Longitudinal proteomic analysis of GCF in regenerative healing of molar furcation degree II defects treated with OFD, EMD, or A-PRF+: a pilot study

Longitudinal proteomic analysis of GCF in regenerative healing of molar furcation degree II defects treated with OFD, EMD, or A-PRF+: a pilot study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Longitudinal proteomic analysis of GCF in regenerative healing of molar furcation degree II defects treated with OFD, EMD, or A-PRF+: a pilot study Luciano Pitzurra, E. Stamatelou, D. Vasdravellis, S. Bizzarro, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8065385/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract This study evaluated associations between protein expression and postoperative healing scores following three periodontal regenerative procedures (Open Flap Debridement–OFD, Enamel Matrix Derivative–EMD, Advanced Platelet Rich Fibrin–A-PRF+) in molar degree II furcations over six months. Associations between baseline systemic biochemical parameters and six-month clinical outcomes were also assessed. Eighteen patients were randomly assigned to control (OFD), EMD, or A-PRF + groups. Baseline plasma biomarkers were modeled on probing depth (PD), clinical attachment level (CAL), and alveolar bone level (ABL) at six months. Gingival crevicular fluid (GCF) samples were analyzed using the Olink® Target 96 Immuno-Oncology panel at day 3, weeks 1, 2, 6, and months 3 and 6. Multivariable linear regressions identified proteins associated with the modified Early Wound Healing Index (mEHI) per treatment. Seventeen patients completed follow-up. HDL- and LDL-cholesterol and creatinine were negatively associated with PD, CAL, and ABL improvements (p < 0.05). At early timepoints, CASP-8, IL-8, and LAP TGF-β1 showed stronger associations with mEHI in A-PRF + than OFD or EMD, while ARG-1 and TRAIL demonstrated treatment-specific patterns over time. No differences were observed at month 6. High baseline cholesterol and creatinine may impair periodontal regeneration. Distinct proteomic signatures suggest differential biological pathways underlying healing across OFD, EMD, and A-PRF + procedures. Health sciences/Biomarkers Biological sciences/Cancer Health sciences/Diseases Health sciences/Medical research Platelet rich fibrin (PRF) Periodontal regeneration Furcation defect Enamel matrix derivative (EMD) Proteomics Figures Figure 1 Figure 2 Figure 3 Figure 4 1 | Introduction The inflammatory process in periodontitis can lead to diverse patterns of periodontal ligament and alveolar bone destruction. One such pattern is the formation of intraosseous bone defects, creating irregular angular configurations in the alveolar bone structure along the tooth root 1 – 3 . In molar teeth, these destructive patterns often result in deep furcation degree II or III defects. Regenerative procedures of furcation degree II or III defects is most challenging in periodontal therapy 4 – 8 . Different techniques and materials have been explored for periodontal tissue regeneration, with positive results. Platelet-Rich Fibrin (PRF) has shown promising potential due to its ability to provide a scaffold rich in growth factors, cytokines and bioactive molecules which promote cell proliferation and migration of periodontal regenerative key players, such as fibroblasts and osteoblasts 5 , 9 – 15 . Therefore, PRF preparations may be promising for regeneration of furcation defects. Enamel Matrix Derivative (EMD) is a widely used material in regenerative periodontal surgery 16 , 17 . A vast body of literature on preclinical and clinal studies supports EMD as the material of choice, especially in the treatment of infrabony defects, in association with the correct microsurgical technique 16 , 18 , 19 . Despite the widespread use of regenerative materials, understanding the healing patterns following their application in the early wound healing phase remains challenging. In addition to well-known local factors 6 , periodontal healing may be modulated by a disbalance in circulatory biomarkers which reflects body’s overall state of health and which could affect tissue repair and angiogenesis. For example, Low-Density Lipoprotein (LDL) has been associated with delayed healing and increased risk of complications 20 – 25 . Thus, such systemic markers may also have an impact on wound healing of intra-osseous defects with or without regenerative approaches. Previously, we reported on the clinical potential of A-PRF + in the regenerative treatment of molars with furcation II defects, compared to the use of EMD and open flap debridement (OFD) 26 . The quality of healing was scored using a validated periodontal wound healing index. Clinical differences in the healing patterns among the treatment modalities were observed, leading to the need to better understand how the timing and the presence of different wound healing related proteins are involved. The analysis of gingival crevicular fluid (GCF), obtained at postoperative appointments, can discover key biomarkers and molecular patterns representing periodontal healing and tissue regeneration in defects treated using various regenerative approaches, therefore investigating healing beyond clinical observations 27 . Current proteomics techniques are sensitive to the smallest amount of proteins in minimal volumes of GCF 28 ,and allow to study and possibly identify a spectrum of proteins representing biological patterns related to periodontal healing after regenerative surgery 29 – 39 . Therefore, the aim of the current study is to explore whether certain protein expressions differed across three regenerative procedures (A-PRF+, EMD, OFD) in molar furcation degree II and corresponding wound healing scores during a postoperative follow-up of 6 months. In addition, we investigated whether selected systemic biochemical parameters at baseline could be associated with the clinical outcomes at 6 months postoperatively. 2 | Results 2.1 | Study population From 18 included patients, one (EMD) dropped out due to an endodontic complication. 17 patients were included for the final analysis (OFD: n = 6; EMD: n = 5; A-PRF+: n = 6) (Table 1). The median age was 60 years, 10 were the females, 6 were the smokers. Thirteen maxillary molars (OFD: 3, EMD: 3, A-PRF+: 6) and four mandibular molars (OFD: 2, EMD: 2) were included. Buccal furcations were present in 5 patients (OFD: 3, EMD: 2) and interproximal furcations in 12 (OFD: 3, EMD: 3, A-PRF+: 6). Horizontal furcation involvement ≥ 6 mm was observed in 3 OFD, 3 EMD, and 1 A-PRF + cases. Baseline mean Pocket Depth (PD) were 6.7 ± 1.9 mm (OFD), 7.4 ± 1.3 mm (EMD), and 5.7 ± 0.8 mm (A-PRF+); mean Clinical Attachment Level (CAL): 8.5 ± 2.4 mm, 8.4 ± 1.6 mm, and 6.5 ± 0.8 mm; and mean Alveolar Bone Level (ABL): 9.3 ± 2.6 mm, 9.0 ± 1.6 mm, and 8.2 ± 1.2 mm, respectively. 2.2 | Early Healing analysis The clinical healing outcomes have been published before 26 . Notably, 3 out 6 furcation II defects treated with A-PRF + regressed to I or 0, and one defect treated with EMD regressed to I. Figure 1 shows the modified Early Healing Index (mEHI) scores of the treated furcation sites per group for each follow-up examination. A-PRF + group presented a trend of higher healing scores, indicating delayed healing. A trend towards complete wound healing over the post-op period is visible in all groups, with A-PRF + showing higher heterogeneity in the healing patterns. The individual mEHI scores per patient at each time point are tabulated in Supplementary Table S1. 2.3 | Baseline systemic biochemical parameters and associations with 6-months postoperative results Table S2 lists the measured systemic parameters at baseline for the 3 groups. The median values of LDL and triglycerides were above the normal range for OFD, of LDL for EMD and of HbA1c, total cholesterol and LDL for A-PRF + cases. Table S3 presents the individual values and values above or below the normal range for each systemic parameter. We explored first in univariate linear regression modeling whether age, sex and smoking or any of the systemic biomarkers were associated with the final clinical outcomes, the 6 months results for PD, CAL and ABL (Table S4). Table 2 presents the results of the final multivariable linear regression models for PD, CAL and ABL, adjusted for treatment modality and the baseline PD. Baseline High-Density Lipoprotein (HDL) showed a significant association with 6-month PD and ABL (p = 0.018 and p = 0.023. respectively), 6-month CAL showed a significant association with baseline LDL and creatinine levels (p = 0.016 and p = 0.009). 2.4 | Proteins with significant interactions at postoperative timepoints The list of initially investigated proteins is shown in Table S5. Figure 2 shows the various data-processing steps for reaching the final dataset for proteomic analyses. resulting in a total of 46 proteins (Table S6). Figure 3 presents the interaction effects between clinical healing and surgical treatment at the various postoperative time points using volcano plots and corresponding regression lines for the protein Normalized Protein Expression (NPX) values having significant interactions with treatment and mEHI are shown in Fig. 4 . The significant interaction effects (Beta values) and p-values are presented in Table 3 . Thus, at day 3, CASP-8 and IL-8 show significant interaction effects between mEHI and A-PRF + treatment compared to EMD. Both have higher expressions at high values of mEHI (representing poorer wound healing) in A-PRF + compared to EMD (β = 4.74, p adj = 0.019; β = 6.53, p adj = 0.045, respectively). At week 1, CASP-8 and LAP TGF-beta-1, showed higher NPX in the high mEHI scores in A-PRF + compared to OFD (β = 6.7, p adj = 0.039; β = 6.14, p adj = 0.043, respectively). At week 6, ARG-1 levels were associated with less favorable wound healing in both A-PRF + and EMD when compared to OFD (β = 2.77, p adj = 0.041 for A-PRF + vs OFD; β = 2.48, p adj = 0.027 for EMD vs OFD). At month 3, multiple proteins (ARG-1, CD40, HGF, LAP TGF-β1, TRAIL, TWEAK and VEGFR-2) showed lower NPX in higher mEHI values in EMD versus OFD (β= − 1.15, p adj = 0.041; β= − 2.04, p adj = 0.037; β= − 3.52, p adj = 0.008; β= − 3.25, p adj = 0.013; β= − 3.17, p adj = 0.003; β= − 2.42, p adj = 0.009; β= − 0.84, p adj = 0.023). Additionally, at month 3, TRAIL showed higher NPX values in higher mEHI in A-PRF + compared to EMD (β = 2.82, p adj = 0.017). At month 6, no interaction effects for any protein between mEHI and treatment modality were found. 