Concentration-Dependent Effects of Advanced Platelet-Rich Fibrin Combined with A Collagen Membrane on Human Periodontal Ligament Stem Cells: An In Vitro Study

Concentration-Dependent Effects of Advanced Platelet-Rich Fibrin Combined with A Collagen Membrane on Human Periodontal Ligament Stem Cells: An In Vitro 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 Research Article Concentration-Dependent Effects of Advanced Platelet-Rich Fibrin Combined with A Collagen Membrane on Human Periodontal Ligament Stem Cells: An In Vitro Study Meo Nguyen, Minh Bao Nguyen Quoc, Truong Tran Thien, Ha Le Bao Tran, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9154707/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract Background Advanced platelet-rich fibrin (A-PRF) is used in periodontal regeneration due to its autologous fibrin matrix and bioactive content. Collagen membranes are widely used in guided tissue regeneration. However, it remains unclear whether combining A-PRF with a collagen membrane alters early cellular responses. The present in vitro study compared the effects of A-PRF alone and A-PRF combined with a collagen membrane on the viability, proliferation-related metabolic activity, and migration-related wound closure of human periodontal ligament stem cells (hPDLSCs) in different extract concentrations. Methods hPDLSCs were exposed to A-PRF or A-PRF mixed with a porcine collagen membrane (A-PRF-Col) extract at 100% and 20% concentrations prepared from the blood of four donors. Cell viability was assessed after 24 h using an MTT assay and morphological observation. Cell proliferation-related metabolic activity was evaluated by MTT assay on days 1, 3, 5, and 7. Migration-related wound closure was evaluated using a scratch assay after 24 h. Data were analyzed using repeated-measures ANOVA, followed by post hoc multiple-comparison tests. p < 0.05 is considered statistically significant. Results Early responses of hPDLSCs were influenced by A-PRF concentration and further modified by the collagen membrane. Only 100% A-PRF-Col showed cytotoxicity, with a low mean relative growth rate of 32.12%. Proliferation-related metabolic activity increased and peaked around day 5, but different concentrations and formulations affected the patterns. At 100% concentration, A-PRF-Col had lower activity but surpassed A-PRF in subsequent days. At 20% concentration, the A-PRF-Col group showed higher activity at days 3 and 5 but lower activity at day 7. Migration-related wound closure was primarily influenced by the concentration of the extract. 20% extracts outperformed their 100% counterparts, and 20% A-PRF achieved better closure than 20% A-PRF-Col. Conclusions The early effects of A-PRF on hPDLSCs were concentration-dependent and were modified by combination with a collagen membrane. Diluted extracts may support more favorable early cellular responses, while the collagen membrane modified the biological profile in a dependent manner rather than providing consistent enhancements. Advanced platelet-rich fibrin Cell proliferation Cell viability Collagen membrane Periodontal ligament stem cells Periodontal regeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 BACKGROUND Periodontitis is a chronic inflammatory disease that damages tooth-supporting tissues, including the periodontal ligament and alveolar bone, and manifests as periodontal pockets, gingival recession, or both [ 1 ]. Advances in periodontal treatment may help control inflammation and slow disease progression, but predictable periodontal attachment apparatus regeneration still presents a challenge [ 2 ]. Guided tissue regeneration is a well-established periodontal regenerative strategy that uses a barrier membrane to prevent epithelial downgrowth and gingival connective tissue invasion while maintaining a space for tissue formation [ 3 ]. Among available barrier materials, collagen membranes are popular due to their biocompatibility, resorbability, and structural similarity to the extracellular matrix [ 4 ]. However, collagen membranes function primarily as barrier devices, and their capacity to provide sufficient biological stimulation to optimize early cellular regenerative responses may be limited [ 3 , 5 ]. Platelet-rich fibrin (PRF) is a blood concentrate rich in fibrin, platelets, and leukocytes that aids in wound healing [ 6 , 7 ]. Advanced platelet-rich fibrin (A-PRF), a PRF variant produced by low-speed centrifugation, has a distinct clot composition that enhances the release of growth factors [ 6 , 8 ]. These properties suggest that combining A-PRF with a collagen membrane may help integrate the structural function of the membrane with the biological activity of the A-PRF, thereby modulating early cellular responses to periodontal wounds [ 4 , 8 ]. Human periodontal ligament stem cells (hPDLSCs) are valuable for tissue repair due to their clonogenicity, proliferative capacity, and differentiation potential [ 9 ]. Therefore, evaluating the effects of materials on hPDLSC viability, proliferation, and migration is a useful in vitro approach for assessing their compatibility and effectiveness in promoting periodontal wound healing [ 7 ]. Previous in vitro studies have shown that PRF-based formulations can promote cell proliferation, migration, and differentiation. In our previous in vitro study, A-PRF combined with xenogenic bone substitute material enhanced hPDLSC proliferation and migration. However, it remains unclear how the A-PRF and collagen membrane combination affects early hPDLSC responses and whether A-PRF concentration influences them. Therefore, the aim of the present study was to compare the effects of A-PRF and A-PRF combined with a collagen membrane on hPDLSC viability, metabolic activity related to proliferation, and migration during wound closure at 100% and 20% extract concentrations. We hypothesized that the presence of a collagen membrane would modify initial hPDLSC responses to A-PRF, and that these effects would vary with different extract concentrations. METHODS This donor-matched in vitro experimental study compared the effects of A-PRF alone and A-PRF combined with a collagen membrane on hPDLSC viability, proliferation-related metabolic activity, and migration-related wound closure at two extract concentrations (100% and 20%). Human periodontal ligament stem cell culture Human periodontal ligament stem cells (hPDLSCs) at passage 3 were obtained from the Tissue Engineering and Biomedical Materials Laboratory, Ho Chi Minh City University of Science, Viet Nam National University Ho Chi Minh City. These cells were originally isolated by the outgrowth method, and the original tissue collection and cell characterization were ethically approved and previously reported [ 12 ]. These cells had been previously characterized as periodontal ligament-derived cells with mesenchymal stem stromal cell-like properties, showing high expression of CD44, CD73, and CD90, low expression of CD34, CD45, and HLA-DR, and in vitro osteogenic and adipogenic differentiation potential [ 12 ]. Cells were cultured in complete culture medium, consisting of Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F12; Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin-streptomycin solution (10,000 U/mL penicillin and 10 mg/mL streptomycin; Sigma-Aldrich, St. Louis, MO, USA), at 37°C in a humidified atmosphere containing 5% CO₂. The medium was changed every 2 days, and passage 4 cells were used in all assays. A-PRF extract and A-PRF with a collagen membrane extract preparation Peripheral blood was collected from four healthy volunteers aged 20–30 years who were non-smokers, did not consume alcohol, had no systemic disease, were not taking any medications or dietary supplements, had no periodontal disease or active inflammatory condition, and had platelet counts within the normal range. The study protocol was approved by the Biomedical Research Ethics Committee of the University of Medicine and Pharmacy at Ho Chi Minh City (Approval number 1973/DHYD-HĐĐĐ). All participants were informed about the study, and written informed consent was obtained prior to blood collection. Blood was drawn into 10 mL A-PRF tubes without anticoagulant and immediately centrifuged at 1300 rpm (approximately 200 × g) for 14 minutes using a DUO Quattro System centrifuge (Process for PRF, Nice, France) to obtain A-PRF clots. The clots were retrieved using sterile forceps, and the red blood cell fraction at the base of each clot was removed. For the A-PRF group, each clot was gently compressed for 30 seconds using a PRF Box (Process for PRF, Nice, France) to obtain an A-PRF membrane. For the A-PRF with a collagen membrane group (A-PRF-Col), a sterile porcine collagen membrane (Geistlich Bio-Gide®, Geistlich Pharma AG, Wolhusen, Switzerland) with an original size of 30 × 40 mm was trimmed to 20 × 30 mm. The A-PRF clot was placed over the collagen membrane to fully cover the surface, and both materials were compressed together for 30 seconds using the PRF Box, following a method adapted from Blatt et al. (2020) [ 13 ]. For extract preparation, each A-PRF membrane and A-PRF-Col construct was processed separately. The wet mass of each A-PRF membrane, or of the A-PRF component in each A-PRF-Col construct, was measured before incubation. 100% extracts were prepared by incubating the materials in serum-free DMEM/F12 at an extraction ratio of 0.2 g/mL for 24 h at 37°C, following a protocol adapted from ISO 10993-12. In the A-PRF-Col group, the extraction ratio was calculated based on the wet mass of the A-PRF component to standardize the biological dose of A-PRF across formulations. After incubation, the conditioned media were centrifuged at 3,000 rpm (approximately 1,065 × g) for 5 minutes to remove residual cells and debris. The supernatants were collected and defined as 100% extracts. 20% extracts were prepared separately under the same conditions using an extraction ratio of 0.04 g/mL in serum-free DMEM/F12. The experimental groups in the study included 100% A-PRF, 100% A-PRF-Col, 20% A-PRF, and 20% A-PRF-Col, with each extract processed independently from each donor and each donor treated as one biological replicate (n = 4). Effect of A-PRF and A-PRF-Col extracts on hPDLSC viability The effects of A-PRF and A-PRF-Col extracts on hPDLSC viability were assessed via metabolic activity using an MTT assay adapted from ISO 10993-5:2009. Basal serum-free DMEM/F12 was used as the negative control, and the basal serum-free DMEM/F12 with 20% DMSO was used as the positive control for assay validation and comparison. hPDLSCs were seeded in 96-well plates at a density of 2 × 10^4 cells/well and cultured for 24 h at 37°C in a humidified atmosphere containing 5% CO₂. The culture medium was then replaced with 100 µL of the corresponding extract or control medium. Four technical replicate wells were used for each group in each experiment. After 24 h of incubation, cell morphology was qualitatively examined under an inverted microscope (CKX53; Olympus, Japan) using CellSens imaging software (Olympus, Japan) and compared with the control groups. The test media were removed and replaced with 100 µL of MTT solution (0.5 mg/mL; Sigma-Aldrich, St. Louis, MO, USA). Cells were incubated for 4 h under the same conditions. The MTT solution was then removed, and the resulting formazan crystals were dissolved in 100 µL of ethanol-DMSO solution (70:30, v/v). The plates were incubated for 30 minutes, and the contents of each well were mixed thoroughly by pipetting. Optical density was measured at 570 nm (OD 570 ) using an EZ Read 400 microplate reader (Biochrom Ltd., Cambridge, UK), with blank wells containing no cells used for background correction. Relative growth rate (RGR) was then calculated as follows: RGR (%) = (mean blank-corrected OD of the experimental group / mean blank-corrected OD of the negative control group) × 100%. Samples with RGR values below 70% of the negative control were considered cytotoxic. Effect of A-PRF and