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pISSN 2093-2278   eISSN 2093-2286
Open Access, Peer-reviewed
    J Periodontal Implant Sci. 2023;53:e53. Forthcoming. English.
    Published online Nov 13, 2023.
    https://doi.org/10.5051/jpis.2303080154
    Copyright © 2023. Korean Academy of Periodontology
    Original Article

    Comparison of early wound healing using modified papilla preservation technique between enamel matrix derivative and recombinant human fibroblast growth factor

    Yohei Nakayama,1,2 Shinichi Tabe,1 Kazuma Igarashi,1 Satoshi Moriya,1 Tsuyoshi Katsumata,1 Ryo Kobayashi,1 Shuta Nakagawa,1 Tomoko Nishino,1 Namiko Fukuoka,1 Kota Hosono,1 Mai Yamasaki,1 Yosuke Yamazaki,1 Moe Ogihara-Takeda,1 Shoichi Ito,1 Yumi Saito,1 Arisa Yamaguchi,1 Yuto Tsuruya,1 Mizuho Yamazaki-Takai,1,2 Shoichi Yoshino,1 Hideki Takai,1,2 and Yorimasa Ogata1,2
      • 1Department of Periodontology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan.
      • 2Research Institute of Oral Science, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan.
    • Correspondence: Yohei Nakayama. Department of Periodontology and Research Institute of Oral Science, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakaecho-nishi, Matsudo, Chiba 271-8587, Japan. Email: nakayama.youhei@nihon-u.ac.jp , Tel: +81-47-360-9363, Fax: +81-47-360-9364
    Received July 04, 2023; Revised August 30, 2023; Accepted October 05, 2023.

    This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/).

    Abstract

    Purpose

    Enamel matrix derivative (EMD) has demonstrated beneficial effects on wound healing following surgery. However, the effects of recombinant human fibroblast growth factor 2 (rhFGF-2) in periodontal regeneration therapy have not been extensively studied. This retrospective study was conducted to compare the wound healing outcomes of the modified papilla preservation technique (mPPT) between EMD and rhFGF-2 therapies.

    Methods

    A total of 79 sites were evaluated for early wound healing using the modified early wound healing index (mEHI), which included 6 items: incision, fibrin clotting, step, redness, swelling, and dehiscence. A numeric analog scale, along with postoperative images of the 6 mEHI items, was established and used for the evaluations. The inter-rater reliability of the mEHI was assessed via intraclass correlation coefficients (ICCs). After adjusting for factors influencing the mPPT, the differences in mEHI scores between the EMD and rhFGF-2 groups were statistically analyzed. Additionally, radiographic bone fill (RBF) was evaluated 6 months after surgery.

    Results

    The ICC of the mEHI was 0.575. The mEHI, redness score, and dehiscence scores were significantly higher in the rhFGF-2 group (n=33) than in the EMD group (n=46). Similar results were observed in the subgroup of patients aged 50 years or older, but not in those younger than 50 years. In the subgroup with non-contained bone defects, related results were noted, but not in the subgroup with contained bone defects. However, early wound healing did not correlate with RBF at 6 months after surgery.

    Conclusions

    Within the limitations of this study, the findings suggest that early wound healing following the use of mPPT with rhFGF-2 is somewhat superior to that observed after mPPT with EMD. However, mEHI should be improved for use as a predictive tool for early wound healing and to reflect clinical outcomes after surgery.

    Keywords
    Fibroblast growth factor 2; Inflammation; Wound healing

    INTRODUCTION

    Even with the emergence of numerous periodontal regeneration therapies [1, 2, 3, 4], dental professionals are increasingly seeking procedures with high predictability, given the technical sensitivity of these treatments. To achieve highly predictive periodontal tissue regeneration, several approaches involving minimally invasive flap design have been established. These include the simplified papilla preservation flap technique [5, 6], the papilla preservation technique [7], the modified papilla preservation technique (mPPT) [8, 9], and the single flap approach [10]. Research has indicated that wound healing following the mPPT is faster than that after application of the modified Widman flap, with quicker recovery of gingival blood flow [11]. Other techniques, such as the minimally invasive surgical technique (MIST) [12] and the modified MIST [13], have been developed to minimize the surgical area and increase flap stability. However, in cases involving multiple complex and large bone defects, the mPPT is widely used to secure the operative field.