3 | Discussion This study was initially designed as a RCT evaluating clinical and biological healing in molar furcation degree II sites treated with OFD, EMD and A-PRF+. Findings from 17 cases followed for 6 months revealed distinct healing patterns. EMD showed the fastest early wound closure, A-PRF + showed a delayed response, but eventually had favorable clinical outcomes 26 . Patients having high levels of LDL-cholesterol and creatinine at baseline showed deeper residual PD and CAL at 6 months. A-PRF + showed delayed early healing, which coincided with the early overexpression of CASP-8 and IL-8 as well as with LAP TGF-β1 and ARG-1 patterns compared with OFD. In contrast, EMD was associated with faster healing (lower mEHI scores), reflected by lower levels of TRAIL, HGF, TWEAK, VEGFR-2, ARG-1, LAP TGF-β1, and CD40 at 3 months compared with OFD, suggesting a biologically advantageous trajectory. The association between higher cholesterol and LDL levels with deeper residual PD and less gain in CAL at six months. suggest that systemic lipid metabolism may influence periodontal wound healing 25 , 40 , 41 . These findings indicate that hyperlipidemia may impair periodontal healing, potentially through mechanisms involving oxidative stress and endothelial dysfunction 42 , 43 . Furthermore, elevated creatinine levels were associated with ABL at 6 months postoperatively, suggesting that also renal function is somehow involved or functions as a surrogate marker for the modulation of periodontal bone remodeling 44 , 45 . The observed associations corroborate previous findings about the importance of systemic health in the context of periodontal regeneration. The clinical assessment highlighted heterogeneity in postoperative healing patterns among treatment groups and the proteomic investigation was employed to better understand the clinical findings. The immuno-oncology panel from Olink® includes proteins linked to angiogenesis, proliferation, and inflammation, which guided its selection. At the earliest postoperative time point (day 3). OFD and EMD displayed similar healing patterns, whereas A-PRF + demonstrated delayed wound healing. reflected in higher mEHI scores. This was associated with elevated CASP-8 and IL-8 levels, suggesting a transient pro-apoptotic and pro-inflammatory environment 46 . CASP-8 is an initiator of extrinsic apoptosis and its downregulation after injury helps trigger healing. Its presence may indicate premature programmed cell death in response to the dense fibrin scaffold of A-PRF+, while IL-8 elevation highlights neutrophil recruitment and acute inflammatory activity 47 – 49 . Although a certain degree of IL-8 signaling is necessary for early matrix remodeling, excessive or prolonged expression is more consistent with tissue breakdown, which aligns with higher mEHI scores observed in the A-PRF + group 50 . The early elevation of these mediators in the A-PRF + treated defects may potentially be related to host resorption of the dense fibrin matrix and/or mediator release from A-PRF + itself. At week 1 postoperatively, clinical differences between the treatment groups became more pronounced. OFD and EMD showed lower mEHI scores, while A-PRF + continued to exhibit higher mEHI scores, with CASP-8 and LAP TGF-β1 strongly associated with inflammation persistence. TGF-β, normally a pivotal regulator of fibroblast activity and angiogenesis, was predominantly detected in its latent form (LAP–Latency-Associated Peptide), suggesting limited activation at this stage. This pattern may reflect delayed progression from inflammation to proliferation 30 . A complicating factor may contribute: we occasionally noted slight interdental papilla dehiscence after surgery in some A-PRF + treated sites, potentially sustaining inflammation and favoring undesired bacterial colonization before regeneration proceeds. At week 2, none of the proteins exhibited a significant association with any of the procedures. Likely, relevant proteins at this stage were not part of the analyzed set. At week 6 postoperatively, OFD and EMD still present better healing scores than A-PRF+. The emergence of ARG-1 as a key marker indicated a shift toward resolution of inflammation and tissue remodeling, particularly in A-PRF + and EMD. ARG-1 is associated with alternatively activated M2 macrophages and promotes healing by suppressing nitric oxide production and enhancing matrix deposition 51 , 52 . This suggests that, despite slower initiation, both biologically active treatments eventually engaged reparative immune pathways more effectively than OFD 53 . At month 3, although the wound healing was mostly completed, proteomic differences remained evident. OFD and EMD showed lower mEHI scores compared with A-PRF+. indicating more favorable outcomes. In EMD, a cluster of proteins including TRAIL, HGF, VEGFR-2, TWEAK, ARG-1, and CD40 reflected remodeling activity rather than unresolved inflammation. TRAIL and HGF, in particular, are known to support angiogenesis, epithelial proliferation, and tissue maturation when appropriately regulated 54 . HGF, a pleiotropic factor involved in epithelial proliferation, angiogenesis, and mesenchymal remodeling, has been widely associated with wound closure and scar attenuation 55 . VEGFR-2 signaling further reinforced the angiogenic response, while CD40 reflected immune activation tightly linked to tissue repair processes. CD40, expressed on fibroblasts, epithelial, and immune cells, links inflammation to tissue repair through NF-κB–mediated induction of IL-6. IL-8, and ICAM-1. While this signaling supports immune activation during early healing, excessive or prolonged CD40 activity may perpetuate inflammation and hinder repair 56 . Pro-regenerative factors such as FGF-2 can suppress CD40 signaling, promoting resolution and tissue remodeling. In acute wounds, platelet-derived CD40L may act as an early alarmin. The stronger correlation of TRAIL with A-PRF + suggests its function to be context-dependent; the regenerative potential of EMD, mediated by amelogenins, appears to facilitate not only early fibroblast proliferation but also a more rapid and controlled transition to the remodeling stage, consistent with previous reports of accelerated periodontal regeneration. In fact, previous studies have demonstrated that EMD can accelerate periodontal regeneration by enhancing the recruitment and differentiation of mesenchymal cells, while also suppressing the inflammatory response in a favorable manner for wound stabilization 16 , 57 . At the 6-month follow-up, no protein–procedure associations were observed, suggesting that wound healing was complete in all cases. In our data, the association between low levels of early pro-inflammatory biomarkers such as TRAIL, HGF, LAP TGF-β1, VEGFR-2, C040 and ARG-1, aligned with lower wound healing scores, supporting a quicker transition to reparative and remodeling stages 58 , 59 . By contrast, although PRF is commonly viewed as a fibrin scaffold that gradually releases PDGF, VEGF, and TGF-β to support angiogenesis and osteogenesis 15 , 60 , our findings in molar furcation sites showed a different early biology: A-PRF + was linked to stronger acute-phase signaling and slower early closure. While this slower initial healing response may not be ideal for cases requiring rapid wound stabilization, it could be beneficial for long-term periodontal regeneration, particularly in cases where gradual tissue remodeling is desirable 61 . In fact, in our cohort, 3 cases out of 6 treated with A-PRF + showed furcation degree reduction, making this material apparently promising. The main limitations of this study are the small sample size and the uneven distribution of age and furcation types due to interrupted allocation. Strengths include its prospective design, randomization, standardized surgical protocol, and the exploratory integration of systemic and proteomic biomarkers with clinical outcomes. These preliminary findings highlight proteins potentially involved in wound healing and may guide future larger trials on periodontal regeneration in furcation defects. To conclude, elevated baseline cholesterol and creatinine levels may adversely influence PD reduction, as well as CAL and ABL gain. at 6 months following surgery in degree II molar furcation defects. Overall, our findings indicate that, across the OFD, EMD, and A-PRF + procedures, distinct protein expression profiles were associated with different postoperative healing scores. Although A-PRF + exhibited a slower and more heterogeneous early healing response characterized by stronger acute-phase signaling, the improvement in furcation degree observed in A-PRF + treated sites suggests that A-PRF + may offer promising regenerative potential. 4 | Materials and Methods 4.1 | Ethics. study design. patient inclusion The current study is a further analysis, based on Pitzurra et al. 26 , which received approval from the medical ethics committee of the VU University Medical Center Amsterdam (study protocol number: 6265602917). All experimental procedures were performed in accordance with relevant guidelines and regulations (Declaration of Helsinki). Written informed consent was obtained from all participants prior to their inclusion in the study. Inclusion and exclusion criteria, and a comprehensive description of the study protocol, randomization and allocation are previously published 26 . The study was initially planned as a fully powered clinical randomized controlled trial (RCT); however, the study was prematurely terminated due to the COVID-19 pandemic. The study was registered with the ISRCTN registry (ISRCTN13520922) on 17 October 2018. We report here the results of 17 cases randomized into three groups. 4.2 | Clinical procedures. GCF collection and sample processing Briefly, in 18 patients, the selected molar furcation degree II site underwent periodontal surgery, either with an OFD approach (n = 6) or with the application of EMD (n = 6) or A-PRF+ (n = 6). Sites were monitored at different time points from baseline to 6 months postoperatively. Paper strips for periodontal use (Periopaper™, Oraflow, Smithtown, NY, USA) were used to collect GCF. First, the tooth was isolated with cotton rolls. Supragingival plaque was removed with curettes and the tooth was gently dried with a gentle stream of air. The paper strip was then inserted in the sulcus and left in place to soak GCF for 30 seconds, avoiding any contact with saliva. Next, GCF strips were transferred into 1.5 mL centrifuge tubes, placed on ice and within two hours stored at -80°C. Samples were taken at the postoperative time points day 3, weeks 1, 2, 6, month 3 and 6. 