A-PRF-Col extracts on hPDLSC proliferation-related metabolic activity The effects of A-PRF and A-PRF-Col extracts on hPDLSC proliferation-related metabolic activity were assessed using an MTT assay at four different time points. Basal serum-free DMEM/F12 was used as the negative control, and complete culture medium was used as the positive control for assay validation and comparison. hPDLSCs were seeded in 96-well plates at a density of 2 × 10^4 cells/well and cultured for 24 h at 37°C in a humidified atmosphere containing 5% CO₂ to allow cell attachment and spreading. The medium was then replaced with the corresponding extracts or control media. Four technical replicate wells were used for each group at each time point. Growth-related metabolic activity was assessed on days 1, 3, 5, and 7 using the MTT assay. Each time point used a separate 96-well plate with no medium replacement during the assay period. Blank wells without cells were included for background correction. The MTT assay was performed as described in the viability assay section, and blank-corrected OD values at 570 nm were used for analysis. Effect of A-PRF and A-PRF-Col extracts on hPDLSC migration-related wound closure To evaluate migration-related wound closure, hPDLSCs were seeded at a density of 3 × 10^5 cells/well in six-well plates and cultured until a near-confluent monolayer was achieved. Then, the culture medium was replaced with 3 mL of basal, serum-free DMEM/F12, and the cells were incubated for 24 h at 37°C in a humidified atmosphere containing 5% CO₂ to induce serum deprivation. After this period, the medium was removed, and a single linear scratch was created in each well using a sterile 100–1000 µL pipette tip. Detached cells were removed by washing once with 1× phosphate-buffered saline, and 3 mL of the corresponding extract or control medium was immediately added to each well. Basal serum-free DMEM/F12 was used as the negative control, and complete culture medium was used as the positive control. These reference controls were not included in the primary factorial analysis. Four technical replicate wells were used for each group. Images of the scratched area were captured at baseline (0 h) and after 24 h using an Olympus CKX-RCD inverted microscope equipped with a DP2-BSW microscopy camera at 4× objective magnification. Each plate was marked to define the same field imaged at both time points, with a single field per well analyzed. Wound closure was quantified using ImageJ 1.52a software and expressed as the percentage of wound closure relative to baseline, calculated as [(A0 − A24) / A0] × 100, where A0 and A24 represent the cell-free area at 0 and 24 h, respectively. Image analysis was performed in a blinded manner. Because scratch closure reflects a composite response influenced by collective migration, cell spreading, and proliferation-related activity, this outcome was interpreted as migration-related wound closure rather than direct measurement of cell migration alone. Statistical analysis Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software, San Diego, CA, USA). The donor was treated as the matched biological unit, and four technical replicate wells were averaged before analysis. Data are presented as mean ± standard deviation of donor-level values. Model assumptions were evaluated by residual plots and Q-Q plots. In repeated-measures analyses with within-donor factors having more than two levels, sphericity was not assumed, and the Greenhouse-Geisser correction was used. For factors with only two levels, sphericity was not applicable. No a priori sample size calculation was performed because this was an exploratory in vitro study using a fixed number of donor-derived biological replicates. Viability and migration-related wound closure data were analyzed using a two-way repeated-measures ANOVA with concentration and formulation as within-donor factors, followed by Šídák-adjusted pairwise comparisons. In secondary analyses, relative growth rate (RGR) values and wound closure percentages were analyzed using repeated-measures one-way ANOVA with Dunnett’s multiple-comparison test versus the negative control. Proliferation-related metabolic activity was analyzed using three-way repeated-measures ANOVA, with time, concentration, and formulation as within-donor factors. Prespecified simple-effects analyses were then performed using two-way repeated-measures ANOVA within each concentration and within each formulation, followed by Šídák-adjusted pairwise comparisons at each time point. Differences among the four experimental groups and the negative control across time points were analyzed in a secondary analysis, using a two-way repeated-measures ANOVA followed by Dunnett’s post hoc test. Assay-specific reference controls were included for validation and interpretation, but were not included in the primary factorial models. p < 0.05 was considered statistically significant. RESULTS Effect of A-PRF and A-PRF-Col extracts on hPDLSC viability The mean relative growth rate (RGR) values were 72.43 ± 9.08% for 100% A-PRF, 32.12 ± 5.30% for 100% A-PRF-Col, 87.53 ± 7.55% for 20% A-PRF, and 108.0 ± 7.69% for 20% A-PRF-Col after 24 h of exposure. Only 100% A-PRF-Col showed a mean RGR below the 70% cytotoxicity threshold (Fig. 1 b). Microscopic observations were consistent with the viability findings (Fig. 2 ). Cells in the negative control showed the typical spindle-shaped morphology and relatively uniform distribution (Fig. 2 a). In contrast, the positive control showed marked deterioration, with many rounded, shrunken, and poorly attached cells (Fig. 2 b). In the 100% A-PRF group, cell morphology was largely preserved. However, cell distribution appeared less uniform and more contracted cells were observed (Fig. 2 c). The 100% A-PRF-Col group showed the most pronounced adverse changes, characterized by reduced adherent cell density, irregular distribution, and increased cell contraction and detachment (Fig. 2 d). In both 20% extract groups, most cells remained elongated and adherent, resembling the negative control (Fig. 2 e, f). Analysis of blank-corrected OD570 values in the four experimental groups showed a significant main effect of concentration (p = 0.0009), no significant main effect of formulation (p = 0.0785), and a significant concentration × formulation interaction (p = 0.0101) (Fig. 1 a). Šídák-adjusted comparisons showed a significant difference between A-PRF and A-PRF-Col at 100% concentration (p = 0.0239), but not at 20% concentration (p = 0.1361). Within the A-PRF condition, the difference between 100% and 20% was not significant (p = 0.2498), whereas within the A-PRF-Col condition, viability was significantly lower at 100% than at 20% (p = 0.0039). When donor-level RGR values were analyzed together with the negative control, a significant overall group effect was observed (p < 0.0001). Compared with the negative control, both 100% A-PRF (p = 0.0004) and 100% A-PRF-Col (p < 0.0001) showed significantly lower RGR values, whereas 20% A-PRF (p = 0.0755) and 20% A-PRF-Col (p = 0.3311) did not differ significantly from the negative control (Fig. 1 b). Effect of A-PRF and A-PRF-Col extracts on hPDLSC proliferation-related metabolic activity Blank-corrected OD570 values of hPDLSCs cultured with 100% A-PRF, 100% A-PRF-Col, 20% A-PRF, or 20% A-PRF-Col extracts were measured on days 1, 3, 5, and 7. Basal serum-free medium (-) is shown as a reference control and was included only for secondary analysis, not in the primary factorial model. The four experimental groups were analyzed using three-way repeated-measures ANOVA, with time, concentration, and formulation as within-donor factors. Data are presented as mean ± standard deviation (n = 4). Proliferation-related metabolic activity increased over time in all four experimental groups, peaked around day 5, and diverged at day 7 (Fig. 3 ). Three-way repeated-measures ANOVA showed significant effects of time (p < 0.0001) and concentration (p = 0.0017), but not formulation (p = 0.0852). Significant interactions were observed for time × concentration (p < 0.0001), time × formulation (p < 0.0001), concentration × formulation (p = 0.0232), and time × concentration × formulation (p = 0.0222). At 100% concentration, two-way repeated-measures ANOVA showed significant effects of time (p < 0.0001), formulation (p = 0.0004), and time × formulation interaction (p < 0.0001). Compared with 100% A-PRF, 100% A-PRF-Col showed lower activity at day 1 (p = 0.0024), but higher activity at days 3, 5, and 7 (p = 0.0300, p = 0.0019, and p = 0.0016, respectively). At 20% concentration, there was a significant effect of time (p < 0.0001) and a significant time × formulation interaction (p = 0.0010), whereas the main effect of formulation was not significant (p = 0.2653). Compared with 20% A-PRF, 20% A-PRF-Col showed higher activity at days 3 and 5 (p = 0.0011 and p = 0.0090, respectively), did not differ at day 1 (p = 0.0951), and showed lower activity at day 7 (p = 0.0166). Within the A-PRF condition, two-way repeated-measures ANOVA showed significant effects of time (p < 0.0001), concentration (p = 0.0004), and time × concentration interaction (p = 0.0002). Compared with 100% A-PRF, 20% A-PRF showed significantly higher activity at all time points (day 1, p = 0.0428; day 3, p = 0.0331; day 5, p = 0.0001; day 7, p = 0.0047). Within the A-PRF-Col condition, significant effects were also observed for time (p < 0.0001), concentration (p = 0.0010), and time × concentration interaction (p < 0.0001). Compared with 100% A-PRF-Col, 20% A-PRF-Col showed higher activity at days 1, 3, and 5 (p < 0.0001, p = 0.0083, and p = 0.0005, respectively), whereas 100% A-PRF-Col was higher at day 7 (p = 0.0185). In the secondary analysis, including basal medium (-), significant effects of time, condition, and time × condition interaction were observed (all p < 0.0001). At day 1, both 100% A-PRF and 100% A-PRF-Col showed lower activity than basal medium (-) (p = 0.0018 and p < 0.0001, respectively), whereas 20% A-PRF and 20% A-PRF-Col did not differ significantly from it (p = 0.2965 and p = 0.1750, respectively). At day 3, 100% A-PRF remained comparable with basal medium (-) (p = 0.6114), while 100% A-PRF-Col, 20% A-PRF, and 20% A-PRF-Col showed significantly higher activity (p = 0.0041, p = 0.0002, and p = 0.0001, respectively). By days 5 and 7, all four experimental groups showed significantly higher activity than basal medium (-) (all p ≤ 0.0015). Effect of A-PRF and A-PRF-Col extracts on hPDLSC migration-related wound closure The wound closure was primarily influenced by extract concentration after 24 h of incubation (Fig. 4 a). Two-way repeated-measures ANOVA of the four experimental groups showed a significant main effect of concentration (p = 0.0004), no significant main effect of formulation (p = 0.3369), and a significant concentration × formulation interaction (p = 0.0134). In both formulations, 20% extracts produced significantly greater wound closure than 100% extracts, for A-PRF (p = 0.0002) and A-PRF-Col (p = 0.0004). At 20% concentration, A-PRF induced significantly greater wound closure than A-PRF-Col (p = 0.0389), whereas no significant difference between formulations was observed at 100% concentration (p = 0.1238). In the control-based analysis, a significant overall group effect was observed (p < 0.0001) (Fig. 4 b). Compared with the negative control, 100% A-PRF did not significantly increase wound closure (p = 0.5370), whereas 100% A-PRF-Col produced a modest but significant increase (p = 0.0135). Both 20% A-PRF and 20% A-PRF-Col significantly increased wound closure relative to the negative control (both p < 0.0001). Representative scratch images supported the quantitative findings (Fig. 5 ). After 24 h, the cell-free gap remained relatively wide in the negative control and in both 100% extract groups. In contrast, it narrowed more in the positive control and, more markedly, in both 20% extract groups. Among the experimental groups, 20% A-PRF showed the greatest apparent wound closure, followed by 20% A-PRF-Col, while the difference between 100% A-PRF and 100% A-PRF-Col appeared less pronounced. DISCUSSION The current study demonstrated that the early effects of A-PRF on hPDLSCs depend on the concentration of A-PRF and were modified by the addition of a collagen membrane. Overall, the 20% extracts were more favorable than the 100% extracts across the evaluated early cellular responses. Meanwhile, the effect of the collagen membrane influenced the outcomes differently, depending on the specific biological aspect. 