The granulation tissue preservation technique in regenerative periodontal surgery—a randomized controlled clinical trial

Abstract Objectives To investigate if the application of the granulation tissue preservation technique (GTPT) in regenerative therapy of infrabony periodontal defects results in more clinical attachment level (CAL) gain and more radiographic bone gain (RBG) than the conventional resective approach 12 months after surgery. Materials and methods Forty patients exhibiting at least one infrabony defect with a probing pocket depth (PPD) ≥6 mm and a radiographic infrabony component (INFRAX‐ray) ≥3 mm were randomly treated with the GTPT (test group) or the double‐flap approach with resection of the defect‐filling granulation tissue (control group). Enamel matrix derivatives were applied in both groups. Clinical and radiographic parameters were recorded at baseline (t0), 6 months (t1), and 12 months (t2) after surgery. The primary outcome variable was CAL gain between t0 and t2. Results When all patients were considered, ΔCALt0–t2 did not differ significantly between the two groups (p = .160). Significant PPD reduction (test group: 4.38 ± 1.36 mm; control group: 4.06 ± 2.38 mm), CAL gain (test group: 3.75 ± 1.24 mm; control group: 2.88 ± 2.09 mm), and RBG (test group: 3.06 ± 1.74 mm; control group: 3.27 ± 2.19 mm) were achieved at t2 in both groups. Using multivariate linear regression, PPDt0 and group were identified as variables with the greatest influence on ΔCALt0–t2. PPDt0 and INFRAX‐ray were identified as variables with the greatest influence on RBGt0–t2. Patients with a defect angle >22° showed significantly more CAL gain in the test group (t0–t1: 3.08 ± 1.38 mm; t0–t2: 3.62 ± 0.96 mm) than in the control group (t0–t1: 1.77 ± 1.54 mm; t0–t2: 2.18 ± 1.83 mm). Conclusions Regarding all patients, the study failed to show significant differences between the test and control groups. However, the GTPT appears to lead to more CAL gain in noncontaining infrabony defects.


| BACKGROUND
Minimally invasive surgical techniques have been developed to limit the extent of the surgical area, to achieve a stable primary wound closure, and thus to avoid failures in wound healing particularly in the area of the interdental papilla (Cortellini & Tonetti, 2007Harrel, 1998;Harrel & Rees, 1995;Trombelli et al., 2009). All these flap designs have in common that the defect-filling granulation tissue is resected and discarded.
However, advanced infrabony defects are often not limited to the interdental space but extend to the oral and buccal sites. In these cases, it is not sufficient to prepare only one flap, but a double flap approach must be used to ensure sufficient visibility of the root surface to be instrumented. In addition, advanced complex defects usually lack soft tissue support, which is essential for the success of the regenerative procedure. Therefore, bone substitutes are frequently used to fill the space previously occupied by granulation tissue and thus to preserve space for regeneration (Kao et al., 2015;Reynolds et al., 2003). It has been shown for different graft materials that the space created for regeneration leads to new bone formation only to a limited extent (Stavropoulos et al., 2003). Furthermore, graft materials carry the risk of microbial contamination compromising the treatment outcome.
Recently, we introduced the granulation tissue preservation technique (GTPT) for regenerative therapy of infrabony periodontal and peri-implant defects (Günay et al., 2013. Preservation of the defect-filling granulation tissue is hypothesized to serve as soft tissue support, to make the use of bone substitutes dispensable, and to enable increased wound stability particularly in the area of the interdental papilla. Moreover, the existing vascular network and the precursor cells contained in the granulation tissue can be preserved and subsequently promote wound healing. In vitro and in vivo studies have shown that cells with properties of mesenchymal stem cells reside in both inflamed periodontal and peri-implant tissues (Adam et al., , 2020Gousopoulou et al., 2021;Park et al., 2011). From this point of view, it seems to make sense to preserve the defect-filling granulation tissue in regenerative periodontal surgery. Preservation of granulation tissue was first described by Lindhe and Nyman (1985). They already stated that granulation tissue removal during access flap surgery is not a mandatory measure for creating suitable conditions for the proper healing of periodontal tissues.
The aim of the present study was to compare the GTPT with the conventional double-flap approach, in which the defect-filling granulation tissue is resected. Since soft tissue collapse was to be expected particularly in defects with missing bony support, the statistical examination focused on noncontaining defects defined by large radiographic defect angles. The hypothesis of the present study was that infrabony periodontal defects treated with the GTPT would result in more clinical attachment level (CAL) gain and more radiographic bone gain (RBG) than those treated with the conventional approach.