    Multiple methods for evaluating wound healing have been reported in the context of periodontal regeneration. These include techniques designed to assess horizontal incision closure on interdental bone defects in the MIST. One such technique is the wound closure (WC) assessment, which is used to evaluate early wound healing 1 week after surgery [14]. Under the WC assessment, WC is assigned one of 3 scores (0, 1, or 2) based on the closure of the incision line, the presence of fibrin clots in the incision area, the observation of tissue necrosis in the interproximal area, and the exposure of the regenerative biomaterial. Another method is the early wound healing index (EHI), which consists of 5 levels [15]. While fibrin clots and dehiscence are common components of these methods, they do not include assessments of inflammation, such as redness and swelling, which can also be indicative of WC.

    Enamel matrix derivative (EMD; 0.3 mL of Emdogain gel, Straumann Holding AG, Basel, Switzerland) is widely used in periodontal regeneration therapy [1, 2]. The primary component of EMD is amelogenin, which is extracted from porcine tooth germs and crudely purified. Other components include enamel proteins and growth factors [16]. Originally, EMD was designed for the regeneration of periodontal tissue with intrabony defects. However, its use has expanded to include other applications, such as adjunct treatments for root coverage with connective tissue grafts [17], coronally positioned flaps [18], and the acceleration of wound healing [19]. The diverse potential applications of EMD can be attributed to the numerous biological components present in the substance [20].

    Recombinant human fibroblast growth factor 2 (rhFGF-2; REGROTH Dental Kit, 600 μg in hydroxypropyl cellulose, Kaken Pharmaceutical, Tokyo, Japan) is extensively utilized for periodontal regeneration therapy in Japan [4]. This product was derived from a clinically applied rhFGF-2 treatment, employed in the treatment of bedsores, cutaneous ulcers, and other wounds [21]. FGF-2 induces the proliferation and differentiation of undifferentiated mesenchymal cells [22, 23] and promotes osteogenesis [24]. A study that assessed linear bone growth, bone filling, and clinical attachment gain in periodontal intrabony defects indicated that the application of 0.3% rhFGF-2 was more effective than the use of EMD after surgery [4]. Both EMD and rhFGF-2 applications enhance angiogenesis, which positively induces WC [25, 26, 27]; however, no studies have yet established the difference in wound healing between EMD and rhFGF-2 therapies.

    The aim of this study was to compare rhFGF-2 and EMD with regard to early wound healing after the mPPT. We developed a modified EHI (mEHI) method to evaluate early wound healing following mPPT with either EMD or rhFGF-2, using subjective images. After confirming the moderate-to-high inter-rater reliability of the mEHI, we compared the differences in early wound healing between the groups using this mEHI approach.

    MATERIALS AND METHODS

    Participants

    This retrospective cohort study adhered to the Strengthening the Reporting of Observational Studies in Epidemiology guidelines and received approval from the Institutional Internal Review and Ethics Board at the Nihon University School of Dentistry at Matsudo (EC21-028). Prior to surgery, written consent was secured from all patients.

    Periodontal regeneration therapies using either rhFGF-2 or EMD [1, 2] were applied to selected sites at Nihon University Hospital in Matsudo, Chiba, Japan between September 2011 and March 2022. The inclusion criteria were as follows: 1) the patient must be systemically healthy; and 2) the patient must have a confirmed probing depth (PD) of ≥4 mm [4] and a radiographic bone defect depth of ≥3 mm [4] after initial periodontal therapy. The exclusion criteria included: 1) systemic diseases with coagulation disorders and diabetes mellitus, 2) a history of cancer, 3) pregnancy, 4) a plaque control record of ≤20%, 5) periodontal abscesses, 6) untreated occlusal trauma, and 7) being a smoker at the time of the operation. To compare the early wound healing of interdental papillae between the EMD and rhFGF-2 procedures, the sutures were scheduled for removal at 13 to 15 days after surgery (Figure 1).