4.3 | Blood sampling and processing The blood drawing procedure was performed by venipuncture of the antecubital fossa immediately before the surgery to prepare A-PRF+. Four tubes of blood (40 ml) were taken from each patient. Two of them were used for the preparation of the A-PRF+ (sterile plain glass-based vacuum tubes). Two other tubes (with EDTA), not to be used for the preparation of the A-PRF+, were transported within 1 hour to the clinical-biochemical laboratory of the Amsterdam University Medical Center (AUMC) (Study ID: P17-148. PIN 2712681). From the venous blood samples, the following were measured: blood cell counts (total leukocytes, including leuko-differentiations [lymphocytes, monocytes, neutrophils, eosinophils, basophils] and platelets), HbA1c, albumin, ferritin, hemoglobin, total cholesterol plus low-density lipoproteins (LDL) and high-density lipoproteins (HDL) levels, triglycerides, creatinine, and estimated glomerular filtration rate (eGFR). 4.4 | Surgical treatment and wound healing evaluation The complete surgical procedures are previously reported 26 . Surgeries were carried out by an experienced periodontist (SB) using a microsurgical approach (simple and modified papilla preservation). During surgery, patients were randomly assigned to receive either A-PRF+, EMD, or open flap debridement (OFD). For A-PRF+, membranes were minced, placed into the furcation defect, and then stabilized. For EMD, roots were dried first and the material was applied and left undisturbed for two minutes prior to flap closure. Tension-free sutures (6 − 0 polypropylene) were placed allowing primary closure. Patients avoided brushing the surgical area and rinsed with hydrogen peroxide (1.5%) and chlorhexidine (0.12%) for four weeks; sutures were removed at day 14 with polishing at day 7, week 2, and week 6. From week 4, mechanical plaque control was resumed, and a 3-month maintenance visit included supragingival instrumentation and hygiene reinforcement. Clinical measurements of the treated furcation sites took place at baseline and at six months. Early wound healing was scored at all postoperative time points using a modification on the Early Healing Index (mEHI). The original EHI consists of five categories for the degree of flap closure: ‘1’ complete flap closure—no fibrin line in the interproximal area; ‘2’ complete flap closure—fine fibrin line in the interproximal area; ‘3’complete flap closure—fibrin clot in the interproximal area; ‘4’ incomplete flap closure—partial necrosis of the interproximal tissue; ‘5’ incomplete flap closure—complete necrosis of the interproximal tissue. For the current study. score ‘0’ was added to this index. representing no wound visible. The examiner (DV) was calibrated for reproducibility (kappa = 0.80). 4.5 | GCF sample preparation and protein analysis GCF samples were transported on dry ice to the Arcadia Core Facility, University Medical Center Utrecht (The Netherlands) for proteomic analysis. Each strip was incubated with 15 µL 1X PBS, pH 7.4 (DPBS, Corning Life Science, Tewksbury, MA, USA) containing protease inhibitors (Complete Mini, EDTA free, Sigma-Aldrich, St. Louis, MO, USA), vortexed, centrifuged, and incubated with an additional 15 µL PBS for 15 min at room temperature. After centrifugation (10.000 × g. 4°C. 5 min), supernatants were collected and stored at − 80°C until analysis. Proteomic profiling was performed using Proximity Extension Assay (PEA) technology with the Olink® Target 96 Immuno-Oncology panel (Olink Proteomics AB. Uppsala. Sweden) following the manufacturer’s protocol. This panel was chosen for its collection of proteins related to angiogenesis. cell proliferation and inflammation. Four spiked-in internal controls were included to monitor assay and sample performance. Protein levels were reported as Normalized Protein Expression (NPX) values on a log2 scale. 4.6 | Data analysis 4.6.1 | Systemic biomarkers and clinical outcomes We assessed associations between baseline systemic blood biomarkers and 6-month clinical outcomes (PD, CAL, ABL) using linear regression. Treatment group and baseline PD were always retained in all models to adjust for potential confounding. Univariable analyses were performed for each biochemical parameter individually, as well as for age, sex and smoking, followed by multivariable linear regression with backward selection ( p > 0.05 for removal), incorporating variables with p 25% missing frequency among all samples were excluded to ensure assay reliability 62 . Associations between proteins, mEHI, and treatment groups per timepoint were assessed using multivariable linear regression with interaction terms between the mEHI and treatment group, using OFD or EMD as reference categories against A-PRF+. Benjamini–Hochberg correction was performed for multiple testing ( α = 0.05). Analyses were conducted in Python v3.9.6 with Pandas, Numpy, Statsmodels, Plotly, and Seaborn. 4.6.3 | Data availability The raw data and code is available from the corresponding author ( [email protected] ) on reasonable request. Declarations Author contributions L.P., S.B., B.L.: Conceptualization and design of study. L.P., E.S., B.L,.: Data Analysis.L.P., S.B., E.S. , B.L.: Wrote the main manuscript text. S.B., B.L.: Supervision, Reviewing and editing the manuscript. L.P., D.V.: Recruitment, Administrative and IRB submission. Funding information and conflict of interest This study was funded in part by (1) the Department of Periodontology. Academic Centre for Dentistry Amsterdam (ACTA). Amsterdam. The Netherlands; (2) the ORANGEForce project within the ORANGEHealth consortium funded by Health-Holland.nl. div. Life Sciences & Health with grant number LSHM21064; (3) self-funded. 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Periodontol . 30 , 496–504. https://doi.org:10.1034/j.1600-051x.2003.00013.x (2003). Tables Table 1. Patient and periodontal characteristics at baseline. Values represent number or median and percentage (%) or range. Abbreviations: N, number of patients; OFD, open flap debridement; EMD, enamel matrix derivatives; A-PRF+, advanced platelet rich fibrin +. Further description of patient characteristics in 26 . Treatment Group Number or Median % or Range OFD (n=6) Age 51.5 33-60 Sex (female) 3 50% Smoking 2 33% EMD (n=5) Age 57 41-61 Sex (female) 4 80% Smoking 2 40% A-PRF+ (n=6) Age 66.5 61-78 Sex (female) 3 50% Smoking 2 33% Total Age 60 33-78 Sex (female) 10 58.8% Smoking 6 (5.5 sig/day) 35% Table 2. Multivariable linear regression modeling for the association between baseline biochemical parameters and 6-month Pocket Depth (PD). 6 months Clinical Attachment Level (CAL) and 6 months Alveolar bone level (ABL). P value < 0.05. The variables treatment group and baseline PD were always forced into the univariable and multivariable models. Abbreviations: HDL, high-density lipoprotein; LD,. low-density lipoprotein. Final multivariable models 6 months PD 6 monthts CAL 6 months ABL Beta Std. Error P-value Beta Std. Error P-value Beta Std. Error P-value H DL 2.163 0.787 0.018 3.144 1.203 0.023 LDL 0.746 0.262 0.016 Creatinine 0.084 0.027 0.009 Table 3. Proteins with significant interaction effects between treatment and mEHI at different postoperative timepoints. P value < 0.05. Beta (corresponding 95%CI, p-value, adjusted p-value) quantify the interaction effect between treatment and mEHI in the multivariate linear regression model. Abbreviations: CI, confidence interval; mEHI, modified early healing index; OFD, open flap debridement; EMD, enamel matrix derivatives; A-PRF+, advanced platelet rich fibrin +; vs, versus representing the reference treatment. Full names of proteins can be found in Table 2. Protein Timepoint Beta CI p - value Adjusted p - value T reatment × m EHI interaction effect CASP-8 Day 3 4.74 1.98; 7.49 0.0033 0.0199 A-PRF+ (vs EMD) IL-8 Day 3 6.53 2.17; 10.88 0.0075 0.0450 A-PRF+ (vs EMD) CASP-8 Week 1 6.70 2.28; 11.11 0.0066 0.0397 A-PRF+ (vs OFD) LAP TGF-beta-1 Week 1 6.14 2.03; 10.25 0.0072 0.0432 A-PRF+ (vs OFD) ARG-1 Week 6 2.77 0.88; 4.67 0.0081 0.0418 EMD (vs OFD) ARG-1 Week 6 2.49 0.95; 4.03 0.0045 0.0272 A-PRF+ (vs OFD) TRAIL Month 3 -3.17 -4.08; -2.26 0.00004 0.0003 EMD (vs OFD) HGF Month 3 -3.52 -5.22; -1.82 0.0014 0.0084 EMD (vs OFD) LAP TGF-beta-1 Month 3 -3.25 -4.93; -1.56 0.0022 0.0131 EMD (vs OFD) VEGFR-2 Month 3 -0.84 -1.32; -0.35 0.0040 0.0238 EMD (vs OFD) TWEAK Month 3 -2.42 -3.62; -1.23 0.0016 0.0096 EMD (vs OFD) ARG-1 Month 3 -1.15 -1.99; -0.30 0.0139 0.0418 EMD (vs OFD) CD40 Month 3 -2.04 -3.31; -0.76 0.0062 0.0371 EMD (vs OFD) TRAIL Month 3 2.82 1.29; 4.35 0.0028 0.0171 A-PRF+ (vs EMD) Additional Declarations No competing interests reported. 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16:18:23","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46669,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8065385/v1/88978de5ebc68d6d937b10c9.png"},{"id":97260899,"identity":"134960e2-c3bc-46c4-8a40-c0c13aa07abe","added_by":"auto","created_at":"2025-12-02 13:59:43","extension":"png","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":103994,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8065385/v1/fd65a138189b8afb89214a05.png"},{"id":97260890,"identity":"d85f91be-c907-4630-8a51-7d6ef9f7348a","added_by":"auto","created_at":"2025-12-02 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13:59:43","extension":"xml","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":427678,"visible":true,"origin":"","legend":"","description":"","filename":"e3e6734c6b744c969345fbd58f1ed7ad1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8065385/v1/5a43ae24703c428f1dd773b8.xml"},{"id":97260901,"identity":"6629661c-e6bd-49e9-b9cc-57dcfd6e285f","added_by":"auto","created_at":"2025-12-02 13:59:43","extension":"html","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":438601,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8065385/v1/841a5af614563dd3ba0e9516.html"},{"id":97260864,"identity":"ed97404a-f0b3-4dd9-927b-5d3909ef5bab","added_by":"auto","created_at":"2025-12-02 13:59:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":157155,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBox and whisker plot of the mEHI\u003c/strong\u003e \u003csup\u003e63\u003c/sup\u003e of treated sites for each group across follow-up intervals (Day 3, week 1, week 2, week 6, month 3, month 6). For each group (OFD, EMD, A-PRF+), the box represents the interquartile range (25th–75th percentiles), the line inside the box marks the \u003cstrong\u003emedian\u003c/strong\u003e. 1 patient (EMD group) did not attend the 1 week follow-up examination because of reported illness.\u003c/p\u003e\n\u003cp\u003eAbbreviations: OFD, open flap debridement; EMD, enamel matrix derivatives; A-PRF+, advanced platelet rich fibrin +.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8065385/v1/561625ccfe18b54e048511d1.png"},{"id":97260863,"identity":"c36e3368-a828-4a64-aaa1-e111934ae8a9","added_by":"auto","created_at":"2025-12-02 13:59:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":53617,"visible":true,"origin":"","legend":"\u003cp\u003eFlowchart and data cleaning of the proteomic analysis of the study. Abbreviations: mo, month.