| Experimental design
The present clinical trial was performed in the Department of Conservative Dentistry, Periodontology, and Preventive Dentistry of Hannover Medical School (MHH) and had a prospective, randomized, controlled, and double-blinded (patients, investigator) design. In total, 40 patients with 40 deep infrabony periodontal defects received regenerative periodontal surgery using the GTPT (test group;n = 20) or the conventional double flap approach with resection of the defect-filling granulation tissue (control group; n = 20). In both groups, enamel matrix derivatives (EMDs) were used as bioactive molecules to promote periodontal regeneration. The clinical and radiographic outcomes of both groups were longitudinally followed for 12 months (Figure 1)

| Patient and defect eligibility
Systemically healthy individuals presenting with advanced periodontitis (Stage III or IV) and at least one isolated deep, mostly interproximal infrabony defect were considered appropriate for this trial.
The inclusion criteria were (1) Probing pocket depth (PPD) ≥ 6 mm (2) Radiographic infrabony component (INFRA X-ray ) ≥ 3 mm (3) Positive response to sensitivity test with refrigerant spray (4) Hygiene index (HI, see below) ≥ 40% recorded during the first step of periodontal therapy When more than one eligible defect was available, the one with the largest INFRA X-ray (see below) was selected. The exclusion criteria comprised: (1) Heavy smokers (>10 cigarettes daily) (2) Pregnancy (3) Breastfeeding (4) Intake of antibiotics and/or nonsteroidal antirheumatic drugs within the previous 3 months (5) Systemic diseases with known impact on periodontal health Before baseline examination, all patients received the first and second steps of periodontal therapy consisting of supragingival dental biofilm control, oral hygiene instructions, professional mechanical plaque removal, elimination of possible plaque-retentive factors, and subgingival instrumentation (Sanz et al., 2020). Six weeks after completion of the second step of periodontal therapy, clinical and radiographic baseline examinations were conducted.
2.3 | Clinical and radiographic parameters were recorded at baseline (t0) and 6-months (t1) and 12-months (t2) follow-up visits The clinical parameters recorded at baseline (t0), 6 months (t1), and 12 months (t2) after surgery included bleeding on probing (BOP, Ainamo & Bay, 1975), full mouth bleeding score (FMBS), HI, PPD, recession depth (RED), and CAL. BOP, FMBS, PPD, RED, and CAL were assessed at six sites per tooth (mesiobuccal, buccal, distobuccal, mesio-oral, oral, disto-oral) using a WHO periodontal probe. BOP was assessed dichotomously (yes/no) and subsequently used to calculate the FMBS (Cortellini et al., 1993). The FMBS was calculated using the formula: sum of bleeding sites/sum of all sites × 100 in percent. The HI is a modification of the plaque control record (O'Leary et al., 1972) and was used to assess the quality of oral hygiene measures at home.
X-rays were taken at t0, t1, and t2. X-ray film holders (Super-Bite, Kerr) were individualized using addition-curing silicone (Silagum-Putty, DMG) to warrant a reproducible beam path and best possible comparability of the radiographic images ( Figure 2). The following distances were measured at sites affected by the infrabony defect using a software program for dental imaging (byzzKlinik, orangedental): (1) Distance from the cementoenamel junction to the bottom of the defect (CEJ-BD X-ray ) (2) Distance from the cementoenamel junction to the root tip (CEJ-RT X-ray ) The RBG at t1 and t2 was calculated using the formula: Besides this, (INFRA X-ray = distance from the bone crest to the bottom of the defect) and the radiographic defect angle were determined using the byzzKlinik software.

| Surgical procedure
Analgesia was achieved through infiltration or block anesthesia using an epinephrine-containing local anesthetic (Ultracain D-S forte, Sanofi-Aventis). Circumferential, strictly intrasulcular incisions were conducted at the defect-related teeth using a microsurgical blade In the test group, special focus was placed on the exact repositioning of the granulation tissue into its original position within the infrabony defect ( Figure 3). In the control group, the granulation tissue was F I G U R E 2 Application of the granulation tissue preservation technique on tooth 36. Clinical view: (a) before surgery, (b) after mobilization of the mucoperiosteal flap and instrumentation of the defect-related root surface, and (c) 12 months after surgery. Note the completely preserved height of the interdental papilla. (d) Radiographic view of the infrabony defect using an individualized X-ray film holder. Significant radiographic bone gain was observed (e) 6 months and (f) 12 months after surgery completely resected before the interdental papillae were repositioned and fixed. Finally, the operating area was gently compressed with saline-soaked gauze for 1 min.

