Evaluation of the effect of dose-dependent platelet-rich fibrin membrane on treatment of gingival recession: a randomized, controlled clinical trial

Abstract Miller's class I gingival recessions (GR) have been treated using coronally advanced flap (CAF) with platelet-rich fibrin membrane (PRF membrane) or connective tissue graft (CTG). Objective: The aim of this study was to evaluate the effect of different multiple layers of PRF membranes for the treatment of GR compared with the CTG procedure. Material and Methods: Sixty-three Miller class I GR were treated in this study. Twenty-one GR selected randomly were treated with two layers of PRF membranes+CAF in 2PRF+CAF (test group-1), four layers of PRF membranes+CAF in 4PRF+CAF (test group-2), and CTG+CAF in the control group. The plaque index (PI), gingival index (GI), probing depth (PD), keratinized tissue thickness (KTT), clinical attachment level (CAL), recession depth (RD), recession width (RW), and keratinized tissue height (KTH) measurements were performed at baseline and 1, 3, and 6 months after surgery. The post-operative discomfort of patients, assessed with the visual analog scale (VAS) and healing index (HI), was recorded after surgery. Results: PI, GI, and PD scores were similar for all patients at all times. RD and RW scores were similar for each patient at 1 month, but these values were significantly increased in the subsequent periods in test group-1. The increase in KTT was significantly higher in the control group compared with the test groups. Similar root coverage scores were obtained in the test group-2 and control groups, and these scores were significantly higher compared with test group-1. Conclusions: The PRF membrane+CAF technique may be an alternative to the CTG+CAF technique for postoperative patient comfort. However, PRF membranes should use as many layers as possible.


Introduction
Gingival recession (GR) is the apical migration of the marginal tissue beyond the cementoenamel junction (CEJ) 29 . GR occurs because of traumatic tooth brushing, excessive frenulum attachments, and malposition of teeth. In addition, it may result in dentinal hypersensitivity, root caries, and unaesthetic gingival appearance [25][26][27][28][29] . There are various techniques that provide coverage of the exposed roots 3  for Miller Class I and II recession defects 13,25 and recent studies have reported that the percent root coverage (RC) ranges from 79.9% to 89.6% 4,9,13 . The advantage of this technique is the enhancement of keratinized tissue width, which can be explained by the determination of CTG surface epithelium characteristics 13 . Moreover, there are many disadvantages, such as postoperative pain or bleeding and a second surgical site after CTG operations 28 .
Platelet-rich fibrin (PRF) is the second generation of platelet concentrates 7 . A PRF membrane contains many growth factors, such as platelet-derived growth factor (PDGF), which is effective in the soft tissue wound healing process because it stimulates angiogenesis, granulation tissue formation, and epithelial migration.
These substances have been used together with GR treatment techniques, but there are limited data about factors affecting root coverage in human studies 9,24 . Therefore, the aim of this study was to evaluate the different amounts of PRF membranes for the treatment of Miller Class I gingival recessions compared with the CTG procedure, considered the gold standard.

Randomization
Subjects were assigned to one of the three groups Evaluation of the effect of dose-dependent platelet-rich fibrin membrane on treatment of gingival recession: a randomized, controlled clinical trial J Appl Oral Sci. 2018;26:e20170278 3/10 using a computer-generated randomization scheme.
Allocation concealment was obtained using numberlabeled opaque envelopes that were opened just before the surgery.

Initial therapy
Patients received oral hygiene instructions and initial periodontal therapy, including scaling and professional tooth cleaning using a low abrasive polishing paste. They were instructed to use the "Roll" brushing technique and a soft toothbrush to protect the soft tissue from trauma.

Preparation of the PRF membrane
Before the surgery, intravenous blood samples (10 ml) were collected from patients in the test groups.
In test group-1, 2 tubes of 10 ml venous blood were collected and in the test group-2, 4 tubes of 10 ml venous blood were collected from each patient.
Samples were centrifuged at 2700 rpm for 12 minutes (PC-02 machine, Process Ltd., France) and PRF clots were obtained according to the procedure described by Choukroun 7-9 . After coagulation, each PRF clot was prepared in fragment or membrane form using a "PRF Box" 7-13 . The surgical technique in the CTG+CAF group was the "envelope technique", as described by Raetzke 18 (1985). A horizontal incision at the level of the CEJ was designed and a split thickness flap was raised without any vertical incision. The papillae were de-epithelialized. The root was planned, and hard accumulations were removed with no chemical root treatment performed. The connective tissue graft was harvested from the palate using the "trap-door technique", described by Edel 10 (1974). The epithelial layer was elevated with one horizontal and two vertical incisions. The connective tissue graft was harvested at 1 mm using a standard caliper, and the epithelial layer was then sutured using a resorbable suture. The connective tissue graft was sutured to the recipient area using a resorbable suture at the level of the CEJ.
A split thickness flap was coronally advanced and sutured using a resorbable suture ( Figure 3). Finally, a periodontal dressing was fixed on the recipient's