    Figure 1
    Flow chart of the study. In total, 171 participants underwent periodontal regeneration therapy using EMD or rhFGF-2 during the survey period. The following individuals were excluded: 1) those who did not undergo the mPPT; 2) those whose suture removal was not recorded in images; and 3) those who fell outside of the period for suture removal. In total, 79 sites were assessed using the mEHI. To examine the influence of early wound healing on the clinical outcomes of EMD and rhFGF-2 at 6 months after surgery, the following individuals were excluded: 1) those with unacceptable periods for radiographic examination and 2) those with complicated bone shapes and unclear bone crests that prevented evaluation of bone defects. In total, 56 sites were included in the analysis.
    EMD: enamel matrix derivative, rhFGF-2: recombinant human fibroblast growth factor 2, mPPT: modified papilla preservation technique, mEHI: modified early wound healing index, PD: probing depth, CAL: clinical attachment level.

    Periodontal regeneration therapies

    All patients received initial periodontal therapy starting on their first visit. Periodontal examinations were carried out by a trained periodontist (Y. N.) using a periodontal probe (PCP-11, Hu-Friedy, Chicago, IL, USA). The clinical parameters assessed included PD, clinical attachment level (CAL), bleeding on probing (BOP), tooth mobility, and plaque index. These measurements were taken in the same way as in a previous study [28]. All periodontal regeneration therapies were applied by the same trained operator (Y. N.). The mPPT was selected to access vertical intrabony defects with a mesiodistal width of ≥2 mm at the interdental spaces [8]. The PPT U shape [7] was used for horizontal incisions at all mPPT operation sites. In brief, after the mucoperiosteal flap was elevated, granulation tissue was removed from intrabony defects, and scaling and root planing was performed. Following root surface etching with citric acid for 10 seconds and site rinsing with saline, 0.3% rhFGF-2 or EMD was applied to all surgical sites. When autologous bone was harvested from the same operative sites, autogenous bone grafts (AGs) were additionally performed, irrespective of bone wall classification [29]. The flaps were then repositioned and sutured using a combination of Teflon-coated holding sutures (5-0, TefdesserII, WASHIESU MEDICAL, Tokyo, Japan) and Nylon closing sutures (5-0 or 6-0 Nylon, WASHIESU MEDICAL) at the mPPT sites (Figure 2). Postoperative infection control was implemented, and antibiotics (either 500 mg of azithromycin per day or 300 mg of cefcapene pivoxil hydrochloride hydrate per day) were prescribed for 3 days. Analgesics (60 mg of loxoprofen natrium) were also prescribed as needed. Approximately 1 week after the operation, the surgical sites were sanitized using 0.025 w/v% benzalkonium chloride and rinsed with diluted 0.05% chlorhexidine gluconate (ConCool F, Weltech Corp., Osaka, Japan). All sutures were removed at approximately 2 weeks after surgery, and images were captured to document the evaluation sites.

    Figure 2
    Surgical procedure for a case. Preoperative (A, B), intraoperative (C, D, E), disinfection (F), suture removal (G), and 6-month post-surgery (H, I) images are presented. (A) Palatal view. (B) Radiographic examination showing clear vertical bone defects at the distal side of tooth 24. (C) A 1-wall bone defect was observed. (D) Recombinant human fibroblast growth factor 2 (0.3%) was applied to the root surface with an autogenous bone graft on the bone defect. (E) One inverting vertical mattress suture of the papilla (interdental 24–25) was utilized as a holding suture using TefdesserII, and 3 simple sutures of the papilla were employed as closing sutures using Nylon. (F) All sutures were retained at 7 days after surgery. (G) Sutures were removed 14 days after surgery. (H) Palatal view. (I) Radiographic examination showing remarkable improvement of bone defects.