| POSTOPERATIVE CARE
Patients were instructed to spare the surgical area, to refrain from mechanical plaque control, and to use instead a mouth rinse containing 0.2% chlorhexidine digluconate twice daily. Patients attended weekly control sessions during the first 3 weeks. At each visit, the surgical area was carefully cleaned and epithelial wound healing of the interdental papilla was assessed using the early healing index (Wachtel et al., 2003). The sutures were removed 2 weeks after surgery. After this initial 3-week wound healing phase, patients were again allowed to brush their teeth with a very soft toothbrush. The use of interdental brushes was permitted depending on the progress of the papillary soft tissue healing. Supportive periodontal therapy was given at 3-monthly intervals. This included professional supra-and subgingival tooth cleaning and remotivation and re-instruction of the patients to maintain the best possible oral hygiene.

| Statistical analysis
The Institute of Biostatistics (MHH) conducted the sample size calculation (nQuery Advisor 7.0) and randomized patient allocation. The difference of CAL between t0 and t2 (ΔCAL t0-t2 ) was defined as the primary outcome variable. A mean difference of 1.5 mm was expected between the test and control groups. The sample size calculation assumed that there was an unrelated problem, a Type I error of 5% (two-sided), and a standard deviation of 1.5 mm. For this setting, a power of 92% was calculated for 20 patients per group. The values used for the sample size calculation were based on data obtained from studies using the modified Widman flap as resective approach (Heitz-Mayfield et al., 2002) and using the simplified papilla preservation flap as a tissue-preserving approach (Cortellini et al., 2001).
The randomized patient allocation was carried out by telephone on  attendance to the follow-up visits (n = 2; test group), and need for retreatment after abscess formation (n = 1; control group). Besides this, incomplete data sets were generated in two patients due to new pregnancy (n = 1; control group) and nonattendance to the follow-up visit after 12 months (n = 1; control group). Thus, 35 patients were included in the statistical evaluation at t1, and 33 patients at t2.

| Clinical and radiographic outcome at 6-months and 12-months follow-up visits
When considering the entire study population, no significant differences were found between the test and control group for the primary  (Table 3).
We hypothesized that especially noncontaining defects with a large defect angle would benefit from the GTPT. Considering patients with baseline radiographic defect angle >22°, there was a significantly greater CAL gain in the test group than in the control group.
Thus, ΔCAL t0-t1 was 3.08 ± 1.38 mm in the test group and 1.77 ± 1.54 mm in the control group (p = .032; t test for independent samples), and ΔCAL t0-t2 was 3.62 ± 0.96 mm in the test group and 2.18 ± 1.83 mm in the control group (p = .034; t test for independent samples). When evaluating patients with a baseline radiographic defect angle ≤22°, no significant differences between the two groups were found for any of the examined parameters (Table S4).
In the next step, multivariate linear regression with backward elimination was performed (

