defect, the presence or absence of keratinized tissue adjacent to the defect, the width and height of the interdental soft tissue, the depth of the vestibule, type of frenum, and the presence of aesthetic considerations, should all influence surgical technique selection [10].
In smokers, various physiological pathways and cellular functions may be altered/disturbed [13]. Tobacco toxins are abundant and may impede periodontal repair. Smoking also reduces periodontal blood flow and compromises graft vascularization and SCTG outcomes [14]. Miller reported a negative impact on root coverage in smokers [15]. Also, various clinical studies showed reduced root coverage in smokers when compared to non-smokers. In the view of above-mentioned drawbacks such as donor site-related problems, patient morbidity, and compromised vascularization in smokers, an alternative for SCTG is a porcine-derived collagen matrix. The bilayer structure of PDCM is composed of a spongy section (predominant) and a compact layer. The spongy portion is designed to promote cellular ingrowth, this promotes neovascularization and wound healing, resulting in enhanced root attachment and gingival thickness [16]. Cairo et al. in 2008 reported a 90% mean percent of root coverage with the use of additive biomaterials and 60% MRC without additivities in non-smokers. However, in smokers, destruction of the extracellular matrix is seen due to the release of proinflammatory cytokines and compromised connective tissue and healing. Therefore, the current study was aimed at evaluating the root coverage in smokers using PDCM. The gingival and plaque index scores decreased at a statistically significant level in both groups. It is possible that this is related to the regular maintenance of oral hygiene practices. These differences were in accordance with the previous study [9] where in the plaque and gingival index scores reduced significantly from baseline to 6 months and 1 year. Similarly, there was a reduction in bleeding scores in both groups from baseline to 6 months re-evaluation which shows reduced inflammation and complete healing of recipient sites during re-evaluation. However, in the intergroup comparison, the PDCM group had lower bleeding scores due to higher gains in WKT and TKG, which might have enabled better patient aided maintenance and reduce likelihood of gingival inflammation.
Significant improvements in periodontal probing depth were exhibited in both groups throughout the study periods which was in accordance to a study conducted by Vincent et al. [17] and Dragana et al. [18]. Also, significant clinical attachment level gain was observed from baseline to 6 months in both groups. The CAL gain in the present study was in agreement with a previous study conducted by Cardaropoli et al. [16].
Earlier study by Erley et al. [13] reported a greater final recession depth of 1.00 ± 0.85 mm in smokers treated with SCTG as compared to our study where root coverage with SCTG showed 1.03 ± 0.46 mm at 6 months. However, the better results in our study in the PDCM group, i.e., RD of 0.55 ± 0.50 mm, can be attributed to the availability of thick bilayered dense collagen which provides resistance to masticatory forces and allowing ingrowth of fibroblasts for regeneration. Reino et al. [14] in their clinical trial comparing use of PDCM with different surgical techniques reported a greater reduction in recession depth in extended flap technique (0.63 ± 0.44 mm) compared to coronally advanced flap (1.28 ± 0.54 mm). Thus, greater improvement in recession depth in our current study (0.55 ± 0.50 mm) could be attributed to the minimally invasive MCAT technique. However, in terms of recession width, both the groups showed significant improvement from baseline to 6 months which was in accordance with an earlier study [19]. On intergroup comparison, no statistical significant difference was seen during the study period which was similar to the study by Dragana et al. [18]. A significant improvement in keratinized tissue dimensions, i.e., WKT and TKG, were observed in both groups. However, at 6 months re-evaluation, the dimensions are significantly greater in the PDCM group compared to the SCTG group. These findings are contrary to previous studies by Pietruska et al. [20], Vincent-Bugnas et al. [17], and Cosgarea et al. [21] where they have obtained similar gain in keratinized tissue dimensions in both groups. The thickness of keratinized gingiva was improved in both groups which is contrary to the study by Roberto et al. [16] where they reported greater increase in TKG in the SCTG groups. The gain in the PDCM group could be explained by the collagen matrix acting as a 3D scaffold to build pure types I and III collagen which enables fibroblast cell ingrowth and repopulation and merges with the surrounding tissue and blood vessels. The three dimensional interconnected architecture of PDCM might have enabled host cell migration and microvessel formation and angiogenesis and also keratinization [22,23]. Schmitt et al. conducted a histological and immunological analysis and found that 90 days after PDCM implantation, there is an increase in the presence of keratinization tissue markers CKs 5/6, 13, and 14, showing that PDCM is effective in inducing keratinization [24].
The %RC obtained in our study at 3 months is similar to an earlier study done by Rakasevic et al. [18] where they have obtained a similar %RC in both the PDCM and SCTG groups. However, in the current study, 3 to 6 months of follow-up evaluation in the PDCM group showed a greater gain in the %RC compared to SCTG. This could be due to the creeping attachment phenomenon which is usually seen after root coverage procedures. Creeping attachment is a process usually seen with SCTG where the CTG usually shows a tendency towards a gradual increase in keratinized tissue dimensions as reported in earlier studies. However, it remains interesting that in the current study, the PDCM group showed a greater creeping attachment in contrary to the previous study by Aroca et al. [19]. The %RC obtained in the current study is converse to that of previous studies done by Aroca et al. [19] and Pietruska et al. [20] where they observed a greater %RC in SCTG compared to the collagen matrix groups.
The mean %RC in the PDCM and SCTG groups were 79% and 56% respectively which is far less compared to previous studies by Cieślik-Wegemund et al. [25] who reported 91% and 95% in the collagen matrix and SCTG groups, respectively. Also, a similar study by Rakasevic et al. [18] reposted 87% and 83% root coverage in the collagen matrix and SCTG groups. This reduced percentage of root coverage in the current study could be due to the inclusion of smokers who show reduced healing tendency as compared to the previously discussed studies done in non-smokers. Also, the current study included multiple gingival recessions as opposed to the study by Rakasevic et al. [18] which included single tooth recessions.
In the current study, CRC was achieved in 45% of defect sites among the test group which was contrary to the study by Aroca et al. [19] who demonstrated that complete root coverage was obtained in 59% of defect sites within the  [26] which showed higher mean RCES (7.9) in the CTG group.
In terms of patient discomfort, the PDCM group showed less discomfort than the SCTG group which is not in agreement with the study [27]. This better result in terms of satisfaction in the PDCM group could be ascribed to the minimally traumatic surgery and improved postoperative healing compared to the SCTG group which requires a second surgical site to harvest the graft. The duration of surgery was higher in the SCTG group than the PDCM group which can be correlated to the study by Aroca et al. [19], this could be due to longer time taken for harvesting SCTG.
The clinically significant gain in CAL, reduction in recession depth, and increase in keratinized tissue thickness would be beneficial for the patient and could validate the treatment of gingival recession with MCAT technique and PDCM in adequate root coverage. PDCM can be used as a potential alternative to gold standard subepithelial connective tissue graft in smokers with better patient compliance.

Limitations
Only light to moderate smokers were included and further research including heavy smokers may provide more value. Long-term evaluation may be necessary to evaluate the stability of the treatment.

Conclusion
MCAT in conjunction with PDCM serves as an alternative to SCTG minimizing surgical time and patient morbidity. PDCM offered better root coverage than SCTG and a substantial increase in the width of keratinized tissue and thickness of keratinized gingiva in smokers. PDCM could provide a viable and predictable approach to treat multiple gingival recessions in smokers with no uneventful healing.