    Periodontal re-evaluation

    Periodontal re-examinations (performed by Y. N.) and radiographic examinations were conducted both preoperatively and 6 months after surgery. These were done to measure radiographic bone defect depth and the radiographic bone fill (RBF) [30]. The dental x-ray paralleling technique was employed using a universal device to standardize the direction of the radiographs. To maintain blinding in the study, all radiographic analyses were performed by a single periodontist (S.T.), who was not privy to the specific treatment provided. All procedures were meticulously documented in the electronic medical records by Y. N. before proceeding with statistical analysis.

    Evaluation of early wound healing using the mEHI

    The EHI is used to assess fibrin clots and dehiscence as indicators of early wound healing [15]. To evaluate inflammation, we developed the mEHI, which includes 6 items not found in the EHI: incision line, fibrin clotting, step (between buccal and lingual/palatal flaps), redness, swelling, and dehiscence. Each item was scored on a numeric analog scale from 0 to 10, with corresponding images for scores 0, 5, and 10 (Figure 3). Higher scores suggest better early wound healing, and both individual item scores and the total mEHI were recorded. The mEHI was assessed using a panel of 4 images showing the removal of granulation tissue, the sutured incision, the sanitized incision, and the removal of sutures (Figure 4). The assessments were carried out by 20 evaluators, excluding the operator (Y. N.), and each evaluation took approximately 20 seconds per participant using electronic files to display the panels. To minimize evaluator bias, only the affected sites were revealed. However, it was not possible to blind the evaluators to photographs of bone defects from a given direction at the relevant sites or to the number of sutures. Scores were recorded as whole numbers and used for statistical analysis.

    Figure 3
    Guide for wound healing evaluation. Six items were included in the modified early wound healing index: the incision line, fibrin clotting, step, redness, swelling, and dehiscence. Numeric analog scales were attached to the postoperative images for each item.

    Figure 4
    Panels of sites treated with the modified papilla preservation technique for use in the mEHI assessment. Participant numbers are indicated on the upper left of each panel. Surgical information is shown at the bottom of the panels. Individual surgical records were deleted to avoid evaluator bias. All 20 evaluators assessed the wound healing of 79 participants using the mEHI.
    mEHI: modified early wound healing index.

    Selection of outcomes, variables, and potential confounders of the mPPT

    The primary outcomes of the mPPT consisted of 6 mEHI items (each scored from 0 to 10) and the total mEHI score. Patient profiles and clinical statuses were used to distinguish between host and technical factors (Table 1). Bone defect shape was recorded during periodontal surgery, and the number of bone walls was confirmed using images captured after the removal of granulation tissue. Scores of 1, 2, 2.5, and 3 were assigned to bone defects with 1 wall, 2 walls, a combination of 2 and 3 walls, and 3 walls, respectively. Incisions were made horizontally from the buccal, lingual, or palatal directions, with the side having the higher bone wall serving as the incision site. For non-contained bone defects, the side for horizontal incision was determined based on ease of handling. The number of closing sutures was determined by the interdental distance. In cases where autologous bone was harvested from the same operative site, AGs were optionally performed with rhFGF-2 or EMD. Three AG states were defined: full filling, partial filling, and non-graft. Full filling involved filling the entire bone defect with autogenous bone, while partial filling involved filling bone defects to a lesser extent than the standard line connecting points of adjacent cemento-enamel junctions [20].

    Table 1
    Profiling of periodontal status for analyses of early wound healing by modified early wound healing index

    Potential confounders of the mPPT were identified based on associations with the retention of blood clots at the mPPT sites and the severity of periodontal disease. The PD, CAL, BOP, plaque index, and radiographic bone defect depth at base line (BL) were measured before the surgical procedures, and the patient’s age was noted at the time of surgery. The location of the tooth was considered, as this can influence surgical handling. Other potential confounders of the mPPT included the bone defect shape, the side of the incision, the number of sutures, and the use of AGs. Stratified analyses were performed to determine whether these factors could potentially confound the mPPT before examining differences in mEHI between the EMD and rhFGF-2 groups.

