Synergistic Effects of Folic Acid and Dental Pulp Stem Cell-Derived Exosomes on Gene Expression in a Periodontal Injury Model

Synergistic Effects of Folic Acid and Dental Pulp Stem Cell-Derived Exosomes on Gene Expression in a Periodontal Injury Model | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Natural Sciences This is a preprint and has not been peer reviewed. Data may be preliminary. 8 May 2025 V1 Latest version Share on Synergistic Effects of Folic Acid and Dental Pulp Stem Cell-Derived Exosomes on Gene Expression in a Periodontal Injury Model Authors : Saghar Zarei , Mostafa Montazeri 0000-0001-6364-2658 [email protected] , Nader Tanideh , Shahrokh Zare , and Mahintaj Dara Authors Info & Affiliations https://doi.org/10.22541/au.174669680.07803112/v1 300 views 124 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background: Periodontitis, a prevalent inflammatory disease, leads to the destruction of periodontal tissues, and current treatments often fail to achieve complete regeneration. Stem cell-derived exosomes, nanoscale vesicles mediating intercellular communication, have emerged as a promising cell-free therapeutic approach. This study aimed to investigate the potential of exosomes derived from dental pulp mesenchymal stem cells (DP-MSCs) and folic acid (FA), both individually and in combination, to improve periodontal injury in an in vitro model. Materials and Methods: DP-MSCs were isolated from human deciduous teeth, cultured, and characterized by their differentiation potential into adipocytes and osteocytes, as well as flow cytometry analysis of mesenchymal stem cell markers. Exosomes were extracted from DP-MSC conditioned media and characterized using scanning electron microscopy (SEM) and Bradford assay for protein quantification. Human gingival fibroblasts (hGF) were cultured and subjected to an in vitro injury model by exposure to H2O2. The injured hGF cells were then treated with FA (50 µM) and/or DP-MSC-Exos (20 µg/ml). Expression of anti-inflammatory, antioxidant, apoptotic, and osteogenic genes was measured using RT-qPCR. Results: DP-MSCs exhibited fibroblastic morphology, differentiated into adipocytes and osteocytes, and expressed mesenchymal markers CD73 and CD90. SEM confirmed the spherical structure of the extracted exosomes, and the Bradford assay revealed a protein concentration of 850 µg/ml. In injured hGF cells, treatment with DP-SC-Exos significantly decreased the Bax/Bcl2 ratio (p < 0.0001) and reduced the expression of antioxidant genes CATA, Gpx, and SOD, and RIPK3. FA treatment reduced IL-6 and TNF-α expression, while both FA and DP-MSC-Exos treatments resulted in diminished TGF-β expression. Conclusion: DP-MSC-derived exosomes demonstrated a notable capacity to modulate gene expression in an in vitro periodontal injury model, particularly by reducing apoptotic and antioxidant gene expression. These findings suggest that exosomes hold promise as a potential therapeutic agent for periodontal regeneration, offering advantages over cell-based therapies. INTRODUCTION Periodontitis, a highly prevalent inflammatory disease, ranks as the sixth most common disorder globally (1). This condition is a primary driver of the irreversible destruction of the periodontium, the intricate apparatus that anchors teeth, encompassing the periodontal ligament (PDL), cementum, and alveolar bone. The subsequent loss of teeth is a frequent and debilitating outcome of untreated or inadequately managed periodontitis (2). While current periodontal therapies can effectively manage the pathogenic state, they often fall short in reliably regenerating the lost periodontal tissues, thus highlighting a critical unmet clinical need (3). Stem cells, with their remarkable capacity for self-renewal and differentiation into diverse cell types, hold immense promise for regenerative medicine. Consequently, stem cell therapy has been explored across various disease contexts. However, despite promising results in certain clinical scenarios, the widespread translation of stem cell transplantation into routine clinical practice has been hindered (4). This lag is largely attributed to biosafety concerns associated with stem cell therapy, including infusion toxicity, immunological reactions, the potential for oncological complications, and ethical considerations (5). Mesenchymal stem cells (MSCs) have emerged as a particularly attractive option