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8065385/v1/34cd4f2e4cc861218da4e1d1.png"},{"id":97366991,"identity":"dfdfce79-35b0-444b-841a-60e3d3aa47d5","added_by":"auto","created_at":"2025-12-03 16:15:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":391242,"visible":true,"origin":"","legend":"\u003cp\u003eVolcano plot representing the interaction between treatment group and mEHI. The x-axis represents the beta coefficients andthe y-axis by the corresponding negative logarithm of the adjusted p-values. Each protein is depicted trice in each panel. Panel A: day 3; panel B: week 1; panel C: 2 week 2; panel D: week 6; panel E: month 3; panel F: month 6. A significance threshold of α = 0.05 (corresponding to -log10(p) ≈ 1.3) is visually indicated by the red dashed line. Points above the red dashed line (corresponding to adjusted p \u0026lt;0.05) represent statistically significant interactions. Abbreviations: mEHI, modified early wound healing index; full names of proteins in the panels can be retrieved in Supplementary Table 2.\u003c/p\u003e","description":"","filename":"FIGURE3.png","url":"https://assets-eu.researchsquare.com/files/rs-8065385/v1/3eb6e15f2e73c22d6042f578.png"},{"id":97260865,"identity":"db9ef853-8bc7-4149-bd5c-62e5c17c8527","added_by":"auto","created_at":"2025-12-02 13:59:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":180320,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8065385/v1/087ba87f3937d360930e644a.png"},{"id":103251146,"identity":"c2c832d1-149d-46fc-9e3c-07aaf35ba261","added_by":"auto","created_at":"2026-02-23 16:05:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1753214,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8065385/v1/a827ff2d-7892-4697-a908-944bdbf3f3b6.pdf"},{"id":97260862,"identity":"81a17789-649e-420d-9cba-97cad4e25b7f","added_by":"auto","created_at":"2025-12-02 13:59:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":57413,"visible":true,"origin":"","legend":"","description":"","filename":"SuppTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8065385/v1/b7e0c0ddf640edbc78eafd29.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Longitudinal proteomic analysis of GCF in regenerative healing of molar furcation degree II defects treated with OFD, EMD, or A-PRF+: a pilot study","fulltext":[{"header":"1 | Introduction","content":"\u003cp\u003eThe inflammatory process in periodontitis can lead to diverse patterns of periodontal ligament and alveolar bone destruction. One such pattern is the formation of intraosseous bone defects, creating irregular angular configurations in the alveolar bone structure along the tooth root\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In molar teeth, these destructive patterns often result in deep furcation degree II or III defects. Regenerative procedures of furcation degree II or III defects is most challenging in periodontal therapy\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6 CR7\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDifferent techniques and materials have been explored for periodontal tissue regeneration, with positive results. Platelet-Rich Fibrin (PRF) has shown promising potential due to its ability to provide a scaffold rich in growth factors, cytokines and bioactive molecules which promote cell proliferation and migration of periodontal regenerative key players, such as fibroblasts and osteoblasts\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13 CR14\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Therefore, PRF preparations may be promising for regeneration of furcation defects. Enamel Matrix Derivative (EMD) is a widely used material in regenerative periodontal surgery\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. A vast body of literature on preclinical and clinal studies supports EMD as the material of choice, especially in the treatment of infrabony defects, in association with the correct microsurgical technique\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDespite the widespread use of regenerative materials, understanding the healing patterns following their application in the early wound healing phase remains challenging. In addition to well-known local factors\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, periodontal healing may be modulated by a disbalance in circulatory biomarkers which reflects body\u0026rsquo;s overall state of health and which could affect tissue repair and angiogenesis. For example, Low-Density Lipoprotein (LDL) has been associated with delayed healing and increased risk of complications\u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Thus, such systemic markers may also have an impact on wound healing of intra-osseous defects with or without regenerative approaches.\u003c/p\u003e\u003cp\u003ePreviously, we reported on the clinical potential of A-PRF\u0026thinsp;+\u0026thinsp;in the regenerative treatment of molars with furcation II defects, compared to the use of EMD and open flap debridement (OFD)\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The quality of healing was scored using a validated periodontal wound healing index. Clinical differences in the healing patterns among the treatment modalities were observed, leading to the need to better understand how the timing and the presence of different wound healing related proteins are involved. The analysis of gingival crevicular fluid (GCF), obtained at postoperative appointments, can discover key biomarkers and molecular patterns representing periodontal healing and tissue regeneration in defects treated using various regenerative approaches, therefore investigating healing beyond clinical observations\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Current proteomics techniques are sensitive to the smallest amount of proteins in minimal volumes of GCF\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e,and allow to study and possibly identify a spectrum of proteins representing biological patterns related to periodontal healing after regenerative surgery\u003csup\u003e\u003cspan additionalcitationids=\"CR30 CR31 CR32 CR33 CR34 CR35 CR36 CR37 CR38\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTherefore, the aim of the current study is to explore whether certain protein expressions differed across three regenerative procedures (A-PRF+, EMD, OFD) in molar furcation degree II and corresponding wound healing scores during a postoperative follow-up of 6 months. In addition, we investigated whether selected systemic biochemical parameters at baseline could be associated with the clinical outcomes at 6 months postoperatively.\u003c/p\u003e"},{"header":"2 | Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 | Study population\u003c/h2\u003e\n \u003cp\u003eFrom 18 included patients, one (EMD) dropped out due to an endodontic complication. 17 patients were included for the final analysis (OFD: n\u0026thinsp;=\u0026thinsp;6; EMD: n\u0026thinsp;=\u0026thinsp;5; A-PRF+: n\u0026thinsp;=\u0026thinsp;6) (Table\u0026nbsp;1). The median age was 60 years, 10 were the females, 6 were the smokers. Thirteen maxillary molars (OFD: 3, EMD: 3, A-PRF+: 6) and four mandibular molars (OFD: 2, EMD: 2) were included. Buccal furcations were present in 5 patients (OFD: 3, EMD: 2) and interproximal furcations in 12 (OFD: 3, EMD: 3, A-PRF+: 6). Horizontal furcation involvement\u0026thinsp;\u0026ge;\u0026thinsp;6 mm was observed in 3 OFD, 3 EMD, and 1 A-PRF\u0026thinsp;+\u0026thinsp;cases. Baseline mean Pocket Depth (PD) were 6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 mm (OFD), 7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 mm (EMD), and 5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 mm (A-PRF+); mean Clinical Attachment Level (CAL): 8.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 mm, 8.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 mm, and 6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 mm; and mean Alveolar Bone Level (ABL): 9.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 mm, 9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 mm, and 8.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 mm, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 | Early Healing analysis\u003c/h2\u003e\n \u003cp\u003eThe clinical healing outcomes have been published before \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Notably, 3 out 6 furcation II defects treated with A-PRF\u0026thinsp;+\u0026thinsp;regressed to I or 0, and one defect treated with EMD regressed to I. Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e shows the modified Early Healing Index (mEHI) scores of the treated furcation sites per group for each follow-up examination. A-PRF\u0026thinsp;+\u0026thinsp;group presented a trend of higher healing scores, indicating delayed healing. A trend towards complete wound healing over the post-op period is visible in all groups, with A-PRF\u0026thinsp;+\u0026thinsp;showing higher heterogeneity in the healing patterns. The individual mEHI scores per patient at each time point are tabulated in Supplementary Table S1.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 | Baseline systemic biochemical parameters and associations with 6-months postoperative results\u003c/h2\u003e\n \u003cp\u003eTable S2 lists the measured systemic parameters at baseline for the 3 groups. The median values of LDL and triglycerides were above the normal range for OFD, of LDL for EMD and of HbA1c, total cholesterol and LDL for A-PRF\u0026thinsp;+\u0026thinsp;cases. Table S3 presents the individual values and values above or below the normal range for each systemic parameter.\u003c/p\u003e\n \u003cp\u003eWe explored first in univariate linear regression modeling whether age, sex and smoking or any of the systemic biomarkers were associated with the final clinical outcomes, the 6 months results for PD, CAL and ABL (Table S4). Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e presents the results of the final multivariable linear regression models for PD, CAL and ABL, adjusted for treatment modality and the baseline PD. Baseline High-Density Lipoprotein (HDL) showed a significant association with 6-month PD and ABL (p\u0026thinsp;=\u0026thinsp;0.018 and p\u0026thinsp;=\u0026thinsp;0.023. respectively), 6-month CAL showed a significant association with baseline LDL and creatinine levels (p\u0026thinsp;=\u0026thinsp;0.016 and p\u0026thinsp;=\u0026thinsp;0.009).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 | Proteins with significant interactions at postoperative timepoints\u003c/h2\u003e\n \u003cp\u003eThe list of initially investigated proteins is shown in Table S5. Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e shows the various data-processing steps for reaching the final dataset for proteomic analyses. resulting in a total of 46 proteins (Table S6). Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e presents the interaction effects between clinical healing and surgical treatment at the various postoperative time points using volcano plots and corresponding regression lines for the protein Normalized Protein Expression (NPX) values having significant interactions with treatment and mEHI are shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. The significant interaction effects (Beta values) and p-values are presented in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. Thus, at day 3, CASP-8 and IL-8 show significant interaction effects between mEHI and A-PRF\u0026thinsp;+\u0026thinsp;treatment compared to EMD. Both have higher expressions at high values of mEHI (representing poorer wound healing) in A-PRF\u0026thinsp;+\u0026thinsp;compared to EMD (\u0026beta;\u0026thinsp;=\u0026thinsp;4.74, p\u003csub\u003eadj\u003c/sub\u003e= 0.019; \u0026beta;\u0026thinsp;=\u0026thinsp;6.53, p\u003csub\u003eadj\u003c/sub\u003e= 0.045, respectively). At week 1, CASP-8 and LAP TGF-beta-1, showed higher NPX in the high mEHI scores in A-PRF\u0026thinsp;+\u0026thinsp;compared to OFD (\u0026beta;\u0026thinsp;=\u0026thinsp;6.7, p\u003csub\u003eadj\u003c/sub\u003e= 0.039; \u0026beta;\u0026thinsp;=\u0026thinsp;6.14, p\u003csub\u003eadj\u003c/sub\u003e= 0.043, respectively). At week 6, ARG-1 levels were associated with less favorable wound healing in both A-PRF\u0026thinsp;+\u0026thinsp;and EMD when compared to OFD (\u0026beta;\u0026thinsp;=\u0026thinsp;2.77, p\u003csub\u003eadj\u003c/sub\u003e= 0.041 for A-PRF\u0026thinsp;+\u0026thinsp;vs OFD; \u0026beta;\u0026thinsp;=\u0026thinsp;2.48, p\u003csub\u003eadj\u003c/sub\u003e= 0.027 for EMD vs OFD). At month 3, multiple proteins (ARG-1, CD40, HGF, LAP TGF-\u0026beta;1, TRAIL, TWEAK and VEGFR-2) showed lower NPX in higher mEHI values in EMD versus OFD (\u0026beta;= \u0026minus;\u0026thinsp;1.15, p\u003csub\u003eadj\u003c/sub\u003e= 0.041; \u0026beta;= \u0026minus;\u0026thinsp;2.04, p\u003csub\u003eadj\u003c/sub\u003e= 0.037; \u0026beta;= \u0026minus;\u0026thinsp;3.52, p\u003csub\u003eadj\u003c/sub\u003e= 0.008; \u0026beta;= \u0026minus;\u0026thinsp;3.25, p\u003csub\u003eadj\u003c/sub\u003e= 0.013; \u0026beta;= \u0026minus;\u0026thinsp;3.17, p\u003csub\u003eadj\u003c/sub\u003e= 0.003; \u0026beta;= \u0026minus;\u0026thinsp;2.42, p\u003csub\u003eadj\u003c/sub\u003e= 0.009; \u0026beta;= \u0026minus;\u0026thinsp;0.84, p\u003csub\u003eadj\u003c/sub\u003e= 0.023). Additionally, at month 3, TRAIL showed higher NPX values in higher mEHI in A-PRF\u0026thinsp;+\u0026thinsp;compared to EMD (\u0026beta;\u0026thinsp;=\u0026thinsp;2.82, p\u003csub\u003eadj\u003c/sub\u003e= 0.017). At month 6, no interaction effects for any protein between mEHI and treatment modality were found.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3 | Discussion","content":"\u003cp\u003eThis study was initially designed as a RCT evaluating clinical and biological healing in molar furcation degree II sites treated with OFD, EMD and A-PRF+. Findings from 17 cases followed for 6 months revealed distinct healing patterns. EMD showed the fastest early wound closure, A-PRF\u0026thinsp;+\u0026thinsp;showed a delayed response, but eventually had favorable clinical outcomes \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Patients having high levels of LDL-cholesterol and creatinine at baseline showed deeper residual PD and CAL at 6 months. A-PRF\u0026thinsp;+\u0026thinsp;showed delayed early healing, which coincided with the early overexpression of CASP-8 and IL-8 as well as with LAP TGF-β1 and ARG-1 patterns compared with OFD. In contrast, EMD was associated with faster healing (lower mEHI scores), reflected by lower levels of TRAIL, HGF, TWEAK, VEGFR-2, ARG-1, LAP TGF-β1, and CD40 at 3 months compared with OFD, suggesting a biologically advantageous trajectory.\u003c/p\u003e\u003cp\u003eThe association between higher cholesterol and LDL levels with deeper residual PD and less gain in CAL at six months. suggest that systemic lipid metabolism may influence periodontal wound healing\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. These findings indicate that hyperlipidemia may impair periodontal healing, potentially through mechanisms involving oxidative stress and endothelial dysfunction\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Furthermore, elevated creatinine levels were associated with ABL at 6 months postoperatively, suggesting that also renal function is somehow involved or functions as a surrogate marker for the modulation of periodontal bone remodeling\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. The observed associations corroborate previous findings about the importance of systemic health in the context of periodontal regeneration.\u003c/p\u003e\u003cp\u003eThe clinical assessment highlighted heterogeneity in postoperative healing patterns among treatment groups and the proteomic investigation was employed to better understand the clinical findings. The immuno-oncology panel from Olink\u0026reg; includes proteins linked to angiogenesis, proliferation, and inflammation, which guided its selection. At the earliest postoperative time point (day 3). OFD and EMD displayed similar healing patterns, whereas A-PRF\u0026thinsp;+\u0026thinsp;demonstrated delayed wound healing. reflected in higher mEHI scores. This was associated with elevated CASP-8 and IL-8 levels, suggesting a transient pro-apoptotic and pro-inflammatory environment\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. CASP-8 is an initiator of extrinsic apoptosis and its downregulation after injury helps trigger healing. Its presence may indicate premature programmed cell death in response to the dense fibrin scaffold of A-PRF+, while IL-8 elevation highlights neutrophil recruitment and acute inflammatory activity\u003csup\u003e\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Although a certain degree of IL-8 signaling is necessary for early matrix remodeling, excessive or prolonged expression is more consistent with tissue breakdown, which aligns with higher mEHI scores observed in the A-PRF\u0026thinsp;+\u0026thinsp;group\u003csup\u003e50\u003c/sup\u003e. The early elevation of these mediators in the A-PRF\u0026thinsp;+\u0026thinsp;treated defects may potentially be related to host resorption of the dense fibrin matrix and/or mediator release from A-PRF\u0026thinsp;+\u0026thinsp;itself.\u003c/p\u003e\u003cp\u003eAt week 1 postoperatively, clinical differences between the treatment groups became more pronounced. OFD and EMD showed lower mEHI scores, while A-PRF\u0026thinsp;+\u0026thinsp;continued to exhibit higher mEHI scores, with CASP-8 and LAP TGF-β1 strongly associated with inflammation persistence. TGF-β, normally a pivotal regulator of fibroblast activity and angiogenesis, was predominantly detected in its latent form (LAP\u0026ndash;Latency-Associated Peptide), suggesting limited activation at this stage. This pattern may reflect delayed progression from inflammation to proliferation\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. A complicating factor may contribute: we occasionally noted slight interdental papilla dehiscence after surgery in some A-PRF\u0026thinsp;+\u0026thinsp;treated sites, potentially sustaining inflammation and favoring undesired bacterial colonization before regeneration proceeds.\u003c/p\u003e\u003cp\u003eAt week 2, none of the proteins exhibited a significant association with any of the procedures. Likely, relevant proteins at this stage were not part of the analyzed set. At week 6 postoperatively, OFD and EMD still present better healing scores than A-PRF+. The emergence of ARG-1 as a key marker indicated a shift toward resolution of inflammation and tissue remodeling, particularly in A-PRF\u0026thinsp;+\u0026thinsp;and EMD. ARG-1 is associated with alternatively activated M2 macrophages and promotes healing by suppressing nitric oxide production and enhancing matrix deposition\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. This suggests that, despite slower initiation, both biologically active treatments eventually engaged reparative immune pathways more effectively than OFD\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAt month 3, although the wound healing was mostly completed, proteomic differences remained evident. OFD and EMD showed lower mEHI scores compared with A-PRF+. indicating more favorable outcomes. In EMD, a cluster of proteins including TRAIL, HGF, VEGFR-2, TWEAK, ARG-1, and CD40 reflected remodeling activity rather than unresolved inflammation. TRAIL and HGF, in particular, are known to support angiogenesis, epithelial proliferation, and tissue maturation when appropriately regulated\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. HGF, a pleiotropic factor involved in epithelial proliferation, angiogenesis, and mesenchymal remodeling, has been widely associated with wound closure and scar attenuation\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. VEGFR-2 signaling further reinforced the angiogenic response, while CD40 reflected immune activation tightly linked to tissue repair processes. CD40, expressed on fibroblasts, epithelial, and immune cells, links inflammation to tissue repair through NF-κB\u0026ndash;mediated induction of IL-6. IL-8, and ICAM-1. While this signaling supports immune activation during early healing, excessive or prolonged CD40 activity may perpetuate inflammation and hinder repair\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Pro-regenerative factors such as FGF-2 can suppress CD40 signaling, promoting resolution and tissue remodeling. In acute wounds, platelet-derived CD40L may act as an early alarmin. The stronger correlation of TRAIL with A-PRF\u0026thinsp;+\u0026thinsp;suggests its function to be context-dependent; the regenerative potential of EMD, mediated by amelogenins, appears to facilitate not only early fibroblast proliferation but also a more rapid and controlled transition to the remodeling stage, consistent with previous reports of accelerated periodontal regeneration. In fact, previous studies have demonstrated that EMD can accelerate periodontal regeneration by enhancing the recruitment and differentiation of mesenchymal cells, while also suppressing the inflammatory response in a favorable manner for wound stabilization\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. At the 6-month follow-up, no protein\u0026ndash;procedure associations were observed, suggesting that wound healing was complete in all cases.