100% A-PRF-Col showed the least favorable viability profile, 20% A-PRF-Col showed the highest proliferation-related metabolic activity at the intermediate time points, and 20% A-PRF showed the greatest migration-related wound closure. These results suggest that incorporating a collagen membrane alters how hPDLSCs respond to A-PRF, and the effect varies based on the concentration of the extract used. Therefore, A-PRF with a collagen membrane should not be considered simply as a stronger version of A-PRF but with its own unique properties. This finding is relevant to periodontal regeneration because collagen membranes are commonly used as barrier materials in guided tissue regeneration. Therefore, the current study aimed to explore a clinical question: whether combining the structural role of a collagen membrane with the biological activity of A-PRF influences how cells behave during the early stages of wound healing. The results suggest that this combination dose impacts early cellular behavior related to wound healing, but the effects are not consistent across all observed outcomes. The viability assay data indicate that adding a collagen membrane did not consistently improve the early cell compatibility of A-PRF, particularly at the undiluted concentration. After 24 h, only 100% A-PRF-Col dropped below the 70% cytotoxicity threshold, consistent with the marked morphological deterioration observed under the microscope. By contrast, both 20% extract groups maintained a spindle-shaped, adherent morphology similar to the negative control. This pattern suggests that increasing extract concentration did not simply amplify a beneficial biological effect. Rather, a more concentrated preparation, especially when combined with collagen, may have created an environment less conducive to the metabolic activities vital for the survival of hPDLSC. This aligns with existing research on PRF, which generally reports beneficial effects of PRF on proliferation, migration, and differentiation, but also emphasizes substantial heterogeneity across formulations, cell types, and assay designs [ 7 ]. A concentration-dependent effect is biologically reasonable, as platelet-derived preparations do not necessarily exhibit their most favorable cellular effects at maximal concentrations [ 14 , 15 ]. For proliferation-related metabolic activity, the overall pattern favored the 20% extracts, particularly during the first 5 days. All experimental groups increased until day 5, but their trends diverged afterward. Within the A-PRF condition, 20% A-PRF consistently exceeded 100% A-PRF, whereas for the A-PRF-Col groups, 20% A-PRF-Col was higher at days 1, 3, and 5, but not at day 7. These data suggest that a lower concentration may provide a more nurturing environment for sustained cellular metabolic activity, while the collagen membrane may affect the timing of this response rather than producing a uniform enhancement. These findings should be interpreted in the context of an MTT-based assay conducted under conditions of sustained exposure to serum-free extracts. Thus, these findings specifically examine proliferation-related metabolic activity, rather than counting the actual number of cells. These findings align with our previous study, in which exudates from A-PRF combined with xenogenic bone substitute material promoted hPDLSC proliferation, with 20% outperforming 100% at several time points and with a general peak around day 5 [ 11 ]. This contrasts with research by Li et al., which used PRF exudate and reported that both 100% and 20% promoted stronger proliferation than a 4% concentration, suggesting a dose-dependent relationship [ 15 ]. The present data align more closely with our previous model than with Li et al., which suggests that a lower concentration may better support sustained growth-related responses. This difference is biologically plausible because the biomaterial, extract preparation, and target cell context were not identical across studies [ 7 , 11 , 15 ]. The wound closure assays further support that concentration was the primary factor in the present study. Both A-PRF and A-PRF-Col induced significantly greater wound closure at 20% than at 100%, and at the lower concentration, A-PRF had better results than A-PRF-Col. These results suggest that the collagen membrane did not improve migration-related wound closure under the present conditions and may have attenuated the effect of A-PRF at 20%. This specific effect aligns with previous studies showing that different platelet-concentrate formulations can favor different biological processes. Thanasrisuebwong et al. showed that red and yellow i-PRF exerted differential effects on PDLSCs, with red i-PRF favoring proliferation and migration and yellow i-PRF promoting earlier osteogenic differentiation [ 16 ]. Zheng et al. reported that liquid-PRF enhanced the proliferation, migration, and osteogenic differentiation of hPDLCs [ 10 ]. Similarly, Pitzurra et al. found that both L-PRF and A-PRF+ stimulated wound closure in periodontal fibroblasts, with A-PRF+ showing a more sustained effect [ 17 ]. A possible reason for the differing behaviors observed in the three assays is that early cellular responses to A-PRF-based preparations depend not only on the amount of released bioactive mediators but also on how these factors are made available over time and how they interact with the surrounding materials. A-PRF was developed within the low-speed centrifugation process, which has been shown to enhance the release of certain growth factors and produce better cellular responses compared to more conventional platelet-concentrate protocols [ 6 ]. Additionally, PRF may act as a local delivery system for these beneficial mediators, which suggests that the effectiveness of their biological activity relies on both the timing of their release and their concentrations [ 18 ]. Recent in vitro evidence suggests that A-PRF under certain conditions might release specific growth factors more and may stimulate fibroblast proliferation more effectively than some other platelet formulations [ 19 ]. The role of the collagen membrane likely involves changing how A-PRF interacts with the biomaterial itself and how the growth factors are released. Collagen membranes are not biologically inert carriers, and their physicochemical properties can influence absorption, penetration, retention, and release kinetics [ 3 ]. In an ex vivo study, Al-Maawi et al. showed that liquid-PRF interacted differently with different collagen materials, with only partial absorption into a specific type of collagen membrane (Bio-Gide®) [ 20 ]. Additionally, Mozgan et al. reported that collagen barrier membranes supplemented with platelet secretome displayed a strong early release of total protein, TGF-β1, and PDGF-BB [ 21 ]. Similarly, Blatt et al. reported that collagen matrices biofunctionalized with PRF improved the early release of growth factors and stimulated angiogenic activity [ 13 ]. Together, these studies suggest that the collagen membranes may change the presentation and availability of signals derived from A-PRF rather than simply providing a passive scaffold. The differences between the viability assay and the scratch assay in the 100% A-PRF-Col group need to be highlighted. Although 100% A-PRF-Col showed the lowest RGR and the least favorable morphology after 24 h in the viability assay, it still produced a modest but significant increase in wound closure compared with the negative control. This is not necessarily contradictory, as it makes sense given that these two assays were conducted in different settings and evaluated different aspects of cell behavior. As scratch closure reflects a collective response influenced by migration, cell spreading, and, to some extent, proliferation-related activity. Therefore, limited improvement in wound closure may still occur even when the general cell compatibility is less favorable. This is also consistent with the broader trend that shows different regenerative outcomes in PRF-based systems do not always change in a similar manner [ 7 , 16 ]. From a translational perspective, the present findings suggest that the biological performance of PRF-based membrane constructs should not be assumed to improve simply by increasing concentration or by adding a collagen membrane. Instead, both the concentration of the A-PRF used and the type of biomaterial paired with it play a crucial role in balancing early cell compatibility, growth activity, and the behavior of wound closure. This is relevant to periodontal regeneration since current collagen membranes typically act more as barriers and often need additional methods to enhance the biological activity. [ 3 , 4 ]. Several limitations should be acknowledged. One key point is that no collagen membrane-only extract group was included, so the effects of collagen alone could not be separated from the collagen and A-PRF combination. In addition, this was an in vitro study of early responses in a single cell type, and the proliferation was inferred from MTT-derived metabolic activity rather than direct cell counting. There was also no analysis of growth factors or the specific signaling mechanisms involved. Future research should include a control group for collagen alone, investigate how A-PRF and collagen profiles change over time, and expand the examination to more complex, three-dimensional, or in vivo models for periodontal regeneration. In summary, this study shows that the early effects of A-PRF on hPDLSCs are highly dependent on concentration and are further modified by combination with a collagen membrane. Generally, diluted extracts prompted more positive early cellular responses than undiluted extracts. The addition of collagen did not provide a uniform enhancement but rather altered the biological profile of A-PRF based on specific endpoints. The full concentration of the combination seemed to negatively affect early cell viability, while at certain midpoints, it actually enhanced metabolic activity related to cell proliferation. These findings highlight the need for careful and informed optimization of the combination of PRF and membranes, rather than a simple combination alone, before their regenerative potential can be fully interpreted. CONCLUSIONS This in vitro study demonstrates that the effects of A-PRF on hPDLSCs depend on its concentration and are influenced by the presence of a collagen membrane. Generally, diluted extracts elicited more favorable early cellular responses than undiluted extracts. However, the collagen membrane did not uniformly enhance the effects of A-PRF, but altered its biological profile in an endpoint-dependent manner. These findings emphasize the importance of biologically informed optimization of A-PRF and collagen constructs for use in periodontal regeneration. Abbreviations A-PRF: Advanced platelet-rich fibrin A-PRF-Col: Advanced platelet-rich fibrin combined with a collagen membrane DMEM/F12: Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 hPDLSCs: Human periodontal ligament stem cells MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide OD: Optical density RGR: Relative growth rate Declarations Clinical trial number Not applicable. Ethics approval and consent to participate This study was approved by the Biomedical Research Ethics Committee of the University of Medicine and Pharmacy at Ho Chi Minh City (Approval number 1973/ĐHYD-HĐĐĐ). This study was conducted in accordance with the principles of the Declaration of Helsinki. Written informed consent was obtained from all participants prior to blood collection. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding The research was funded by the University of Medicine and Pharmacy at Ho Chi Minh City (Grant number 277/2024/HĐ-ĐHYĐ, dated August 27th, 2024). Author Contribution MN and MBNQ conceived and designed the study. MN, MBNQ, BHTL, and VHN performed the experiments and acquired the data. MBNQ, TTT, and YTNN curated and analyzed the data. MN, TTT, THH, and NNHC interpreted the data. YTNN and NNHC supervised the study. TTT drafted the manuscript. MN, MBNQ, BHTL, YTNN, THH, VHN, and NNHC critically revised the manuscript for important intellectual content. All authors read and approved the final manuscript and agreed to be accountable for all aspects of the work. Acknowledgement We would like to thank the Department of Periodontology, Faculty of Dentistry, University of Medicine and Pharmacy at Ho Chi Minh City, and the Tissue Engineering and Biomedical Materials Laboratory, Ho Chi Minh City University of Science, Viet Nam National University Ho Chi Minh City, for their support of this study. Data Availability The datasets used and analysed during the current study are available from the corresponding author on reasonable request. References Tonetti MS, Greenwell H, Kornman KS. Staging and grading of periodontitis: framework and proposal of a new classification and case definition. J Periodontol. 2018;89(Suppl 1):S159–72. 10.1002/JPER.18-0006 . Liang Y, Luan X, Liu X. Recent advances in periodontal regeneration: a biomaterial perspective. Bioact Mater. 2020;5:297–308. 10.1016/j.bioactmat.2020.02.012 . Ma Y-F, Yan X-Z. Periodontal guided tissue regeneration membranes: limitations and possible solutions for the bottleneck analysis. Tissue Eng Part B Rev. 2023;29:532–44. 10.1089/ten.TEB.2023.0040 . Alqahtani A, Moorehead R, Asencio IO. Guided tissue and bone regeneration membranes: a review of biomaterials and techniques for periodontal treatments. Polymers. 2023;15:3355. 10.3390/polym15163355 . Ren Y, Fan L, Alkildani S, Liu L, Emmert S, Najman S, et al. Barrier membranes for guided bone regeneration (GBR): a focus on recent advances in collagen membranes. Int J Mol Sci. 2022;23:14987. 10.3390/ijms232314987 . Fujioka-Kobayashi M, Miron RJ, Hernandez M, Kandalam U, Zhang Y, Choukroun J. Optimized platelet‐rich fibrin with the low‐speed concept: growth factor release, biocompatibility, and cellular response. J Periodontol. 2017;88:112–21. 10.1902/jop.2016.160443 . Strauss F-J, Nasirzade J, Kargarpoor Z, Stähli A, Gruber R. Effect of platelet-rich fibrin on cell proliferation, migration, differentiation, inflammation, and osteoclastogenesis: a systematic review of in vitro studies. Clin Oral Investig. 2020;24:569–84. 10.1007/s00784-019-03156-9 . Pereira VBS, Lago CAP, Almeida RDAC, Barbirato DDS, Vasconcelos BCDE. Biological and cellular properties of advanced platelet-rich fibrin (A-PRF) compared to other platelet concentrates: systematic review and meta-analysis. Int J Mol Sci. 2023;25:482. 10.3390/ijms25010482 . Tomokiyo A, Wada N, Maeda H. Periodontal ligament stem cells: regenerative potency in periodontium. Stem Cells Dev. 2019;28:974–85. 10.1089/scd.2019.0031 . Zheng S, Zhang X, Zhao Q, Chai J, Zhang Y. Liquid platelet-rich fibrin promotes the regenerative potential of human periodontal ligament cells. Oral Dis. 2020;26:1755–63. 10.1111/odi.13501 . Nguyen M, Nguyen TT, Tran HLB, Tran DN, Ngo LTQ, Huynh NC. Effects of advanced platelet-rich fibrin combined with xenogenic bone on human periodontal ligament stem cells. Clin Exp Dent Res. 2022;8:875–82. 10.1002/cre2.563 . Tran HLB, Doan VN, Le HTN, Ngo LTQ. Various methods for isolation of multipotent human periodontal ligament cells for regenerative medicine. Vitro Cell Dev Biol Anim. 2014;50:597–602. 10.1007/s11626-014-9748-z . Blatt S, Burkhardt V, Kämmerer PW, Pabst AM, Sagheb K, Heller M, et al. Biofunctionalization of porcine-derived collagen matrices with platelet rich fibrin: influence on angiogenesis in vitro and in vivo. Clin Oral Investig. 2020;24:3425–36. 10.1007/s00784-020-03213-8 . Creeper F, Lichanska AM, Marshall RI, Seymour GJ, Ivanovski S. The effect of platelet-rich plasma on osteoblast and periodontal ligament cell migration, proliferation and differentiation. J Periodontal Res. 2009;44:258–65. 10.1111/j.1600-0765.2008.01125.x . Li X, Yang H, Zhang Z, Yan Z, Lv H, Zhang Y, et al. Platelet–rich fibrin exudate promotes the proliferation and osteogenic differentiation of human periodontal ligament cells in vitro. Mol Med Rep. 2018;18:4477–85. 10.3892/mmr.2018.9472 . Thanasrisuebwong P, Kiattavorncharoen S, Surarit R, Phruksaniyom C, Ruangsawasdi N. Red and yellow injectable platelet-rich fibrin demonstrated differential effects on periodontal ligament stem cell proliferation, migration, and osteogenic differentiation. Int J Mol Sci. 2020;21:5153. 10.3390/ijms21145153 . Pitzurra L, Jansen IDC, De Vries TJ, Hoogenkamp MA, Loos BG. Effects of L-PRF and A‐PRF + on periodontal fibroblasts in in vitro wound healing experiments. J Periodontal Res. 2020;55:287–95. 10.1111/jre.12714 . Miron RJ, Zhang Y. Autologous liquid platelet rich fibrin: A novel drug delivery system. Acta Biomater. 2018;75:35–51. 10.1016/j.actbio.2018.05.021 . Ashour SH, Mudalal M, Al-Aroomi OA, Al-Attab R, Li W, Yin L. The effects of injectable platelet-rich fibrin and advanced-platelet rich fibrin on gingival fibroblast cell vitality, proliferation, differentiation. Tissue Eng Regen Med. 2023;20:1161–72. 10.1007/s13770-023-00586-1 . Al-Maawi S, Herrera-Vizcaíno C, Orlowska A, Willershausen I, Sader R, Miron RJ, et al. Biologization of collagen-based biomaterials using liquid-platelet-rich fibrin: new insights into clinically applicable tissue engineering. Materials. 2019;12:3993. 10.3390/ma12233993 . Mozgan E-M, Edelmayer M, Janjić K, Pensch M, Fischer MB, Moritz A, et al. Release kinetics and mitogenic capacity of collagen barrier membranes supplemented with secretome of activated platelets - the in vitro response of fibroblasts of the periodontal ligament and the gingiva. BMC Oral Health. 2017;17:66. 10.1186/s12903-017-0357-6 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 06 May, 2026 Reviews received at journal 29 Apr, 2026 Reviews received at journal 27 Apr, 2026 Reviews received at journal 22 Apr, 2026 Reviewers agreed at journal 20 Apr, 2026 Reviewers agreed at journal 20 Apr, 2026 Reviewers agreed at journal 17 Apr, 2026 Reviews received at journal 15 Apr, 2026 Reviewers agreed at journal 15 Apr, 2026 Reviewers invited by journal 15 Apr, 2026 Editor invited by journal 23 Mar, 2026 Editor assigned by journal 20 Mar, 2026 Submission checks completed at journal 20 Mar, 2026 First submitted to journal 18 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9154707","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":626402677,"identity":"f632bc08-4df9-4b39-a967-c6f0f6abcaa6","order_by":0,"name":"Meo Nguyen","email":"","orcid":"","institution":"University of Medicine and Pharmacy at Ho Chi Minh City","correspondingAuthor":false,"prefix":"","firstName":"Meo","middleName":"","lastName":"Nguyen","suffix":""},{"id":626402680,"identity":"91c1bbf2-ecfc-4ad8-80f7-f93a8be6cda4","order_by":1,"name":"Minh Bao Nguyen Quoc","email":"","orcid":"","institution":"University of Medicine and Pharmacy at Ho Chi Minh City","correspondingAuthor":false,"prefix":"","firstName":"Minh","middleName":"Bao Nguyen","lastName":"Quoc","suffix":""},{"id":626402681,"identity":"d71ed420-2bbc-4f84-9163-db0535984225","order_by":2,"name":"Truong Tran Thien","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYLACxgYJOFsOQrHhVs0DIg6CtEAVGROrBaEosYGQFnvpw88ef9xhIWcu3/tM4uOO2vQN184eYPhQdpjB4HYDdlv40swNDp6RMLZsYzeTnHnmeO6G23kJjDPOAbXcOYBdCw+DmcTBNonEDcfY2KR5244BteQYMPO2HWaQnJGAQwv7N5CWepiWdAOQlr94tfCAbUkwgGipSQBrYQRq4ZfAoeUMT5nE2TYJww3H0pgtZ7YdMJwJ9MvBnnPpPLi0sPewb5OobKuTNzh8jPHGRyCD73buwQc/yqzl2HBoQQeHQTYzHGCAxhgxoI4UxaNgFIyCUTBCAACHv1rFGGfMVgAAAABJRU5ErkJggg==","orcid":"","institution":"University of Medicine and Pharmacy at Ho Chi Minh City","correspondingAuthor":true,"prefix":"","firstName":"Truong","middleName":"Tran","lastName":"Thien","suffix":""},{"id":626402685,"identity":"58ed7497-b622-4680-afb1-fcae64e2a0fc","order_by":3,"name":"Ha Le Bao Tran","email":"","orcid":"","institution":"Vietnam National University Ho Chi Minh City","correspondingAuthor":false,"prefix":"","firstName":"Ha","middleName":"Le Bao","lastName":"Tran","suffix":""},{"id":626402688,"identity":"8e509deb-3a3c-43cd-9063-a306fed20d05","order_by":4,"name":"Yen Thu Nguyen Ngoc","email":"","orcid":"","institution":"University of Medicine and Pharmacy at Ho Chi Minh City","correspondingAuthor":false,"prefix":"","firstName":"Yen","middleName":"Thu Nguyen","lastName":"Ngoc","suffix":""},{"id":626402690,"identity":"583722a4-f187-40a2-8ea7-a6a50f5f47d0","order_by":5,"name":"Thi Hoa Ho","email":"","orcid":"","institution":"University of Medicine and Pharmacy at Ho Chi Minh City","correspondingAuthor":false,"prefix":"","firstName":"Thi","middleName":"Hoa","lastName":"Ho","suffix":""},{"id":626402693,"identity":"c87175d1-25ce-4bde-be6c-30aeb096d5c0","order_by":6,"name":"Viet Ha Nguyen","email":"","orcid":"","institution":"University of Medicine and Pharmacy at Ho Chi Minh City","correspondingAuthor":false,"prefix":"","firstName":"Viet","middleName":"Ha","lastName":"Nguyen","suffix":""},{"id":626402695,"identity":"c2634812-60e5-49ef-bd21-7275cbccdd97","order_by":7,"name":"Nhat Nam Huynh Cong","email":"","orcid":"","institution":"University of Medicine and Pharmacy at Ho Chi Minh City","correspondingAuthor":false,"prefix":"","firstName":"Nhat","middleName":"Nam Huynh","lastName":"Cong","suffix":""}],"badges":[],"createdAt":"2026-03-18 05:08:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9154707/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9154707/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107705892,"identity":"1f5917b6-8425-4fc4-a234-3a1e66e5621b","added_by":"auto","created_at":"2026-04-24 09:15:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":61918,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of A-PRF and A-PRF-Col extracts on hPDLSC viability at 24 h\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Blank-corrected OD570 values of hPDLSCs after 24 h exposure to 100% and 20% A-PRF or A-PRF-Col extracts were analyzed by two-way repeated-measures ANOVA with Šídák pairwise comparisons. (b) Donor-level RGR percentages normalized to the negative control were analyzed with one-way repeated-measures ANOVA and Dunnett’s test versus the negative control. Data are mean ± standard deviation, with individual donor values (n = 4).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9154707/v1/52a5aaffbd72f962ed0d50ea.png"},{"id":107705316,"identity":"5d70771e-0a9e-4c1b-8dff-92ce25e120de","added_by":"auto","created_at":"2026-04-24 09:11:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2329877,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroscopic morphologies of hPDLSCs after 24 h in the viability assay.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Negative control; (b) Positive control; (c) 100% A-PRF; (d) 100% A-PRF-Col; (e) 20% A-PRF; (f) 20% A-PRF-Col. Scale bar = 200 µm.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9154707/v1/d37af7b6e45fceb5f3f11455.png"},{"id":107544710,"identity":"4a0425cd-da5f-441b-936c-c462b2bcf748","added_by":"auto","created_at":"2026-04-22 13:00:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":110297,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of A-PRF and A-PRF-Col extracts on hPDLSC proliferation-related metabolic activity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlank-corrected OD570 values of hPDLSCs cultured with 100% A-PRF, 100% A-PRF-Col, 20% A-PRF, or 20% A-PRF-Col extracts were measured on days 1, 3, 5, and 7. Basal serum-free medium (-) is shown as a reference control and was included only for secondary analysis, not in the primary factorial model. The four experimental groups were analyzed using three-way repeated-measures ANOVA, with time, concentration, and formulation as within-donor factors. Data are presented as mean ± standard deviation (n = 4).