    Statistical analysis

    The sample size for comparing early wound healing between the EMD and rhFGF-2 groups using the mEHI could not be precisely computed through power analysis due to the lack of precedent in previous research. To assess the inter-rater reliability of early wound healing evaluations using the mEHI, intraclass correlation coefficients (ICCs) were computed, and F-tests were used to identify significant differences between groups. To establish the sample size required for ICC assessment, we conducted an a posteriori test of 2 paired means for 2 groups using an effect size with a dose of 0.50 and an alpha risk of 0.05, in a 2-sided test performed with G*Power 3.1.9.7 (University of Düsseldorf, Düsseldorf, Germany). This calculation yielded a sample size of >26 participants in each group. We used the Welch t-distribution test, based on normality and variance, to evaluate statistical differences in the mEHI between the EMD and rhFGF-2 groups. We computed partial correlation coefficients to compare mEHI items between these groups. Quantitative variables, such as age and number of sutures, were stratified into 2 groups based on a previous study (age: <50 and ≥50 years) [31] and median values (number of sutures: ≤2 and ≥3) to investigate potential confounding effects on the mPPT. Categorical variables, such as affected teeth, bone defect shape, number of sutures, and incision side, were stratified into 2 or 3 groups based on category to evaluate potential confounders of the mPPT. Statistically significant differences in mEHI were analyzed between 3 subgroups of potential confounders using the Kruskal-Wallis test with the Steel multiple comparison test, or between 2 subgroups of potential confounders using the Student t-distribution test or the Welch t-distribution test, based on normality and variance. Mann-Whitney U tests were additionally used to compare levels of clinical improvement between BL and re-evaluation for various measures, such as PD and CAL. Differences in clinical parameters between the EMD and rhFGF-2 groups were analyzed using the Welch t-distribution test. The rates of sex and shape of bone defects were assessed using the Fisher exact test. A P value of <0.05 was considered to indicate statistical significance.

    RESULTS

    Characteristics of clinical parameters in the EMD and rhFGF-2 groups

    During the observational period, 171 sites received periodontal regeneration therapy using either EMD or rhFGF-2. For the evaluation of wound healing with the mEHI, 92 sites were excluded from the study. The reasons for exclusion were as follows: 82 sites did not undergo mPPT, 2 sites lacked oral photographs at the time of suture removal, and 8 sites were assessed outside of the suture removal period. Furthermore, when analyzing the relationship between wound healing and clinical outcomes 6 months after surgery, several sites were excluded. Specifically, 16 fell outside of the acceptable period (6 months ± 4 weeks) for radiographic examination (EMD, n=8; rhFGF2, n=8), and 7 could not be evaluated due to unclear bone crests (EMD, n=3; rhFGF2, n=4). Consequently, 79 sites (46 treated with EMD and 33 with rhFGF-2) were evaluated for wound healing using the mEHI (Figure 1). The clinical parameters at BL, which included sex, age, affected teeth, jaw, bone defect shape (contained or non-contained), period of suture removal, incision side, number of sutures, and AG, showed no significant differences between the EMD and rhFGF-2 groups (Table 1).

    Comparison of mEHI between EMD and rhFGF-2 groups

    Early wound healing was estimated using the mEHI by 20 evaluators across 79 patients. Significant differences were observed in the ICCs of the overall mEHI and 6 item scores, with the overall mEHI score demonstrating particularly strong correlation (0.575; 95% confidence interval, 0.485–0.666). The ICCs of the mEHI scores were comparable between the EMD and rhFGF-2 groups. Although the ICCs of the incision line and swelling were relatively weak, they were still deemed sufficient for subsequent analyses (Table 2).

    Table 2
    Inter-rater reliability in evaluations of early wound healing by mEHI

    The rhFGF-2 group demonstrated statistically higher scores for mEHI (38.6±9.40), redness (6.45±1.71), and dehiscence (7.55±1.70) compared to the EMD group (mEHI, 33.6±10.8; redness, 5.32±2.10; dehiscence, 6.34±2.09). However, while the other items in the rhFGF-2 group exhibited positive trends compared to the EMD group, no significant differences were identified (Table 3).