| DISCUSSION
The hypothesis of the present study was that the GTPT would result in significantly more CAL gain than the double flap approach with resection of the defect-filling granulation tissue 12 months after regenerative periodontal surgery. However, the study failed to find a T A B L E 2 Changes of PPD, RED, CAL, and RBG between baseline (t0) and the follow-up visits 6 months (t1) and 12 months (t2) after surgery  (Clementini et al., 2019). The 18 studies included showed a PPD reduction of 4.24 mm, a RED increase of 0.44 mm, and a CAL gain of 3.89 mm, which is comparable to the results of our study. The lower CAL gain and higher RED increase observed in our study may be explained by the fact that the patientrelated factors HI and FMBS were not as good as in other studies, in which MIPS was applied (Cortellini et al., 2017;Ribeiro et al., 2011Ribeiro et al., , 2013 (Nibali et al., 2021). Consistent with these findings, we observed by multivariate linear regression that PPD t0 and particularly INFRA X-ray had the greatest influence on RBG 12 months after surgery. This agrees with the results of other studies that also found a positive correlation between RBG and INFRA X-ray (Ilgenli et al., 2007;Liñares et al., 2006;Meyle et al., 2011). There is evidence that the defect morphology plays a crucial role in the outcome of regenerative periodontal therapy (Cortellini et al., 2008;Losada et al., 2017;Meyle et al., 2011 radiographic defect angle. A large defect angle is known to have a negative impact on the clinical and radiographic outcome (Eickholz et al., 2004;Losada et al., 2017). Accordingly, Tsitoura et al. reported that the probability of achieving CAL gain ≥ 4 mm is 2.5 times higher in defects with a defect angle ≤ 22°than in those with a defect angle ≥ 36° (Tsitoura et al., 2004). Our evaluation of patients with a defect angle >22°revealed that a significantly greater CAL gain was achieved in the test group. This difference was detectable after 6 and 12 months and was mainly a result of PPD reduction. The fact that the multivariate linear regression identified the variables PPD t0 and group as having the greatest influence on ΔCAL t0-t2 also supported this observation. This is consistent with the results reported by Eickholz et al. (2004), who investigated infrabony defects treated by the guided tissue regeneration technique. They also attested the initial PPD to be a significant predictor for CAL gain.
The rationale behind GTPT is that the use of bone substitutes can be avoided because the granulation tissue itself acts as soft tissue support. To achieve this goal, the granulation tissue should be mobilized in its entirety from the defect during the preparation of the mucoperiosteal flap. Special attention should be paid to placing the interdental incision on the bone crest (Figure 3), which sometimes requires bone sounding. Finally, the granulation tissue must be repositioned in its original position at the end of the surgical intervention. At this point, it is important to mention that both mobilization and repositioning of the soft tissue can be realized more easily the better the surgical area has been instrumented during the second step of periodontal therapy. One could assume that more time is needed for the surgical procedure of the GTPT. However, the duration of surgery was not significantly different between the two groups. In the present study, complete mobilization of the defectfilling granulation tissue was not equally achieved in all defects. In general, it was more difficult to mobilize the entire granulation tissue in narrow three-wall defects compared to wide one-and two-wall defects. However, even if it was only possible to mobilize the coronal part of the granulation tissue, while the apical remained in the defect, the preservation and precise repositioning of the coronal part provided additional stability to the papillary soft tissue and facilitated the subsequent wound healing process.
Bone substitutes are frequently applied in regenerative therapy of advanced infrabony defects to prevent soft tissue collapse into the defect and, thus, preserve space for regeneration (Kao et al., 2015).
There is conflicting data on whether EMD + graft material results in more CAL gain and greater PPD reduction than EMD alone. A recently published meta-analysis looked at the treatment of infrabony defects using either EMD or EMD + graft material and differentiated the treatment outcome by flap design (Trombelli et al., 2021). They reported that EMD + graft material resulted in more CAL gain in minimally invasive variants (EMD: 3.69 mm; EMD + graft: 4.10 mm) and papilla preservation variants (EMD: 3.08 mm; EMD + graft: 3.65 mm) than EMD alone. However, other studies provide evidence that the use of EMD + graft material does not lead to more PPD reduction and greater CAL gain in the treatment of noncontaining defects (Hoffmann et al., 2016;Losada et al., 2017;Pietruska et al., 2012). These observations raise the question of whether it would have been better to use EMD + graft material in the control group instead of EMD alone.
Another significant limitation of the present study was that patients with containing defects (three-wall defects, defect angle ≤ 22°) were also included. This was probably the main reason why no significant differences (apart from ΔRED t0-t1 ) were found between the test and control group when all study participants were considered.
Another possible explanation could be that EMDs were used in both groups. Furthermore, the present study does not provide any information on whether periodontal regeneration or repair actually occurred during the wound healing process. Interestingly, there was a smaller RBG in the GTPT group compared to the control group, when all patients were considered. This was the case despite greater PPD reduction, greater CAL gain, and lower RED increase in the GTPT group. In contrast, patients with defect angle >22°tended to have a larger RBG in the test group. These contradictory data lead to the question of what happens to the granulation tissue during wound healing and what influence EMDs play in its maturation process.
Animal studies could provide important information to clarify these questions.

| CONCLUSIONS
We conclude that the GTPT may show its advantages over the conventional technique especially in advanced cases characterized by unfavorable defect morphology, namely few residual bone walls and large defect angle. Conversely, if sufficient residual bone walls and/or small defect angles are still present, removal of the granulation tissue does not represent a disadvantage for the regenerative healing process.