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9154707/v1/397571e8e9e7dc4a30573617.png"},{"id":107544712,"identity":"d2e72c46-f52f-4fdc-a84f-6334dbe59a51","added_by":"auto","created_at":"2026-04-22 13:00:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":56804,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of A-PRF and A-PRF-Col extracts on hPDLSC wound closure at 24 h\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Donor-level wound closure percentages in the four experimental groups after 24 h were analyzed by two-way repeated-measures ANOVA with Šídák pairwise comparisons. (b) Donor-level wound closure percentages, including the negative control, were analyzed by one-way repeated-measures ANOVA with Dunnett’s test versus the negative control. Data are mean ± standard deviation, with individual donor values (n = 4).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9154707/v1/046a40980137e6e45d8b6594.png"},{"id":107706030,"identity":"a98d3183-823a-4186-aa21-f8174db7d4ba","added_by":"auto","created_at":"2026-04-24 09:17:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":799540,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative scratch assay images of hPDLSCs at 0 and 24 h.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative microscopic images of hPDLSCs in the scratch assay at baseline (0 h) and after 24 h. Upper row, 0 h; lower row, 24 h. (a) Negative control; (b) Positive control; (c) 100% A-PRF; (d) 100% A-PRF-Col; (e) 20% A-PRF; (f) 20% A-PRF-Col. Scale bar = 200 µm.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9154707/v1/ea8e1363fed2ca51324c266b.png"},{"id":107709037,"identity":"734ec32a-16b2-4ed3-9e9f-4b21997c724f","added_by":"auto","created_at":"2026-04-24 09:34:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4204524,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9154707/v1/f2b8c818-af2a-4f59-858d-b8e7f8718c9a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Concentration-Dependent Effects of Advanced Platelet-Rich Fibrin Combined with A Collagen Membrane on Human Periodontal Ligament Stem Cells: An In Vitro Study","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003ePeriodontitis is a chronic inflammatory disease that damages tooth-supporting tissues, including the periodontal ligament and alveolar bone, and manifests as periodontal pockets, gingival recession, or both [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Advances in periodontal treatment may help control inflammation and slow disease progression, but predictable periodontal attachment apparatus regeneration still presents a challenge [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGuided tissue regeneration is a well-established periodontal regenerative strategy that uses a barrier membrane to prevent epithelial downgrowth and gingival connective tissue invasion while maintaining a space for tissue formation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Among available barrier materials, collagen membranes are popular due to their biocompatibility, resorbability, and structural similarity to the extracellular matrix [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, collagen membranes function primarily as barrier devices, and their capacity to provide sufficient biological stimulation to optimize early cellular regenerative responses may be limited [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePlatelet-rich fibrin (PRF) is a blood concentrate rich in fibrin, platelets, and leukocytes that aids in wound healing [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Advanced platelet-rich fibrin (A-PRF), a PRF variant produced by low-speed centrifugation, has a distinct clot composition that enhances the release of growth factors [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These properties suggest that combining A-PRF with a collagen membrane may help integrate the structural function of the membrane with the biological activity of the A-PRF, thereby modulating early cellular responses to periodontal wounds [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHuman periodontal ligament stem cells (hPDLSCs) are valuable for tissue repair due to their clonogenicity, proliferative capacity, and differentiation potential [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, evaluating the effects of materials on hPDLSC viability, proliferation, and migration is a useful in vitro approach for assessing their compatibility and effectiveness in promoting periodontal wound healing [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious in vitro studies have shown that PRF-based formulations can promote cell proliferation, migration, and differentiation. In our previous in vitro study, A-PRF combined with xenogenic bone substitute material enhanced hPDLSC proliferation and migration. However, it remains unclear how the A-PRF and collagen membrane combination affects early hPDLSC responses and whether A-PRF concentration influences them. Therefore, the aim of the present study was to compare the effects of A-PRF and A-PRF combined with a collagen membrane on hPDLSC viability, metabolic activity related to proliferation, and migration during wound closure at 100% and 20% extract concentrations. We hypothesized that the presence of a collagen membrane would modify initial hPDLSC responses to A-PRF, and that these effects would vary with different extract concentrations.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003eThis donor-matched in vitro experimental study compared the effects of A-PRF alone and A-PRF combined with a collagen membrane on hPDLSC viability, proliferation-related metabolic activity, and migration-related wound closure at two extract concentrations (100% and 20%).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHuman periodontal ligament stem cell culture\u003c/h2\u003e \u003cp\u003e Human periodontal ligament stem cells (hPDLSCs) at passage 3 were obtained from the Tissue Engineering and Biomedical Materials Laboratory, Ho Chi Minh City University of Science, Viet Nam National University Ho Chi Minh City. These cells were originally isolated by the outgrowth method, and the original tissue collection and cell characterization were ethically approved and previously reported [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These cells had been previously characterized as periodontal ligament-derived cells with mesenchymal stem stromal cell-like properties, showing high expression of CD44, CD73, and CD90, low expression of CD34, CD45, and HLA-DR, and in vitro osteogenic and adipogenic differentiation potential [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCells were cultured in complete culture medium, consisting of Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium/Nutrient Mixture F-12 (DMEM/F12; Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin-streptomycin solution (10,000 U/mL penicillin and 10 mg/mL streptomycin; Sigma-Aldrich, St. Louis, MO, USA), at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂. The medium was changed every 2 days, and passage 4 cells were used in all assays.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eA-PRF extract and A-PRF with a collagen membrane extract preparation\u003c/h3\u003e\n\u003cp\u003ePeripheral blood was collected from four healthy volunteers aged 20\u0026ndash;30 years who were non-smokers, did not consume alcohol, had no systemic disease, were not taking any medications or dietary supplements, had no periodontal disease or active inflammatory condition, and had platelet counts within the normal range. The study protocol was approved by the Biomedical Research Ethics Committee of the University of Medicine and Pharmacy at Ho Chi Minh City (Approval number 1973/DHYD-HĐĐĐ). All participants were informed about the study, and written informed consent was obtained prior to blood collection.\u003c/p\u003e \u003cp\u003eBlood was drawn into 10 mL A-PRF tubes without anticoagulant and immediately centrifuged at 1300 rpm (approximately 200 \u0026times; g) for 14 minutes using a DUO Quattro System centrifuge (Process for PRF, Nice, France) to obtain A-PRF clots. The clots were retrieved using sterile forceps, and the red blood cell fraction at the base of each clot was removed. For the A-PRF group, each clot was gently compressed for 30 seconds using a PRF Box (Process for PRF, Nice, France) to obtain an A-PRF membrane. For the A-PRF with a collagen membrane group (A-PRF-Col), a sterile porcine collagen membrane (Geistlich Bio-Gide\u0026reg;, Geistlich Pharma AG, Wolhusen, Switzerland) with an original size of 30 \u0026times; 40 mm was trimmed to 20 \u0026times; 30 mm. The A-PRF clot was placed over the collagen membrane to fully cover the surface, and both materials were compressed together for 30 seconds using the PRF Box, following a method adapted from Blatt et al. (2020) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor extract preparation, each A-PRF membrane and A-PRF-Col construct was processed separately. The wet mass of each A-PRF membrane, or of the A-PRF component in each A-PRF-Col construct, was measured before incubation. 100% extracts were prepared by incubating the materials in serum-free DMEM/F12 at an extraction ratio of 0.2 g/mL for 24 h at 37\u0026deg;C, following a protocol adapted from ISO 10993-12. In the A-PRF-Col group, the extraction ratio was calculated based on the wet mass of the A-PRF component to standardize the biological dose of A-PRF across formulations. After incubation, the conditioned media were centrifuged at 3,000 rpm (approximately 1,065 \u0026times; g) for 5 minutes to remove residual cells and debris. The supernatants were collected and defined as 100% extracts. 20% extracts were prepared separately under the same conditions using an extraction ratio of 0.04 g/mL in serum-free DMEM/F12.\u003c/p\u003e \u003cp\u003eThe experimental groups in the study included 100% A-PRF, 100% A-PRF-Col, 20% A-PRF, and 20% A-PRF-Col, with each extract processed independently from each donor and each donor treated as one biological replicate (n\u0026thinsp;=\u0026thinsp;4).\u003c/p\u003e\n\u003ch3\u003eEffect of A-PRF and A-PRF-Col extracts on hPDLSC viability\u003c/h3\u003e\n\u003cp\u003eThe effects of A-PRF and A-PRF-Col extracts on hPDLSC viability were assessed via metabolic activity using an MTT assay adapted from ISO 10993-5:2009. Basal serum-free DMEM/F12 was used as the negative control, and the basal serum-free DMEM/F12 with 20% DMSO was used as the positive control for assay validation and comparison.\u003c/p\u003e \u003cp\u003ehPDLSCs were seeded in 96-well plates at a density of 2 \u0026times; 10^4 cells/well and cultured for 24 h at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂. The culture medium was then replaced with 100 \u0026micro;L of the corresponding extract or control medium. Four technical replicate wells were used for each group in each experiment. After 24 h of incubation, cell morphology was qualitatively examined under an inverted microscope (CKX53; Olympus, Japan) using CellSens imaging software (Olympus, Japan) and compared with the control groups. The test media were removed and replaced with 100 \u0026micro;L of MTT solution (0.5 mg/mL; Sigma-Aldrich, St. Louis, MO, USA). Cells were incubated for 4 h under the same conditions. The MTT solution was then removed, and the resulting formazan crystals were dissolved in 100 \u0026micro;L of ethanol-DMSO solution (70:30, v/v). The plates were incubated for 30 minutes, and the contents of each well were mixed thoroughly by pipetting. Optical density was measured at 570 nm (OD\u003csub\u003e570\u003c/sub\u003e) using an EZ Read 400 microplate reader (Biochrom Ltd., Cambridge, UK), with blank wells containing no cells used for background correction.