\u003c/p\u003e\u003cp\u003eIn our data, the association between low levels of early pro-inflammatory biomarkers such as TRAIL, HGF, LAP TGF-β1, VEGFR-2, C040 and ARG-1, aligned with lower wound healing scores, supporting a quicker transition to reparative and remodeling stages\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. By contrast, although PRF is commonly viewed as a fibrin scaffold that gradually releases PDGF, VEGF, and TGF-β to support angiogenesis and osteogenesis\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, our findings in molar furcation sites showed a different early biology: A-PRF\u0026thinsp;+\u0026thinsp;was linked to stronger acute-phase signaling and slower early closure. While this slower initial healing response may not be ideal for cases requiring rapid wound stabilization, it could be beneficial for long-term periodontal regeneration, particularly in cases where gradual tissue remodeling is desirable\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. In fact, in our cohort, 3 cases out of 6 treated with A-PRF\u0026thinsp;+\u0026thinsp;showed furcation degree reduction, making this material apparently promising.\u003c/p\u003e\u003cp\u003eThe main limitations of this study are the small sample size and the uneven distribution of age and furcation types due to interrupted allocation. Strengths include its prospective design, randomization, standardized surgical protocol, and the exploratory integration of systemic and proteomic biomarkers with clinical outcomes. These preliminary findings highlight proteins potentially involved in wound healing and may guide future larger trials on periodontal regeneration in furcation defects.\u003c/p\u003e\u003cp\u003eTo conclude, elevated baseline cholesterol and creatinine levels may adversely influence PD reduction, as well as CAL and ABL gain. at 6 months following surgery in degree II molar furcation defects. Overall, our findings indicate that, across the OFD, EMD, and A-PRF\u0026thinsp;+\u0026thinsp;procedures, distinct protein expression profiles were associated with different postoperative healing scores. Although A-PRF\u0026thinsp;+\u0026thinsp;exhibited a slower and more heterogeneous early healing response characterized by stronger acute-phase signaling, the improvement in furcation degree observed in A-PRF\u0026thinsp;+\u0026thinsp;treated sites suggests that A-PRF\u0026thinsp;+\u0026thinsp;may offer promising regenerative potential.\u003c/p\u003e"},{"header":"4 | Materials and Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e4.1 | Ethics. study design. patient inclusion\u003c/h2\u003e\u003cp\u003eThe current study is a further analysis, based on Pitzurra et al.\u003csup\u003e26\u003c/sup\u003e, which received approval from the medical ethics committee of the VU University Medical Center Amsterdam (study protocol number: 6265602917). All experimental procedures were performed in accordance with relevant guidelines and regulations (Declaration of Helsinki). Written informed consent was obtained from all participants prior to their inclusion in the study. Inclusion and exclusion criteria, and a comprehensive description of the study protocol, randomization and allocation are previously published \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The study was initially planned as a fully powered clinical randomized controlled trial (RCT); however, the study was prematurely terminated due to the COVID-19 pandemic. The study was registered with the ISRCTN registry (ISRCTN13520922) on 17 October 2018. We report here the results of 17 cases randomized into three groups.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e4.2 | Clinical procedures. GCF collection and sample processing\u003c/h2\u003e\u003cp\u003eBriefly, in 18 patients, the selected molar furcation degree II site underwent periodontal surgery, either with an OFD approach (n\u0026thinsp;=\u0026thinsp;6) or with the application of EMD (n\u0026thinsp;=\u0026thinsp;6) or A-PRF+ (n\u0026thinsp;=\u0026thinsp;6). Sites were monitored at different time points from baseline to 6 months postoperatively.\u003c/p\u003e\u003cp\u003ePaper strips for periodontal use (Periopaper\u0026trade;, Oraflow, Smithtown, NY, USA) were used to collect GCF. First, the tooth was isolated with cotton rolls. Supragingival plaque was removed with curettes and the tooth was gently dried with a gentle stream of air. The paper strip was then inserted in the sulcus and left in place to soak GCF for 30 seconds, avoiding any contact with saliva. Next, GCF strips were transferred into 1.5 mL centrifuge tubes, placed on ice and within two hours stored at -80\u0026deg;C. Samples were taken at the postoperative time points day 3, weeks 1, 2, 6, month 3 and 6.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e4.3 | Blood sampling and processing\u003c/h2\u003e\u003cp\u003eThe blood drawing procedure was performed by venipuncture of the antecubital fossa immediately before the surgery to prepare A-PRF+. Four tubes of blood (40 ml) were taken from each patient. Two of them were used for the preparation of the A-PRF+ (sterile plain glass-based vacuum tubes). Two other tubes (with EDTA), not to be used for the preparation of the A-PRF+, were transported within 1 hour to the clinical-biochemical laboratory of the Amsterdam University Medical Center (AUMC) (Study ID: P17-148. PIN 2712681). From the venous blood samples, the following were measured: blood cell counts (total leukocytes, including leuko-differentiations [lymphocytes, monocytes, neutrophils, eosinophils, basophils] and platelets), HbA1c, albumin, ferritin, hemoglobin, total cholesterol plus low-density lipoproteins (LDL) and high-density lipoproteins (HDL) levels, triglycerides, creatinine, and estimated glomerular filtration rate (eGFR).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e4.4 | Surgical treatment and wound healing evaluation\u003c/h2\u003e\u003cp\u003eThe complete surgical procedures are previously reported\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Surgeries were carried out by an experienced periodontist (SB) using a microsurgical approach (simple and modified papilla preservation). During surgery, patients were randomly assigned to receive either A-PRF+, EMD, or open flap debridement (OFD). For A-PRF+, membranes were minced, placed into the furcation defect, and then stabilized. For EMD, roots were dried first and the material was applied and left undisturbed for two minutes prior to flap closure. Tension-free sutures (6\u0026thinsp;\u0026minus;\u0026thinsp;0 polypropylene) were placed allowing primary closure. Patients avoided brushing the surgical area and rinsed with hydrogen peroxide (1.5%) and chlorhexidine (0.12%) for four weeks; sutures were removed at day 14 with polishing at day 7, week 2, and week 6. From week 4, mechanical plaque control was resumed, and a 3-month maintenance visit included supragingival instrumentation and hygiene reinforcement.\u003c/p\u003e\u003cp\u003eClinical measurements of the treated furcation sites took place at baseline and at six months. Early wound healing was scored at all postoperative time points using a modification on the Early Healing Index (mEHI). The original EHI consists of five categories for the degree of flap closure: \u0026lsquo;1\u0026rsquo; complete flap closure\u0026mdash;no fibrin line in the interproximal area; \u0026lsquo;2\u0026rsquo; complete flap closure\u0026mdash;fine fibrin line in the interproximal area; \u0026lsquo;3\u0026rsquo;complete flap closure\u0026mdash;fibrin clot in the interproximal area; \u0026lsquo;4\u0026rsquo; incomplete flap closure\u0026mdash;partial necrosis of the interproximal tissue; \u0026lsquo;5\u0026rsquo; incomplete flap closure\u0026mdash;complete necrosis of the interproximal tissue. For the current study. score \u0026lsquo;0\u0026rsquo; was added to this index. representing no wound visible. The examiner (DV) was calibrated for reproducibility (kappa\u0026thinsp;=\u0026thinsp;0.80).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e4.5 | GCF sample preparation and protein analysis\u003c/h2\u003e\u003cp\u003eGCF samples were transported on dry ice to the Arcadia Core Facility, University Medical Center Utrecht (The Netherlands) for proteomic analysis. Each strip was incubated with 15 \u0026micro;L 1X PBS, pH 7.4 (DPBS, Corning Life Science, Tewksbury, MA, USA) containing protease inhibitors (Complete Mini, EDTA free, Sigma-Aldrich, St. Louis, MO, USA), vortexed, centrifuged, and incubated with an additional 15 \u0026micro;L PBS for 15 min at room temperature. After centrifugation (10.000 \u0026times; g. 4\u0026deg;C. 5 min), supernatants were collected and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis. Proteomic profiling was performed using Proximity Extension Assay (PEA) technology with the Olink\u0026reg; Target 96 Immuno-Oncology panel (Olink Proteomics AB. Uppsala. Sweden) following the manufacturer\u0026rsquo;s protocol. This panel was chosen for its collection of proteins related to angiogenesis. cell proliferation and inflammation. Four spiked-in internal controls were included to monitor assay and sample performance. Protein levels were reported as Normalized Protein Expression (NPX) values on a log2 scale.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e4.6 | Data analysis\u003c/h2\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e4.6.1 | Systemic biomarkers and clinical outcomes\u003c/h2\u003e\u003cp\u003eWe assessed associations between baseline systemic blood biomarkers and 6-month clinical outcomes (PD, CAL, ABL) using linear regression. Treatment group and baseline PD were always retained in all models to adjust for potential confounding. Univariable analyses were performed for each biochemical parameter individually, as well as for age, sex and smoking, followed by multivariable linear regression with backward selection (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05 for removal), incorporating variables with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.1 identified in the univariate analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e4.6.2 | Proteomic analyses\u003c/h2\u003e\u003cp\u003eProteins with \u0026gt;\u0026thinsp;25% missing frequency among all samples were excluded to ensure assay reliability\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Associations between proteins, mEHI, and treatment groups per timepoint were assessed using multivariable linear regression with interaction terms between the mEHI and treatment group, using OFD or EMD as reference categories against A-PRF+. Benjamini\u0026ndash;Hochberg correction was performed for multiple testing (\u003cem\u003eα\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.05). Analyses were conducted in Python v3.9.6 with Pandas, Numpy, Statsmodels, Plotly, and Seaborn.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e4.6.3 | Data availability\u003c/h2\u003e\u003cp\u003eThe raw data and code is available from the corresponding author ([email protected]) on reasonable request.