Analysis of the treatment of RT2 recessions with a xenogeneic collagen matrix vs. connective tissue graft combined with a coronally advanced flap. A double-blinded randomized clinical trial

Objectives To compare the clinical efficacy in terms of mean root coverage in RT2 recession treated with a coronally advanced flap combined with a xenogeneic collagen matrix versus a connective tissue graft. Materials and methods A total of 20 patients were randomized to receive one of two treatments: coronally advanced flap + xenogeneic collagen matrix (test group) and coronally advanced flap + connective tissue graft (control group). Patient-related outcomes measures and professional aesthetic assessment by root esthetic score were performed. A descriptive and analytical statistical analysis of the variables was performed. Results At 12 months, the mean root coverage was 56.48% in the test group and 69.72% in the control group (p = 0.048), with a 35% and 40% complete root coverage in the xenogeneic collagen matrix and connective tissue graft, respectively. Test group presented less pain (3.65 vs. 5.2 VAS units) (p = 0.015) and less surgical time (45 vs. 49.15 min) (p = 0.004) than control group. Conclusion The use of xenogeneic collagen matrix in RT2 recessions was effective for recession reduction to those obtained using autologous grafts; with the advantage that the duration of surgery and patient morbidity decreased. Therefore, xenogeneic collagen matrix in RT2 recessions could be an alternative to autologous grafts. Clinical relevance The use of xenogeneic collagen matrix decreases the surgery time and patient morbidity but connective tissue graft results in significantly better mean root coverage and complete root coverage. Xenogeneic collagen matrix can be used in the treatment of RT2 gingival recessions. Study registration NCT 03344315.


Introduction
Gingival recessions are a very common pathology in the adult population [1,2].The ideal goal of recession treatment is to achieve complete root coverage (CRC) [3] and good aesthetic results of surrounding soft tissues [4].
In terms of the percentage of mean root coverage (MRC) and CRC, the treatment of choice is the combination of a subepithelial connective tissue graft (CTG) with a coronally advanced flap (CAF) [5][6][7].The use of a CTG increases gingival thickness (GT) and achieves long-term stability [8] of the gingival margin [6,9].However, obtaining a CTG may cause postoperative complications [10,11], thus leading to the development of different alternatives, such as membranes [12], biological agents [13], and allografts [14].The objective of these therapies was to find a therapeutic alternative to the use of CTGs, thus allowing the treatment of multiple recessions in a single session, which can reduce surgery times, avoid the need for a second surgery, and improve the color and texture of the tissues [12][13][14][15][16].This is especially important in patients with periodontitis and multiple recessions with interproximal attachment loss whose treatment is more complex [17].
RT2 [20] recessions are very prevalent in the adult population [6], but the CRC is not always predictable [27].Although there are clinical studies in which CRC as well as high percentages of MRC have been achieved [28,29], it is necessary to determine the predictability of treatment with autologous grafts as well as with possible alternative therapies [6,30], such as the CMX.
Hence, this multicenter clinical trial aimed to compare the clinical efficacy of CMX (Geistlich Mucograft®: Geistlich Pharma, AG, Wolhusen Switzerland) versus a subepithelial CTG when combined to CAF [31] for the treatment of RT2 recessions in terms of percentage of MRC.

Trial design
This was a randomized, double-blind, multicenter (2-center) clinical trial with a 12-month follow-up period.The study was registered in clinicaltrials.gov(NCT 03344315) and was conducted following the Consolidated Standards of Reporting Trials (CONSORT) guidelines [32].The study was conducted following the principles of the Declaration of Helsinki (revised in 2013) and was approved by the Euskadi Ethics Committee (CEIC-E) (PI 20,161,008-PS) in February 2017.
The primary aim was to analyze the percentage of MRC associated with the treatment of recessions with CMX (test group) vs. CTG (control group), with the null hypothesis (H0) that both treatments are equally effective at 12 months.The secondary outcomes were the percentage of CRC, the reduction of recession (RECred) measured in mm, the gain in keratinized tissue width (KTW), the change in GT, patient-related outcome measures (PROMs) regarding postsurgical pain, satisfaction with the treatment and aesthetics perceived by the patient, and aesthetics by a blinded clinical monitor (MVF).