\u003c/p\u003e \u003cp\u003eRelative growth rate (RGR) was then calculated as follows: RGR (%) = (mean blank-corrected OD of the experimental group / mean blank-corrected OD of the negative control group) \u0026times; 100%. Samples with RGR values below 70% of the negative control were considered cytotoxic.\u003c/p\u003e\n\u003ch3\u003eEffect of A-PRF and A-PRF-Col extracts on hPDLSC proliferation-related metabolic activity\u003c/h3\u003e\n\u003cp\u003eThe effects of A-PRF and A-PRF-Col extracts on hPDLSC proliferation-related metabolic activity were assessed using an MTT assay at four different time points. Basal serum-free DMEM/F12 was used as the negative control, and complete culture medium was used as the positive control for assay validation and comparison.\u003c/p\u003e \u003cp\u003ehPDLSCs were seeded in 96-well plates at a density of 2 \u0026times; 10^4 cells/well and cultured for 24 h at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂ to allow cell attachment and spreading. The medium was then replaced with the corresponding extracts or control media. Four technical replicate wells were used for each group at each time point. Growth-related metabolic activity was assessed on days 1, 3, 5, and 7 using the MTT assay. Each time point used a separate 96-well plate with no medium replacement during the assay period. Blank wells without cells were included for background correction. The MTT assay was performed as described in the viability assay section, and blank-corrected OD values at 570 nm were used for analysis.\u003c/p\u003e\n\u003ch3\u003eEffect of A-PRF and A-PRF-Col extracts on hPDLSC migration-related wound closure\u003c/h3\u003e\n\u003cp\u003eTo evaluate migration-related wound closure, hPDLSCs were seeded at a density of 3 \u0026times; 10^5 cells/well in six-well plates and cultured until a near-confluent monolayer was achieved. Then, the culture medium was replaced with 3 mL of basal, serum-free DMEM/F12, and the cells were incubated for 24 h at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂ to induce serum deprivation. After this period, the medium was removed, and a single linear scratch was created in each well using a sterile 100\u0026ndash;1000 \u0026micro;L pipette tip. Detached cells were removed by washing once with 1\u0026times; phosphate-buffered saline, and 3 mL of the corresponding extract or control medium was immediately added to each well. Basal serum-free DMEM/F12 was used as the negative control, and complete culture medium was used as the positive control. These reference controls were not included in the primary factorial analysis. Four technical replicate wells were used for each group.\u003c/p\u003e \u003cp\u003eImages of the scratched area were captured at baseline (0 h) and after 24 h using an Olympus CKX-RCD inverted microscope equipped with a DP2-BSW microscopy camera at 4\u0026times; objective magnification. Each plate was marked to define the same field imaged at both time points, with a single field per well analyzed. Wound closure was quantified using ImageJ 1.52a software and expressed as the percentage of wound closure relative to baseline, calculated as [(A0\u0026thinsp;\u0026minus;\u0026thinsp;A24) / A0] \u0026times; 100, where A0 and A24 represent the cell-free area at 0 and 24 h, respectively. Image analysis was performed in a blinded manner.\u003c/p\u003e \u003cp\u003eBecause scratch closure reflects a composite response influenced by collective migration, cell spreading, and proliferation-related activity, this outcome was interpreted as migration-related wound closure rather than direct measurement of cell migration alone.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism 10 (GraphPad Software, San Diego, CA, USA). The donor was treated as the matched biological unit, and four technical replicate wells were averaged before analysis. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of donor-level values. Model assumptions were evaluated by residual plots and Q-Q plots. In repeated-measures analyses with within-donor factors having more than two levels, sphericity was not assumed, and the Greenhouse-Geisser correction was used. For factors with only two levels, sphericity was not applicable. No a priori sample size calculation was performed because this was an exploratory in vitro study using a fixed number of donor-derived biological replicates.\u003c/p\u003e \u003cp\u003eViability and migration-related wound closure data were analyzed using a two-way repeated-measures ANOVA with concentration and formulation as within-donor factors, followed by Š\u0026iacute;d\u0026aacute;k-adjusted pairwise comparisons. In secondary analyses, relative growth rate (RGR) values and wound closure percentages were analyzed using repeated-measures one-way ANOVA with Dunnett\u0026rsquo;s multiple-comparison test versus the negative control. Proliferation-related metabolic activity was analyzed using three-way repeated-measures ANOVA, with time, concentration, and formulation as within-donor factors. Prespecified simple-effects analyses were then performed using two-way repeated-measures ANOVA within each concentration and within each formulation, followed by Š\u0026iacute;d\u0026aacute;k-adjusted pairwise comparisons at each time point. Differences among the four experimental groups and the negative control across time points were analyzed in a secondary analysis, using a two-way repeated-measures ANOVA followed by Dunnett\u0026rsquo;s post hoc test. Assay-specific reference controls were included for validation and interpretation, but were not included in the primary factorial models. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of A-PRF and A-PRF-Col extracts on hPDLSC viability\u003c/h2\u003e\n \u003cp\u003eThe mean relative growth rate (RGR) values were 72.43\u0026thinsp;\u0026plusmn;\u0026thinsp;9.08% for 100% A-PRF, 32.12\u0026thinsp;\u0026plusmn;\u0026thinsp;5.30% for 100% A-PRF-Col, 87.53\u0026thinsp;\u0026plusmn;\u0026thinsp;7.55% for 20% A-PRF, and 108.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.69% for 20% A-PRF-Col after 24 h of exposure. Only 100% A-PRF-Col showed a mean RGR below the 70% cytotoxicity threshold (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e\n \u003cp\u003eMicroscopic observations were consistent with the viability findings (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Cells in the negative control showed the typical spindle-shaped morphology and relatively uniform distribution (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In contrast, the positive control showed marked deterioration, with many rounded, shrunken, and poorly attached cells (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In the 100% A-PRF group, cell morphology was largely preserved. However, cell distribution appeared less uniform and more contracted cells were observed (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The 100% A-PRF-Col group showed the most pronounced adverse changes, characterized by reduced adherent cell density, irregular distribution, and increased cell contraction and detachment (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). In both 20% extract groups, most cells remained elongated and adherent, resembling the negative control (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, f).\u003c/p\u003e\n \u003cp\u003eAnalysis of blank-corrected OD570 values in the four experimental groups showed a significant main effect of concentration (p\u0026thinsp;=\u0026thinsp;0.0009), no significant main effect of formulation (p\u0026thinsp;=\u0026thinsp;0.0785), and a significant concentration \u0026times; formulation interaction (p\u0026thinsp;=\u0026thinsp;0.0101) (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). \u0026Scaron;\u0026iacute;d\u0026aacute;k-adjusted comparisons showed a significant difference between A-PRF and A-PRF-Col at 100% concentration (p\u0026thinsp;=\u0026thinsp;0.0239), but not at 20% concentration (p\u0026thinsp;=\u0026thinsp;0.1361). Within the A-PRF condition, the difference between 100% and 20% was not significant (p\u0026thinsp;=\u0026thinsp;0.2498), whereas within the A-PRF-Col condition, viability was significantly lower at 100% than at 20% (p\u0026thinsp;=\u0026thinsp;0.0039).\u003c/p\u003e\n \u003cp\u003eWhen donor-level RGR values were analyzed together with the negative control, a significant overall group effect was observed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Compared with the negative control, both 100% A-PRF (p\u0026thinsp;=\u0026thinsp;0.0004) and 100% A-PRF-Col (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) showed significantly lower RGR values, whereas 20% A-PRF (p\u0026thinsp;=\u0026thinsp;0.0755) and 20% A-PRF-Col (p\u0026thinsp;=\u0026thinsp;0.3311) did not differ significantly from the negative control (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of A-PRF and A-PRF-Col extracts on hPDLSC proliferation-related metabolic activity\u003c/h2\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eBlank-corrected OD570 values of hPDLSCs cultured with 100% A-PRF, 100% A-PRF-Col, 20% A-PRF, or 20% A-PRF-Col extracts were measured on days 1, 3, 5, and 7. Basal serum-free medium (-) is shown as a reference control and was included only for secondary analysis, not in the primary factorial model. The four experimental groups were analyzed using three-way repeated-measures ANOVA, with time, concentration, and formulation as within-donor factors. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;4).\u003c/p\u003e\n \u003cp\u003eProliferation-related metabolic activity increased over time in all four experimental groups, peaked around day 5, and diverged at day 7 (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Three-way repeated-measures ANOVA showed significant effects of time (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and concentration (p\u0026thinsp;=\u0026thinsp;0.0017), but not formulation (p\u0026thinsp;=\u0026thinsp;0.0852). Significant interactions were observed for time \u0026times; concentration (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), time \u0026times; formulation (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), concentration \u0026times; formulation (p\u0026thinsp;=\u0026thinsp;0.0232), and time \u0026times; concentration \u0026times; formulation (p\u0026thinsp;=\u0026thinsp;0.0222).\u003c/p\u003e\n \u003cp\u003eAt 100% concentration, two-way repeated-measures ANOVA showed significant effects of time (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), formulation (p\u0026thinsp;=\u0026thinsp;0.0004), and time \u0026times; formulation interaction (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Compared with 100% A-PRF, 100% A-PRF-Col showed lower activity at day 1 (p\u0026thinsp;=\u0026thinsp;0.0024), but higher activity at days 3, 5, and 7 (p\u0026thinsp;=\u0026thinsp;0.0300, p\u0026thinsp;=\u0026thinsp;0.0019, and p\u0026thinsp;=\u0026thinsp;0.0016, respectively). At 20% concentration, there was a significant effect of time (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and a significant time \u0026times; formulation interaction (p\u0026thinsp;=\u0026thinsp;0.0010), whereas the main effect of formulation was not significant (p\u0026thinsp;=\u0026thinsp;0.2653). Compared with 20% A-PRF, 20% A-PRF-Col showed higher activity at days 3 and 5 (p\u0026thinsp;=\u0026thinsp;0.0011 and p\u0026thinsp;=\u0026thinsp;0.0090, respectively), did not differ at day 1 (p\u0026thinsp;=\u0026thinsp;0.0951), and showed lower activity at day 7 (p\u0026thinsp;=\u0026thinsp;0.0166).