    Table 3
    Comparison of differences in mEHI between EMD and rhFGF-2 groups

    Comparison of mEHI between stratified potential confounders

    In the age-stratified groups (those under 50 years and those 50 or older), 5 mEHI items (all except swelling) had significantly higher scores in the group under 50 years compared to those aged 50 or older. Similarly, in the subgroup of molars, 5 mEHI items (all except fibrin clotting) had significantly higher scores compared to the premolar subgroup. In the non-contained group, the scores for fibrin clotting, step, and dehiscence were significantly higher than those in the contained group (Table 4). When considering technical factors such as the number of sutures, incision side, and AG, almost no mEHI scores showed statistically significant differences between the stratified confounders (Table 5).

    Table 4
    Differences of evaluations by mEHI in host factors

    Table 5
    Differences of evaluations by mEHI in technical factors

    Comparison of mEHI between EMD and rhFGF-2 subgroups, while accounting for confounders

    Considering the identified confounders for early wound healing (Tables 4 and 5), we compared the differences in mEHI items between the EMD and hrFGF-2 subgroups, stratifying by these confounders. In the subgroup aged under 50 years, the overall mEHI, incision line, redness, and dehiscence scores were significantly higher in the rhFGF-2 subgroup compared to the EMD subgroup. However, among those aged 50 years and older, no significant difference was observed in mEHI scores between the EMD and rhFGF-2 subgroups (as depicted in Figure 5). In the non-contained group, the mEHI, incision line, step, redness, and dehiscence scores were significantly higher in the rhFGF-2 subgroup compared to the EMD subgroup. Within the same non-contained group, however, no significant difference was noted in mEHI scores between the EMD and rhFGF-2 subgroups (Figure 6). No statistically significant differences were observed between the EMD and rhFGF-2 subgroups in the groups of teeth involved in the study (incisors, premolars, and molars; data not shown).

    Figure 5
    Box plots showing differences in the mEHI between the EMD and rhFGF-2 groups in the <50- and ≥50-year age subgroups.
    mEHI: modified early wound healing index, EMD: enamel matrix derivative, rhFGF-2: recombinant human fibroblast growth factor, N.S.: not significant.

    Statistically significant differences were observed between the ≥50-year EMD and other subgroups based on the Kruskal-Wallis test with the Steel multiple comparison test (a)P < 0.05, b)P<0.01) as well as between the EMD and rhFGF-2 groups based on the Welch t-test and normality-based t-test, respectively (a)P<0.05, b)P<0.01).

    Figure 6
    Box plots of mEHI differences between the EMD and rhFGF-2 groups in the contained and non-contained bone defect subgroups.
    mEHI: modified early wound healing index, EMD: enamel matrix derivative, rhFGF-2: recombinant human fibroblast growth factor, N.S.: not significant.

    Statistically significant differences were observed between the contained EMD and other subgroups according to the Kruskal-Wallis test with the Steel multiple comparison test (a)P<0.05, b)P<0.01), and between the EMD and rhFGF-2 groups according to the Welch t-test and normality-based t-test, respectively (a)P<0.05, b)P<0.01).

    Relationship between early wound healing as indicated by mEHI and 6-month postoperative outcomes

    A total of 56 participants were evaluated for RBF 6 months after surgery during the survey period (Figure 1). Significant improvements in PD and CAL at BL were observed in both the EMD and rhFGF-2 groups at the time of reassessment. The RBFs were 57.08%±21.70% and 57.25%±24.49% in the EMD and rhFGF-2 groups, respectively, with no significant differences detected between groups (Table 6). Furthermore, the mEHI evaluations did not reveal any significant correlation with these outcomes in either group at the 6-month postoperative assessment (data not shown).