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.P., S.B., B.L.: Conceptualization and design of study. L.P., E.S., B.L,.: Data Analysis.L.P., S.B., E.S. , B.L.: Wrote the main manuscript text. S.B., B.L.: Supervision, Reviewing and editing the manuscript. L.P., D.V.: Recruitment, Administrative and IRB submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information and conflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded in part by (1) the Department of Periodontology. Academic Centre for Dentistry Amsterdam (ACTA). Amsterdam. The Netherlands; (2) the ORANGEForce project within the ORANGEHealth consortium funded by Health-Holland.nl. div. Life Sciences \u0026amp; Health with grant number LSHM21064; (3) self-funded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare they have no direct or indirect financial interests in the products or information presented in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authos thank Dr. N. Su for the extensive biotstatistical advice and they thank Dr. N.A.M. Rosema for his clinical contributions.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003evan der Velden, U. et al. Java project on periodontal diseases: causes of tooth loss in a cohort of untreated individuals. \u003cem\u003eJ. Clin. 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Clin. Periodontol\u003c/em\u003e. \u003cb\u003e30\u003c/b\u003e, 496\u0026ndash;504. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1034/j.1600-051x.2003.00013.x\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1034/j.1600-051x.2003.00013.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2003).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1. Patient and periodontal characteristics at baseline.\u003c/p\u003e\n\u003cp\u003eValues represent number or median and percentage\u0026nbsp;(%) or range.\u003c/p\u003e\n\u003cp\u003eAbbreviations: N, number of patients; OFD, open flap debridement; EMD, enamel matrix derivatives; A-PRF+, advanced platelet rich fibrin +. Further description of patient characteristics in \u003csup\u003e26\u003c/sup\u003e.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"548\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.8686%;\"\u003e\n \u003cp\u003e\u003cem\u003eTreatment Group\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4453%;\"\u003e\n \u003cp\u003eNumber or Median\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6861%;\"\u003e\n \u003cp\u003e% or Range\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.8686%;\"\u003e\n \u003cp\u003e\u003cem\u003eOFD (n=6)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4453%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6861%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.8686%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4453%;\"\u003e\n \u003cp\u003e51.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6861%;\"\u003e\n \u003cp\u003e33-60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.8686%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003eSex (female)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4453%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6861%;\"\u003e\n \u003cp\u003e50%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.8686%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003eSmoking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4453%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6861%;\"\u003e\n \u003cp\u003e33%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.8686%;\"\u003e\n \u003cp\u003e\u003cem\u003eEMD (n=5)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4453%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6861%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.8686%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4453%;\"\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6861%;\"\u003e\n \u003cp\u003e41-61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.8686%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003eSex (female)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4453%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6861%;\"\u003e\n \u003cp\u003e80%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.8686%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003eSmoking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4453%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6861%;\"\u003e\n \u003cp\u003e40%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.8686%;\"\u003e\n \u003cp\u003e\u003cem\u003eA-PRF+ (n=6)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4453%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6861%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.8686%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4453%;\"\u003e\n \u003cp\u003e66.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6861%;\"\u003e\n \u003cp\u003e61-78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.8686%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003eSex (female)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4453%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6861%;\"\u003e\n \u003cp\u003e50%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.8686%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003eSmoking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4453%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6861%;\"\u003e\n \u003cp\u003e33%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.8686%;\"\u003e\n \u003cp\u003e\u003cem\u003eTotal\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4453%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6861%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.8686%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003eAge\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4453%;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6861%;\"\u003e\n \u003cp\u003e33-78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.8686%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003eSex (female)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4453%;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6861%;\"\u003e\n \u003cp\u003e58.8%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 38.8686%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003eSmoking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4453%;\"\u003e\n \u003cp\u003e6 (5.5 sig/day)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.6861%;\"\u003e\n \u003cp\u003e35%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eTable 2. Multivariable linear regression modeling for the association between baseline biochemical parameters and 6-month Pocket Depth (PD). 6 months Clinical Attachment Level (CAL) and 6 months Alveolar bone level (ABL). P value \u0026lt; \u003cstrong\u003e0.05.\u003c/strong\u003e The variables treatment group and baseline PD were always forced into the univariable and multivariable models.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAbbreviations: HDL, high-density lipoprotein; LD,. low-density lipoprotein.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"700\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eFinal multivariable models\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e6 months PD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e6 monthts CAL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e6 months ABL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cem\u003eBeta\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cem\u003eStd. Error\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cem\u003eBeta\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cem\u003eStd. Error\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003e\u003cem\u003eBeta\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cem\u003eStd. Error\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.1954%;\"\u003e\n \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.9852%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;H\u003c/em\u003e\u003cem\u003eDL\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003e2.163\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.787\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.018\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 51px;\"\u003e\n \u003cp\u003e3.144\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003e1.203\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.1954%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.023\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.9852%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eLDL\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e0.746\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0.262\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.016\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 51px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.1954%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.9852%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cem\u003eCreatinine\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 44px;\"\u003e\n \u003cp\u003e0.084\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0.027\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.009\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 51px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.1954%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.9852%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eTable 3. Proteins with significant interaction effects between treatment and mEHI at different postoperative timepoints. P value \u0026lt; \u003cstrong\u003e0.05.\u003c/strong\u003e Beta (corresponding 95%CI, p-value, adjusted p-value) quantify the interaction effect between treatment and mEHI in the multivariate linear regression model.\u003c/p\u003e\n\u003cp\u003eAbbreviations: CI, confidence interval; mEHI, modified early healing index;\u0026nbsp;OFD, open flap debridement; EMD, enamel matrix derivatives; A-PRF+,\u0026nbsp;advanced platelet rich fibrin +; vs, versus representing the reference treatment. Full names of proteins can be found in Table 2.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"671\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eProtein\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eTimepoint\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eBeta\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eCI\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ep\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e-\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003evalue\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eAdjusted\u0026nbsp;\u003cbr\u003e\u0026nbsp;p\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e-\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003evalue\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 190px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eT\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ereatment\u0026nbsp;\u003cstrong\u003e\u0026times; m\u003c/strong\u003eEHI\u0026nbsp;\u003cbr\u003e\u0026nbsp;interaction effect\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eCASP-8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003eDay 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e1.98; 7.