Reference population
All participants were recruited from two private centers between March 2017 and May 2019.Patients received information about the treatment and the advantages and disadvantages of participating in this study.Informed consent was obtained from all participants before the start of the study.
Inclusion criteria were as follows: (a) patients ≥18 years old; (b) periodontally treated and healthy with at least one or more RT2 buccal gingival recessions in incisors, canines, and premolars, located in the same quadrant or sextant with a minimal depth of ≥ 2 mm; (c) full mouth plaque index had to be under 25% [33].
The exclusion criteria were as follows: (a) active periodontal disease; (b) subjects with severe systemic pathology who took or had taken medication that could interfere with the healing of periodontal tissues during the last 6 months.

Sample size calculation
For sample size calculation, the percentage of MRC was considered as the primary outcome to calculate the sample size.To provide a statistical power of 80%, an α-risk of 5%, and an SD = 14, as previously described in the literature [22], 18 patients would be necessary.To account for anticipated dropouts, an additional 10% was added, resulting in 20 patients.

Randomization
To determine the type of graft (CMX or CTG) to be used, the patients were randomized in blocks of two treatments using statistical software (IBM SPSS® Statistics 20.0) (AMGF).The allocation was kept hidden by a clinical monitor (AMGF) until the time of the intervention in opaque envelopes that were opened immediately after flap elevation.

Control of study bias
The clinical examiner (MVF) and the biostatistician (XMM) were blinded to the type of treatment (CMX or CTG).The reproducibility of the clinical examiner (MVF) was determined by evaluating 4 patients (presenting multiple RT2 recessions) not related to the study, at least twice, with a separation of at least 24 h.An intraclass correlation coefficient > 0.85 was considered acceptable.

Intervention: Surgical procedure
All patients initially completed a plaque control program, including oral hygiene instructions [34] to correct habits related to the etiology as well as a presurgical prophylaxis.All the surgeries were performed by two experienced periodontists (LAAZ and ERGP), being the CAF [31] the technique chosen in all cases.The data related to the chair time, defined as the duration of the surgical procedure (in minutes) from the first incision to the last suture was also registered.
After preparing the recipient bed, a CMX (Geistlich Mucograft®: Geistlich Pharma, AG, Wolhusen Switzerland) (test group) or CTG (control group), which was obtained using the UPV/EHU technique [35], was implanted.Both the CMX and the CTG were sutured with absorbable sutures (Ethicon Vicryl® 5/0; Johnson & Johnson); the flap was sutured with Gore-Tex e-PTFE® 5/0 (W.L. Gore & Associates (UK), LTD, Scotland)) Postoperative care included a single presurgical dose of 2 ml of intramuscular betamethasone (Celestone® Crono-dose®, Schering Plough S.A., Spain), amoxicillin 875 mg/ clavulanic acid 125 mg (Augmentine®, GlaxoSmithKline S.A., Spain) every 8 h for 7 days, analgesics on demand (25 mg dexketoprofen) and rinses with 0.2% chlorhexidine digluconate (2 times a day for 3 weeks).Also, local cold applications for two days, a soft diet and no physical exercise during the first week after the surgery were advised.Sutures were removed from the palate at 7 days and from the recipient bed at 14 days respectively.At 21 days after surgery, daily brushing was resumed [34].

Monitoring and data collection
At 7, 14 days, 4 weeks, 6, and 12 months patients were scheduled to attend follow-up appointments, which included a professional plaque removal with reinforcement of oral hygiene instructions.

Clinical parameters
At baseline, 6 and 12 months, an experienced, blinded, and previously calibrated examiner (MVF) recorded the following parameters using a standardized periodontal probe (PCP-11, Hu-Friedy, Mfg.Co. LLC, Chicago, USA); the measurements were rounded to the nearest 0.5 mm: • Recession depth (REC depth): measured in the mid vestibular from the cemento-enamel junction (CEJ) to the deepest point of the pocket.As non-carious cervical lesions were not excluded, if the CEJ was not present or not detectable, it was determined using the technique described by Cairo and Pini-Prato [36] in 2010.

Patient-related outcomes measures
Regarding esthetics, a PROM was carried out using a visual analogue scale (VAS, ranging from 0 to10) in which patients were asked to score their satisfaction related to their clinical experience (procedure and postoperative pain) at 7 days and esthetic (0 = worst possible aesthetic result and 10 = excellent aesthetic result) at 12 months.Respecting the clinical experience (0 was considered the worst procedure experienced for the patient and 10 the best) and for postoperative pain (0 = no pain and 10 = extreme pain).