Intergroup comparisons
Clinical evaluation of recession-defect characteristics from baseline and 1, 3, and 6 months after the root coverage surgery are shown in Tables 1 and 2. PI, GI, and PD were similar for all groups and the difference was not statistically significant at baseline, 3, and 6 months after surgery (p>0.05). The reduction in RD was significantly higher in 4PRF+CAF and CTG+CAF groups compared with the 2PRF+CAF group at 3 and Despite the similarity in the initial CAL value in all groups, the clinical attachment gain was higher in the 4PRF+CAF and CTG+CAF groups than in the 2PRF+CAF group. RC was higher in the 4PRF+CAF and CTG+CAF groups than in the 2PRF+CAF group at 3 and 6 months after surgery. RC was 56.34%, 69.65%, and 80.13% at 6 months for the 2PRF+CAF, 4PRF+CAF and CTG+CAF groups, respectively. Post-operative pain and the amount of analgesic used after surgery were significantly higher in the CTG+CAF group than in the test groups (Table 3).   Postoperative pain reduced daily in the first week after the surgery. However, the mean pain level increased on the fourth day after surgery, but the difference between the pain level at 4 days and at 3 days was not statistically significant.    The results of another clinical study, in which PRF membranes+CAF procedure was performed, showed results opposite to our study 3  In a different study, in which groups were prepared in the same manner, autologous platelet-rich fibrin (PRF) and CTG were used to treat multiple adjacent gingival recessions that consisted of two or three teeth as in our study 23 . Despite the similarity in the treatment technique of studies, there were two test groups in our study (2PRF+CAF and 4PRF+CAF) and there was one test group (L-PRF+CAF) in the study conducted by Tunali, et al. 23 (2015). Although two or four layers of PRF membranes were used in test groups of our study, decreases in RD values were not only statistically significant in our study, but also in the other study. In the study by Tunali, et al. 23 (2015) Evaluation of the effect of dose-dependent platelet-rich fibrin membrane on treatment of gingival recession: a randomized, controlled clinical trial J Appl Oral Sci. 2018;26:e20170278 9/10 initial gingival recession values were deeper compared to our study. The author stated that the significant decreased RD values for test groups may be explained due to the deep defects that can help the occurrence of statistical difference. In such recent study, CAL gain and increase in KGW values between test and control groups were similar. However, CAL gain in the 4PRF+CAF and CTG+CAF groups was higher than in the 2PRF+CAF group, and KTT in the CTG+CAF group was significantly higher than in our test groups. In our study, the parallel design trial was used because of three study groups. Tunali, et al. 23 (2015) treated multiple bilateral defects, but the author complained due to the limited number of patients.

Intragroup comparisons
In other recent study, PRF membranes treated with CTG in test groups were only compared with the CTG procedure in control groups 13 . The results of this study show a statistically significant difference between the groups, such as RC and tissue thickness at 6 months.
Our clinical trial is a study of parallel design. The small number of patients in split-mouth design trials should be considered as a limitation of this study. In our study, clinical measurements and defect-related characteristics were similar in all groups at baseline, and there were more recession defects than in splitmouth studies.
In this study, the RD and RW values decreased the most in the first month in all groups. However, RD values increased again at 6 months in 2PRF+CAF and 4PRF+CAF, and the increase was statistically significant in 2PRF+CAF but not significant in 4PRF+CAF. RD values continued to decrease at 3 and 6 months compared with baseline, but this decrease was not statistically significant in the control group. Changes in RD values in the control group were associated with graft maturation and "creeping attachment" 12

Conclusions
The results of the GR treatment procedure in which different amounts of PRF membranes were applied in test groups were compared, and better results were obtained when the maximum possible amount was used. Additionally, closure of the root surface was preserved significantly longer in the follow-up period in these patients. The CAF+PRF membrane technique may be used as an alternative to the CAF+CTG procedure when PRF membranes are adequately used, and more successful results can be achieved with longterm follow-up studies.