\u003c/p\u003e\n \u003cp\u003eWithin the A-PRF condition, two-way repeated-measures ANOVA showed significant effects of time (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), concentration (p\u0026thinsp;=\u0026thinsp;0.0004), and time \u0026times; concentration interaction (p\u0026thinsp;=\u0026thinsp;0.0002). Compared with 100% A-PRF, 20% A-PRF showed significantly higher activity at all time points (day 1, p\u0026thinsp;=\u0026thinsp;0.0428; day 3, p\u0026thinsp;=\u0026thinsp;0.0331; day 5, p\u0026thinsp;=\u0026thinsp;0.0001; day 7, p\u0026thinsp;=\u0026thinsp;0.0047). Within the A-PRF-Col condition, significant effects were also observed for time (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), concentration (p\u0026thinsp;=\u0026thinsp;0.0010), and time \u0026times; concentration interaction (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Compared with 100% A-PRF-Col, 20% A-PRF-Col showed higher activity at days 1, 3, and 5 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, p\u0026thinsp;=\u0026thinsp;0.0083, and p\u0026thinsp;=\u0026thinsp;0.0005, respectively), whereas 100% A-PRF-Col was higher at day 7 (p\u0026thinsp;=\u0026thinsp;0.0185).\u003c/p\u003e\n \u003cp\u003eIn the secondary analysis, including basal medium (-), significant effects of time, condition, and time \u0026times; condition interaction were observed (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). At day 1, both 100% A-PRF and 100% A-PRF-Col showed lower activity than basal medium (-) (p\u0026thinsp;=\u0026thinsp;0.0018 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively), whereas 20% A-PRF and 20% A-PRF-Col did not differ significantly from it (p\u0026thinsp;=\u0026thinsp;0.2965 and p\u0026thinsp;=\u0026thinsp;0.1750, respectively). At day 3, 100% A-PRF remained comparable with basal medium (-) (p\u0026thinsp;=\u0026thinsp;0.6114), while 100% A-PRF-Col, 20% A-PRF, and 20% A-PRF-Col showed significantly higher activity (p\u0026thinsp;=\u0026thinsp;0.0041, p\u0026thinsp;=\u0026thinsp;0.0002, and p\u0026thinsp;=\u0026thinsp;0.0001, respectively). By days 5 and 7, all four experimental groups showed significantly higher activity than basal medium (-) (all p\u0026thinsp;\u0026le;\u0026thinsp;0.0015).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of A-PRF and A-PRF-Col extracts on hPDLSC migration-related wound closure\u003c/h2\u003e\n \u003cp\u003eThe wound closure was primarily influenced by extract concentration after 24 h of incubation (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Two-way repeated-measures ANOVA of the four experimental groups showed a significant main effect of concentration (p\u0026thinsp;=\u0026thinsp;0.0004), no significant main effect of formulation (p\u0026thinsp;=\u0026thinsp;0.3369), and a significant concentration \u0026times; formulation interaction (p\u0026thinsp;=\u0026thinsp;0.0134). In both formulations, 20% extracts produced significantly greater wound closure than 100% extracts, for A-PRF (p\u0026thinsp;=\u0026thinsp;0.0002) and A-PRF-Col (p\u0026thinsp;=\u0026thinsp;0.0004). At 20% concentration, A-PRF induced significantly greater wound closure than A-PRF-Col (p\u0026thinsp;=\u0026thinsp;0.0389), whereas no significant difference between formulations was observed at 100% concentration (p\u0026thinsp;=\u0026thinsp;0.1238).\u003c/p\u003e\n \u003cp\u003eIn the control-based analysis, a significant overall group effect was observed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Compared with the negative control, 100% A-PRF did not significantly increase wound closure (p\u0026thinsp;=\u0026thinsp;0.5370), whereas 100% A-PRF-Col produced a modest but significant increase (p\u0026thinsp;=\u0026thinsp;0.0135). Both 20% A-PRF and 20% A-PRF-Col significantly increased wound closure relative to the negative control (both p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\n \u003cp\u003eRepresentative scratch images supported the quantitative findings (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). After 24 h, the cell-free gap remained relatively wide in the negative control and in both 100% extract groups. In contrast, it narrowed more in the positive control and, more markedly, in both 20% extract groups. Among the experimental groups, 20% A-PRF showed the greatest apparent wound closure, followed by 20% A-PRF-Col, while the difference between 100% A-PRF and 100% A-PRF-Col appeared less pronounced.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe current study demonstrated that the early effects of A-PRF on hPDLSCs depend on the concentration of A-PRF and were modified by the addition of a collagen membrane. Overall, the 20% extracts were more favorable than the 100% extracts across the evaluated early cellular responses. Meanwhile, the effect of the collagen membrane influenced the outcomes differently, depending on the specific biological aspect. 100% A-PRF-Col showed the least favorable viability profile, 20% A-PRF-Col showed the highest proliferation-related metabolic activity at the intermediate time points, and 20% A-PRF showed the greatest migration-related wound closure. These results suggest that incorporating a collagen membrane alters how hPDLSCs respond to A-PRF, and the effect varies based on the concentration of the extract used. Therefore, A-PRF with a collagen membrane should not be considered simply as a stronger version of A-PRF but with its own unique properties. This finding is relevant to periodontal regeneration because collagen membranes are commonly used as barrier materials in guided tissue regeneration. Therefore, the current study aimed to explore a clinical question: whether combining the structural role of a collagen membrane with the biological activity of A-PRF influences how cells behave during the early stages of wound healing. The results suggest that this combination dose impacts early cellular behavior related to wound healing, but the effects are not consistent across all observed outcomes.\u003c/p\u003e \u003cp\u003eThe viability assay data indicate that adding a collagen membrane did not consistently improve the early cell compatibility of A-PRF, particularly at the undiluted concentration. After 24 h, only 100% A-PRF-Col dropped below the 70% cytotoxicity threshold, consistent with the marked morphological deterioration observed under the microscope. By contrast, both 20% extract groups maintained a spindle-shaped, adherent morphology similar to the negative control. This pattern suggests that increasing extract concentration did not simply amplify a beneficial biological effect. Rather, a more concentrated preparation, especially when combined with collagen, may have created an environment less conducive to the metabolic activities vital for the survival of hPDLSC. This aligns with existing research on PRF, which generally reports beneficial effects of PRF on proliferation, migration, and differentiation, but also emphasizes substantial heterogeneity across formulations, cell types, and assay designs [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. A concentration-dependent effect is biologically reasonable, as platelet-derived preparations do not necessarily exhibit their most favorable cellular effects at maximal concentrations [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor proliferation-related metabolic activity, the overall pattern favored the 20% extracts, particularly during the first 5 days. All experimental groups increased until day 5, but their trends diverged afterward. Within the A-PRF condition, 20% A-PRF consistently exceeded 100% A-PRF, whereas for the A-PRF-Col groups, 20% A-PRF-Col was higher at days 1, 3, and 5, but not at day 7. These data suggest that a lower concentration may provide a more nurturing environment for sustained cellular metabolic activity, while the collagen membrane may affect the timing of this response rather than producing a uniform enhancement. These findings should be interpreted in the context of an MTT-based assay conducted under conditions of sustained exposure to serum-free extracts. Thus, these findings specifically examine proliferation-related metabolic activity, rather than counting the actual number of cells.\u003c/p\u003e \u003cp\u003eThese findings align with our previous study, in which exudates from A-PRF combined with xenogenic bone substitute material promoted hPDLSC proliferation, with 20% outperforming 100% at several time points and with a general peak around day 5 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This contrasts with research by Li et al., which used PRF exudate and reported that both 100% and 20% promoted stronger proliferation than a 4% concentration, suggesting a dose-dependent relationship [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The present data align more closely with our previous model than with Li et al., which suggests that a lower concentration may better support sustained growth-related responses. This difference is biologically plausible because the biomaterial, extract preparation, and target cell context were not identical across studies [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe wound closure assays further support that concentration was the primary factor in the present study. Both A-PRF and A-PRF-Col induced significantly greater wound closure at 20% than at 100%, and at the lower concentration, A-PRF had better results than A-PRF-Col. These results suggest that the collagen membrane did not improve migration-related wound closure under the present conditions and may have attenuated the effect of A-PRF at 20%. This specific effect aligns with previous studies showing that different platelet-concentrate formulations can favor different biological processes. Thanasrisuebwong et al. showed that red and yellow i-PRF exerted differential effects on PDLSCs, with red i-PRF favoring proliferation and migration and yellow i-PRF promoting earlier osteogenic differentiation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Zheng et al. reported that liquid-PRF enhanced the proliferation, migration, and osteogenic differentiation of hPDLCs [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Similarly, Pitzurra et al. found that both L-PRF and A-PRF+ stimulated wound closure in periodontal fibroblasts, with A-PRF+ showing a more sustained effect [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA possible reason for the differing behaviors observed in the three assays is that early cellular responses to A-PRF-based preparations depend not only on the amount of released bioactive mediators but also on how these factors are made available over time and how they interact with the surrounding materials. A-PRF was developed within the low-speed centrifugation process, which has been shown to enhance the release of certain growth factors and produce better cellular responses compared to more conventional platelet-concentrate protocols [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Additionally, PRF may act as a local delivery system for these beneficial mediators, which suggests that the effectiveness of their biological activity relies on both the timing of their release and their concentrations [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Recent in vitro evidence suggests that A-PRF under certain conditions might release specific growth factors more and may stimulate fibroblast proliferation more effectively than some other platelet formulations [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe role of the collagen membrane likely involves changing how A-PRF interacts with the biomaterial itself and how the growth factors are released. Collagen membranes are not biologically inert carriers, and their physicochemical properties can influence absorption, penetration, retention, and release kinetics [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In an ex vivo study, Al-Maawi et al. showed that liquid-PRF interacted differently with different collagen materials, with only partial absorption into a specific type of collagen membrane (Bio-Gide\u0026reg;) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Additionally, Mozgan et al. reported that collagen barrier membranes supplemented with platelet secretome displayed a strong early release of total protein, TGF-β1, and PDGF-BB [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Similarly, Blatt et al. reported that collagen matrices biofunctionalized with PRF improved the early release of growth factors and stimulated angiogenic activity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Together, these studies suggest that the collagen membranes may change the presentation and availability of signals derived from A-PRF rather than simply providing a passive scaffold.