Professional esthetic evaluation
Besides the patient's esthetic perception, the esthetics were also assessed from a professional point of view.Thus, one blinded and calibrated examiner (MVF) compared the photographs taken at baseline and 12 months postoperatively.

Clinical results
Clinical results at 6 months are summarized in Table 2.
When analyzing the intergroup results, no significant differences were observed in any of the parameters analyzed.Clinical results at 12 months are reported in Tables 2 and 3, and 4; the changes between 6 and 12 months are shown in Table 3.
The percentage of MRC was 56.48% on test and 69.72% on control sites at 12 months (p = 0.048).Between 6 and 12 months, the percentage of MRC decreased from 59.60 to 56.48% in the test group The assessment was made using the Root Coverage Esthetic Score (RES) [37].

Statistical analysis
All the obtained data were analyzed using statistical software (IBM SPSS® Statistics 20.0; IBM, Chicago, IL, USA), with the patient as the unit of analysis.Initially, the Shapiro-Wilk test was used to evaluate if the distribution was normal or not.First, descriptive statistics were performed, and means and standard deviations were provided for quantitative variables, and percentages were determined for categorical variables.Subsequently, in the analysis of statistical relationships, normality tests led to the use of nonparametric tests for both intragroup (Wilcoxon test of the ranges for related samples) and intergroup comparisons (Mann-Whitney U).

Results
The CONSORT diagram, which shows each group and the number of participants, as well as the number of drop-outs, is shown in Fig. 1.  for all parameters (except KTW and GT) in the control group between the upper and lower jaws, with favorable results for the maxilla; in the test group, the differences between the maxilla and mandible were only significant for the increase in GT.

Results of the surgical procedure and PROMs
No postoperative complications were observed after any surgery.The results are reported in Table 5.The mean surgical time was 45 min (SD:9.01),i.e., 41 min (SD:7.6)(CI 95% 37.41-44.59) in the test group and 49.15 min (SD:8.5)(CI 95% 45.15-53.15) in the control group; the differences between groups were significant (p = 0.004).
Regarding the assessment of pain, less pain was reported by patients who received CMX, with a VAS = 3.65 (SD:2.08)(95% CI 2.67-4.63)vs. VAS = 5.2 (SD:2.31)(95% CI 4.12-6.28)(p = 0.015).Patients in the test group were more and an increased in the control group from 68.70 to 69.72%, respectively.The CRC was achieved in 7 patients (35%) in the test group and 8 patients (40%) in the control group Three patients presented with CRC for all treated recessions in the control group.At the recession level, the percentage of CRC decreased in the test group (from 15.5 to 12.1%), and in the control group, it increased from 18.9 to 22.6% from 6 to 12 months.Both treatment groups showed significant post-surgical improvement in all clinical variables, except for the KTW in the test group.The GT increased after the surgical procedures: 0.46 mm (SD:0.54) in the test group and 0.78 mm (SD:0.47) in the control group (p = 0.038).
An analysis was performed by the treatment group (test group or control group) and the results are summarized in Table 4. Statistically significant differences were observed  score in the test group (6.7 (SD:1.97))and in the control group (7.28 (SD:2.24)).Clinical and radiographic characteristics and surgical procedures of one patient from test group and control group at baseline and 12 months are shown in Figs. 2  and 3.