    Table 6
    Profiling of periodontal examination in patients for analysis the influences of evaluations by modified early wound healing index on clinical outcomes 6 months after surgeries

    DISCUSSION

    This study is the first to compare early wound healing between the EMD and rhFGF-2 procedures, while taking into account factors influencing the mPPT procedure. Within the mEHI, the rhFGF-2 group exhibited higher redness and dehiscence scores than the EMD group, implying that the anti-inflammatory effects of rhFGF-2 may be superior to those of EMD. The clinical findings suggest that rhFGF-2 is more effective than EMD in facilitating early wound healing.

    To perform a statistically sound analysis of surgical wounds, we developed a reliable and reproducible method for quantification (Figure 2). Recognizing the need for multiple evaluators to assess numerous incision wounds, we structured this study using retrospective data. Given that the clearly visible horizontal incision line is traced at the buccal (mPPT) or palatal (or lingual) (PPT) gingiva at the base of the papilla, our investigation was confined to mPPT cases.

    The components of WC and EHI were recorded based on the conditions of fibrin clots and dehiscence, and they were categorized into 3 and 5 scales, respectively [14, 15]. However, the quantity and interdental span of fibrin clots, along with the mesial-distal extent of dehiscence, have not been detailed. Consequently, evaluating the EHI can be difficult in complex cases.

    In this study, we developed the mEHI method with the goal of providing assessments that offer appropriate numerical variations for the statistical analysis of wound healing. The panel was allotted 20 seconds per case for evaluation. Given that 79 cases were assessed consecutively, this may have posed challenges in the evaluation process. In fact, during data aggregation, we suspected some misdiagnoses, but these were neither re-evaluated nor corrected. Specifically, the ICCs for the mEHI items of incision line (0.392) and swelling (0.299) were relatively low (Table 2), indicating potential difficulties in assessing these parameters.

    Redness and dehiscence were more common in the rhFGF-2 group than in the EMD group (Table 2). This was also true for the subgroup of participants aged 50 years and above. However, among those aged under 50 years, the efficacy of rhFGF-2 was not evident (Figure 5). Aging is a gradual biological process characterized by a decline in cellular and organismal functions, and it is known to adversely impact wound healing [32]. The process of wound healing includes 4 overlapping phases: hemostasis, inflammation, proliferation, and tissue reconstruction. Hemostasis, which occurs immediately after surgery, results in vasoconstriction and the activation of platelets. These platelets secrete various soluble wound healing factors, leading to the formation of a fibrin clot through the activated coagulation pathway [33]. Delayed gingival wound repair is associated with reduced cellular responses due to aged blood factors [34]. These findings sufficiently support the idea that the notable efficacy of rhFGF-2 on wound healing may have been obscured by active hemostasis in the younger subgroup.

    The analysis of potential mPPT confounders revealed superior early wound healing in the non-contained subgroup compared to the contained subgroup (Table 4). Within the contained subgroup, rhFGF-2 was associated with higher scores for incision line, step, redness, and dehiscence compared to EMD. However, this pattern was not observed in the non-contained subgroup (Figure 6). The present study included 5 cases with 1-wall bone defects, and horizontal incisions of the mPPT at the lingual or palatal side were preferred to avoid aesthetic issues resulting from poor WC. Technically, threading the papilla is somewhat easier in non-contained bone defects than in contained ones, as thicker papillae can be preserved in the former. Consequently, papillae on non-contained bone defects may have an advantage in terms of securing blood supply from wider sections, potentially influencing differences in fibrin clot, step, and dehiscence evaluations. To confirm whether bone defect type was an influencing factor in mPPT wound healing, the distance between the horizontal incision line and thread point, as well as the thickness and width of the papilla, should also have been measured.