\u003c/p\u003e \u003cp\u003eThe differences between the viability assay and the scratch assay in the 100% A-PRF-Col group need to be highlighted. Although 100% A-PRF-Col showed the lowest RGR and the least favorable morphology after 24 h in the viability assay, it still produced a modest but significant increase in wound closure compared with the negative control. This is not necessarily contradictory, as it makes sense given that these two assays were conducted in different settings and evaluated different aspects of cell behavior. As scratch closure reflects a collective response influenced by migration, cell spreading, and, to some extent, proliferation-related activity. Therefore, limited improvement in wound closure may still occur even when the general cell compatibility is less favorable. This is also consistent with the broader trend that shows different regenerative outcomes in PRF-based systems do not always change in a similar manner [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFrom a translational perspective, the present findings suggest that the biological performance of PRF-based membrane constructs should not be assumed to improve simply by increasing concentration or by adding a collagen membrane. Instead, both the concentration of the A-PRF used and the type of biomaterial paired with it play a crucial role in balancing early cell compatibility, growth activity, and the behavior of wound closure. This is relevant to periodontal regeneration since current collagen membranes typically act more as barriers and often need additional methods to enhance the biological activity. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral limitations should be acknowledged. One key point is that no collagen membrane-only extract group was included, so the effects of collagen alone could not be separated from the collagen and A-PRF combination. In addition, this was an in vitro study of early responses in a single cell type, and the proliferation was inferred from MTT-derived metabolic activity rather than direct cell counting. There was also no analysis of growth factors or the specific signaling mechanisms involved. Future research should include a control group for collagen alone, investigate how A-PRF and collagen profiles change over time, and expand the examination to more complex, three-dimensional, or in vivo models for periodontal regeneration.\u003c/p\u003e \u003cp\u003eIn summary, this study shows that the early effects of A-PRF on hPDLSCs are highly dependent on concentration and are further modified by combination with a collagen membrane. Generally, diluted extracts prompted more positive early cellular responses than undiluted extracts. The addition of collagen did not provide a uniform enhancement but rather altered the biological profile of A-PRF based on specific endpoints. The full concentration of the combination seemed to negatively affect early cell viability, while at certain midpoints, it actually enhanced metabolic activity related to cell proliferation. These findings highlight the need for careful and informed optimization of the combination of PRF and membranes, rather than a simple combination alone, before their regenerative potential can be fully interpreted.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThis in vitro study demonstrates that the effects of A-PRF on hPDLSCs depend on its concentration and are influenced by the presence of a collagen membrane. Generally, diluted extracts elicited more favorable early cellular responses than undiluted extracts. However, the collagen membrane did not uniformly enhance the effects of A-PRF, but altered its biological profile in an endpoint-dependent manner. These findings emphasize the importance of biologically informed optimization of A-PRF and collagen constructs for use in periodontal regeneration.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eA-PRF: Advanced platelet-rich fibrin\u003c/p\u003e\n\u003cp\u003eA-PRF-Col: Advanced platelet-rich fibrin combined with a collagen membrane\u003c/p\u003e\n\u003cp\u003eDMEM/F12: Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium/Nutrient Mixture F-12\u003c/p\u003e\n\u003cp\u003ehPDLSCs: Human periodontal ligament stem cells\u003c/p\u003e\n\u003cp\u003eMTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide\u003c/p\u003e\n\u003cp\u003eOD: Optical density\u003c/p\u003e\n\u003cp\u003eRGR: Relative growth rate\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eClinical trial number\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003e This study was approved by the Biomedical Research Ethics Committee of the University of Medicine and Pharmacy at Ho Chi Minh City (Approval number 1973/ĐHYD-HĐĐĐ). This study was conducted in accordance with the principles of the Declaration of Helsinki. Written informed consent was obtained from all participants prior to blood collection.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe research was funded by the University of Medicine and Pharmacy at Ho Chi Minh City (Grant number 277/2024/HĐ-ĐHYĐ, dated August 27th, 2024).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMN and MBNQ conceived and designed the study. MN, MBNQ, BHTL, and VHN performed the experiments and acquired the data. MBNQ, TTT, and YTNN curated and analyzed the data. MN, TTT, THH, and NNHC interpreted the data. YTNN and NNHC supervised the study. TTT drafted the manuscript. MN, MBNQ, BHTL, YTNN, THH, VHN, and NNHC critically revised the manuscript for important intellectual content. All authors read and approved the final manuscript and agreed to be accountable for all aspects of the work.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to thank the Department of Periodontology, Faculty of Dentistry, University of Medicine and Pharmacy at Ho Chi Minh City, and the Tissue Engineering and Biomedical Materials Laboratory, Ho Chi Minh City University of Science, Viet Nam National University Ho Chi Minh City, for their support of this study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTonetti MS, Greenwell H, Kornman KS. 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Materials. 2019;12:3993. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ma12233993\u003c/span\u003e\u003cspan address=\"10.3390/ma12233993\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMozgan E-M, Edelmayer M, Janjić K, Pensch M, Fischer MB, Moritz A, et al. Release kinetics and mitogenic capacity of collagen barrier membranes supplemented with secretome of activated platelets - the in vitro response of fibroblasts of the periodontal ligament and the gingiva. BMC Oral Health. 2017;17:66. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12903-017-0357-6\u003c/span\u003e\u003cspan address=\"10.1186/s12903-017-0357-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Advanced platelet-rich fibrin, Cell proliferation, Cell viability, Collagen membrane, Periodontal ligament stem cells, Periodontal regeneration","lastPublishedDoi":"10.21203/rs.3.rs-9154707/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9154707/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAdvanced platelet-rich fibrin (A-PRF) is used in periodontal regeneration due to its autologous fibrin matrix and bioactive content. Collagen membranes are widely used in guided tissue regeneration. However, it remains unclear whether combining A-PRF with a collagen membrane alters early cellular responses. The present in vitro study compared the effects of A-PRF alone and A-PRF combined with a collagen membrane on the viability, proliferation-related metabolic activity, and migration-related wound closure of human periodontal ligament stem cells (hPDLSCs) in different extract concentrations.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003ehPDLSCs were exposed to A-PRF or A-PRF mixed with a porcine collagen membrane (A-PRF-Col) extract at 100% and 20% concentrations prepared from the blood of four donors. Cell viability was assessed after 24 h using an MTT assay and morphological observation. Cell proliferation-related metabolic activity was evaluated by MTT assay on days 1, 3, 5, and 7. Migration-related wound closure was evaluated using a scratch assay after 24 h. Data were analyzed using repeated-measures ANOVA, followed by post hoc multiple-comparison tests. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 is considered statistically significant.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eEarly responses of hPDLSCs were influenced by A-PRF concentration and further modified by the collagen membrane. Only 100% A-PRF-Col showed cytotoxicity, with a low mean relative growth rate of 32.12%. Proliferation-related metabolic activity increased and peaked around day 5, but different concentrations and formulations affected the patterns. At 100% concentration, A-PRF-Col had lower activity but surpassed A-PRF in subsequent days. At 20% concentration, the A-PRF-Col group showed higher activity at days 3 and 5 but lower activity at day 7. Migration-related wound closure was primarily influenced by the concentration of the extract. 20% extracts outperformed their 100% counterparts, and 20% A-PRF achieved better closure than 20% A-PRF-Col.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe early effects of A-PRF on hPDLSCs were concentration-dependent and were modified by combination with a collagen membrane. Diluted extracts may support more favorable early cellular responses, while the collagen membrane modified the biological profile in a dependent manner rather than providing consistent enhancements.\u003c/p\u003e","manuscriptTitle":"Concentration-Dependent Effects of Advanced Platelet-Rich Fibrin Combined with A Collagen Membrane on Human Periodontal Ligament Stem Cells: An In Vitro Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-22 13:00:49","doi":"10.21203/rs.3.rs-9154707/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-06T07:03:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-29T14:24:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-27T14:43:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-22T06:15:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"109732914983606520393883698446437651257","date":"2026-04-20T14:29:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"159928477909549085194713994033030547443","date":"2026-04-20T12:19:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"47014987936686180497910424812239211485","date":"2026-04-17T13:29:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-15T12:38:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"69200291794825583110626903109967456132","date":"2026-04-15T12:32:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-15T08:24:13+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-23T13:08:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-20T14:57:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-20T14:56:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Oral Health","date":"2026-03-18T05:00:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9583b7ff-d07c-473a-8fb6-a4a2886390bd","owner":[],"postedDate":"April 22nd, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-06T07:03:04+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-17T10:53:08+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-22 13:00:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9154707","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9154707","identity":"rs-9154707","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}