Discussion
To our knowledge, there is no double-blind randomized clinical trial in the literature that compares the use of a soft tissue substitute such as the CMX vs. the CTG for the treatment of RT2 recessions in combination with CAF, thus making it difficult to compare our results with recent evidence [6,27,30].
The results of this study indicated that the percentage of MRC was lower in the test group than in the control group (CMX: 56.48% vs. CTG: 69.72%) (p = 0.048) and that the percentage of CRC was lower in the test group than in the control group (35% vs. 40%) after 12 months; therefore, hypothesis (H0) is rejected.
Our results partially coincide with a recent meta-analysis of Miller classes I and II recessions [18,38], in which lower MRC were observed with CMX compared with CTG, with similar CRC in both study groups.
satisfied with the procedure in general, but the differences between groups were not significant.
The aesthetic evaluation score at 12 months was 6.85 (SD:1.5) in the test group and 7.15 (SD:1.3) in the control group These values were similar to the RES  (root coverage esthetic score), PROMs: clinical experience using a visual analog scale (VAS) (0 being the worst procedure and 10 the best); postoperative pain using a visual analog scale (VAS) (0 being the least pain and 10 being the maximum pain).Aesthetic result as assessed by the patient using a visual analog scale (VAS) (0 = worst possible aesthetic result and 10 = excellent aesthetic result) SD (standard deviation); mm (millimeters); CMX (xenogeneic collagen matrix); CTG (connective tissue graft) 1 3 in our study (35%), they are not comparable due to the type of recessions treated, where the RECred as well as KTW gain should be considered a treatment success in the treatment of gingival recessions associated with loss of interproximal attachment [44,45].
A recent CRC meta-analysis of 37 publications about these types of recessions [27] found wide CRC ranges (32.87 − 63.82%).Achieving CRC for these types of recessions is considered a challenge because of interproximal attachment loss [47], which implies an increase in the avascular surface [26,48].Our results are consistent with those previously reported [49] of Miller class III recessions treated with modified tunnel technique (TTM) in combination with However, the percentage of MRC (CMX:56.48% vs. CTG: 69.72%) (p = 0.048) and the percentage of CRC (CMX:35% vs. CTG: 40%) results for both treatment groups were within the ranges reported in previous studies of RT2 recessions with CAF + CTG at 12 months (54.8-77.57%)[39][40][41].Only, Fernández-Jiménez et al. [40], 2023, evaluated the CRC at 12 months in multiple recessions with results (50%) that were slightly superior to those herein (40%) where the KTW at baseline was higher.
The evidence regarding CMX is limited to RT1 recessions where different treatments have been compared:  [22,23].The percentage of MRC with CMX at 12 months ranged from 76.2% [23] to 94.32% [24].Our results were lower (56.48%)than the current evidence, probably due to the type of recessions treated in this clinical study (RT2) where attachment loss was present.Also, only three studies until now have treated multiple recessions [8,24,25] with CAF + CMX, and CRC was evaluated in five studies previously [8,[24][25][26]42] and it ranged from 36% [42] to 72% [24].Although these results were superior to those obtained step or CEJ whose importance has been evidenced in the recent 2018 classification.Also, the soft tissue outcomes were assessed at 12 months, so long-term observations are necessary to confirm these results.However, this is the first double-blinded RCT in RT2 recessions that compares a CMX and autogenous grafts, including both maxillae (mandible:58.5%),and multiple recessions (96.39%).

Conclusions
Within the limitations of this clinical study, although MRC was greater with CTG we conclude that CMX is a treatment option for these challenging RT2 recessions.The surgical time and patient morbidity were reduced in the test group.Therefore, CMX can be considered an alternative to autologous grafts.
It is necessary to perform more RCTs in patients with periodontitis due to the high prevalence of multiple recessions in these patients, which require large grafts that entail greater postoperative morbidity.

Fig. 3 Fig. 2
Fig. 3 Surgical intervention in a patient allocated to the CAF + CTG group: (g)(h) clinical and radiographic features at baseline, (i) Intra-operative view: CTG (j) Immediate postoperative view: coronally flap advancement and closure (k)(l) Outcome at 12-months follow-up • Recession width (REC width): measured mesio-distally at the level of the CEJ.• Keratinized tissue width (KTW): measured from the gingival margin to the mucogingival junction.• Clinical attachment level (CAL), measured from the CEJ to the gingival margin to the deepest point of the pocket (PD + REC depth).
• Percentage of CRC (the number of treated recessions with REC depth = 0 mm): CRC × 100/number of recessions.•Percentage of MRC (mean preoperative REC depth -mean postoperative REC depth/mean preoperative REC depth × 100) were calculated.• GT: measured 3 mm apical from the center of the gingival margin (at baseline and 12 months) using a 25-gauge K endodontic file with a rubber stopper, measured perpendicular to the tooth axis under local anesthesia.

Table 3
Results of clinical changes at six (T1) and twelve (T2) months

Table 4
Clinical results in maxilla and mandible at 12 months of follow-up

Table 5
Patient based surgical parameters and patient perception dur-