    Redness is a result of increased blood flow due to telangiectasis during inflammation and the initial phase of wound healing. The anti-inflammatory effects of EMD application have been reported to impact early wound healing. In a clinical trial comparing the levels of matrix metalloproteinase-1 and -8, along with tissue inhibitor of metalloproteinase-1, in crevicular fluid 12 days after surgery between flap operation and EMD groups, lower levels of these inflammatory markers were found in the EMD group [35]. Another study that combined EMD with bovine-derived bone grafting materials found that EMD was associated with a significant decrease in interleukin 1 beta expression [36]. Moreover, the adjunctive application of EMD during surgical treatment resulted in a slight increase in serum C-reactive protein level 24 hours after surgery [19]. Regarding the anti-inflammatory effects of FGF-2, prior findings indicate that FGF-2 downregulates the expression of cluster of differentiation (CD) 40 in the periodontal ligament cells (MPDL22). This inhibition of CD40 signaling by FGF-2 results in a decrease in the production of interleukin 6 and tumor necrosis factor alpha, both of which can recruit immune cells to inflamed sites [37]. However, no studies have yet documented the anti-inflammatory effects of rhFGF-2 in clinical trials for periodontal therapies. Additionally, the anti-inflammatory effects of EMD and rhFGF-2 must be compared both in vitro and in vivo.

    Dehiscence can occur in the papilla area following periodontal surgery. This not only stems from clear clinical failures such as incomplete flap closure and suture loss, but also may be associated with cell proliferation and extracellular matrix production subsequent to angiogenesis, both of which are crucial events in wound healing. EMD has been shown to stimulate gingival fibroblast proliferation, promote extracellular matrix production, and inhibit apoptosis [38]. Research has also shown that EMD treatment increases microvascular endothelial cell proliferation and chemotaxis [39] and induces angiogenesis due to elevated expression levels of endothelial vascular endothelial growth factor in gingival tissue biopsies of patients 48 hours after EMD surgery [40]. The angiogenic effects of EMD have been corroborated in an in vivo angiogenesis assay involving silicon tubes implanted subcutaneously in mice [40]. EMD also influences the proliferation, survival, adhesion, and migration of human umbilical vein endothelial cells [27]. While FGF-2 is recognized as one of many inducers of angiogenesis [27], no studies have directly compared the angiogenic effects of EMD and FGF-2. Consequently, it is necessary to compare the anti-inflammatory effects of EMD and rhFGF-2 using angiogenic biomarkers and clinical outcomes.

    In conclusion, the results of this retrospective cohort study indicate that rhFGF-2 demonstrates superior clinical efficacy in promoting early wound healing following mPPT, compared with EMD. The findings suggest that rhFGF-2 may have more potent anti-inflammatory effects than EMD. Within the limitations of this study, improved clinical periodontal outcomes were observed in the rhFGF-2 group, suggesting its potential for broader clinical use. Overall, these findings offer valuable insights into the adjunctive use of rhFGF-2 and EMD in periodontal surgery.

    Notes

    Conflict of Interest:No potential conflict of interest relevant to this article was reported.

    Author Contributions:

    • Conceptualization: Yohei Nakayama, Yorimasa Ogata.

    • Formal analysis: Yohei Nakayama.

    • Investigation: Yohei Nakayama, Shinichi Tabe, Kazuma Igarashi, Satoshi Moriya, Tsuyoshi Katsumata, Ryo Kobayashi, Shuta Nakagawa, Tomoko Nishino, Namiko Fukuoka, Kota Hosono, Mai Yamasaki, Yosuke Yamazaki, Moe Ogihara-Takeda, Shoichi Ito, Yumi Saito, Yuto Tsuruya, Arisa Yamaguchi, Mizuho Yamazaki-Takai, Shoichi Yoshino, Hideki Takai.

    • Methodology: Yohei Nakayama.

    • Project administration: Yorimasa Ogata.

    • Writing - original draft: Yohei Nakayama.

    • Writing - review & editing: Yohei Nakayama, Yorimasa Ogata.

    ACKNOWLEDGEMENTS

    This study received partial support from the Japan Society for the Promotion of Science (Chiyoda, Tokyo, Japan) through the KAKENHI Grants-in-Aid for Scientific Research (C), No. 21K09921, awarded to Y. N. We extend our thanks to Emiri Wada and Ayako Kato for their evaluation of wound healing by modified early wound healing index (mEHI).

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