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0.0033\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0.0199\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 190px;\"\u003e\n \u003cp\u003eA-PRF+ (vs\u0026nbsp;EMD)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eIL-8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003eDay 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e6.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2.17; 10.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0.0075\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0.0450\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 190px;\"\u003e\n \u003cp\u003eA-PRF+ (vs\u0026nbsp;EMD)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eCASP-8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003eWeek 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e6.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2.28; 11.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0.0066\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0.0397\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 190px;\"\u003e\n \u003cp\u003eA-PRF+ (vs\u0026nbsp;OFD)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eLAP TGF-beta-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003eWeek 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e6.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2.03; 10.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0.0072\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0.0432\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 190px;\"\u003e\n \u003cp\u003eA-PRF+ (vs\u0026nbsp;OFD)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eARG-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003eWeek 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e2.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.88; 4.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0.0081\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0.0418\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 190px;\"\u003e\n \u003cp\u003eEMD (vs\u0026nbsp;OFD)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eARG-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003eWeek 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e2.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.95; 4.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0.0045\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0.0272\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 190px;\"\u003e\n \u003cp\u003eA-PRF+ (vs\u0026nbsp;OFD)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eTRAIL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003eMonth 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-3.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e-4.08; -2.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0.00004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0.0003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 190px;\"\u003e\n \u003cp\u003eEMD (vs\u0026nbsp;OFD)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eHGF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003eMonth 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-3.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e-5.22; -1.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0.0014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0.0084\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 190px;\"\u003e\n \u003cp\u003eEMD (vs OFD)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eLAP TGF-beta-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003eMonth 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-3.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e-4.93; -1.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0.0022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0.0131\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 190px;\"\u003e\n \u003cp\u003eEMD (vs\u0026nbsp;OFD)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eVEGFR-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003eMonth 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-0.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e-1.32; -0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0.0040\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0.0238\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 190px;\"\u003e\n \u003cp\u003eEMD (vs OFD)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eTWEAK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003eMonth 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-2.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e-3.62; -1.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0.0016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0.0096\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 190px;\"\u003e\n \u003cp\u003eEMD (vs\u0026nbsp;OFD)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eARG-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003eMonth 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-1.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e-1.99; -0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0.0139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0.0418\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 190px;\"\u003e\n \u003cp\u003eEMD (vs OFD)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eCD40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003eMonth 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e-2.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e-3.31; -0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0.0062\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0.0371\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 190px;\"\u003e\n \u003cp\u003eEMD (vs\u0026nbsp;OFD)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eTRAIL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003eMonth 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e2.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e1.29; 4.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0.0028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0.0171\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 190px;\"\u003e\n \u003cp\u003eA-PRF+ (vs\u0026nbsp;EMD)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Platelet rich fibrin (PRF), Periodontal regeneration, Furcation defect, Enamel matrix derivative (EMD), Proteomics","lastPublishedDoi":"10.21203/rs.3.rs-8065385/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8065385/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study evaluated associations between protein expression and postoperative healing scores following three periodontal regenerative procedures (Open Flap Debridement\u0026ndash;OFD, Enamel Matrix Derivative\u0026ndash;EMD, Advanced Platelet Rich Fibrin\u0026ndash;A-PRF+) in molar degree II furcations over six months. Associations between baseline systemic biochemical parameters and six-month clinical outcomes were also assessed.\u003c/p\u003e\u003cp\u003eEighteen patients were randomly assigned to control (OFD), EMD, or A-PRF\u0026thinsp;+\u0026thinsp;groups. Baseline plasma biomarkers were modeled on probing depth (PD), clinical attachment level (CAL), and alveolar bone level (ABL) at six months. Gingival crevicular fluid (GCF) samples were analyzed using the Olink\u0026reg; Target 96 Immuno-Oncology panel at day 3, weeks 1, 2, 6, and months 3 and 6. Multivariable linear regressions identified proteins associated with the modified Early Wound Healing Index (mEHI) per treatment.\u003c/p\u003e\u003cp\u003eSeventeen patients completed follow-up. HDL- and LDL-cholesterol and creatinine were negatively associated with PD, CAL, and ABL improvements (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At early timepoints, CASP-8, IL-8, and LAP TGF-β1 showed stronger associations with mEHI in A-PRF\u0026thinsp;+\u0026thinsp;than OFD or EMD, while ARG-1 and TRAIL demonstrated treatment-specific patterns over time. No differences were observed at month 6.\u003c/p\u003e\u003cp\u003eHigh baseline cholesterol and creatinine may impair periodontal regeneration. Distinct proteomic signatures suggest differential biological pathways underlying healing across OFD, EMD, and A-PRF\u0026thinsp;+\u0026thinsp;procedures.\u003c/p\u003e","manuscriptTitle":"Longitudinal proteomic analysis of GCF in regenerative healing of molar furcation degree II defects treated with OFD, EMD, or A-PRF+: a pilot study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-02 13:59:37","doi":"10.21203/rs.3.rs-8065385/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-18T12:26:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-12T16:06:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"22302012521208927555879807905171084578","date":"2025-12-12T15:05:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-08T10:56:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"109278137891425168265221570482436148571","date":"2025-11-27T00:53:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"221342814679488684279130319943989409716","date":"2025-11-26T21:34:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4784956225046611734011631649930972823","date":"2025-11-26T12:04:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-26T11:21:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-26T11:04:38+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-21T09:13:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-20T20:21:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-20T20:16:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"45059358-1d8d-4c03-a226-89cede435a77","owner":[],"postedDate":"December 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":58842096,"name":"Health sciences/Biomarkers"},{"id":58842097,"name":"Biological sciences/Cancer"},{"id":58842098,"name":"Health sciences/Diseases"},{"id":58842099,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-02-23T16:01:54+00:00","versionOfRecord":{"articleIdentity":"rs-8065385","link":"https://doi.org/10.1038/s41598-026-39474-8","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-02-19 15:58:26","publishedOnDateReadable":"February 19th, 2026"},"versionCreatedAt":"2025-12-02 13:59:37","video":"","vorDoi":"10.1038/s41598-026-39474-8","vorDoiUrl":"https://doi.org/10.1038/s41598-026-39474-8","workflowStages":[]},"version":"v1","identity":"rs-8065385","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8065385","identity":"rs-8065385","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}