for periodontal regeneration (6). Nevertheless, cell-based therapies involving MSCs face substantial hurdles related to the production, distribution, and storage of cells for transplantation. It is now clear that MSCs exert their therapeutic effects, at least in part, through the secretion of a variety of bioactive factors that promote healing, tissue regeneration, and mitigate tissue damage (7). In line with this, conditioned media derived from bone marrow MSCs and PDL stem cells have demonstrated the capacity to enhance periodontal regeneration (8, 9). Among the various trophic factors released by MSCs, exosomes, nanoscale vesicles with a bilipid membrane, have been identified as key mediators of their therapeutic efficacy (10-13). Exosomes, typically ranging from 40 to 100 nm in size, primarily function in intercellular communication by transporting bioactive molecules between cells, thereby eliciting biological responses in recipient cells (10). Notably, exosomes can precisely deliver a diverse cargo, including proteins, nucleic acids, small molecules, and nanoparticles, to the inflammatory microenvironment (14). A growing body of evidence indicates that MSC-derived exosomes play a significant role in both the development and resolution of chronic inflammation (15, 16), exerting control over the inflammatory microenvironment through the release of anti-inflammatory factors and modulation of gene expression (17, 18). Intriguingly, even brief exposure to stem cell-derived exosomes can initiate sustained regenerative processes (6). For instance, MSC-derived exosomes can rapidly restore homeostasis and initiate natural tissue repair and regeneration (19, 20). Furthermore, exosomes derived from MSCs exhibit potent immunomodulatory and anti-inflammatory properties (21-23). The therapeutic potential of exosomes has been extensively investigated in various chronic inflammatory conditions, including rheumatoid arthritis, osteoarthritis, atherosclerosis, and inflammatory bowel disease (24). However, the precise mechanisms and efficacy of exosome-based therapies in the context of periodontal regeneration remain to be fully elucidated (6). This study aimed to explore the combined effects of folic acid and exosomes derived from dental pulp stem cells (DP-MSCs), a readily accessible source of MSCs, on improving an in vitro model of periodontal injury. The investigation focused on analyzing alterations in the expression of key genes involved in anti-inflammatory responses, antioxidant activity, apoptosis, and osteogenesis. Materials and Methods : Dental Pulp Mesenchymal Stem Cell (DP-MSC) Isolation and Culture This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Islamic Azad University of Shiraz (approval code: IR.IAU.SHIRAZ.REC.1403.063; approval date: May 19, 2024). Healthy children aged 6-8 years, scheduled for deciduous teeth extraction at a designated dental clinic, were included in the study. Children with systemic or local inflammation or a history of medication use within the past month were excluded. Informed written consent was obtained from the parents of all participants. Healthy deciduous teeth were extracted from four children, without regard to sex. A total of six teeth were collected. The extracted teeth were thoroughly washed with povidone-iodine and sterile saline buffer. Dental pulps were carefully extracted using a barbed broach No. 3 and immediately placed in saline buffer supplemented with penicillin, streptomycin, and amphotericin. The samples were transported to the laboratory within two hours. In the laboratory, the pulps were minced into 1-2 mm pieces and enzymatically digested with type I collagenase (3 mg/ml, Thermo Fisher Scientific (Gibco), USA) and dispase (4 mg/ml, Gibco, USA) for 45 minutes at 37°C in a shaker. The digestion was stopped by adding 3 ml of α-MEM culture medium (Thermo Fisher Scientific (Gibco), USA) supplemented with 20% FBS and 1% penicillin/streptomycin. The resulting suspension was centrifuged at 1500 rpm for 5 minutes (Eppendorf Centrifuge 5810R). The supernatant was discarded, and the cell pellet was resuspended and transferred to a T25 culture flask containing complete culture medium. Cells were cultured in a humidified atmosphere at 37°C with 5% CO 2 . The culture medium was replaced every three days until the cells reached 80% confluence, which took approximately 7-14 days. Cells were passaged by treatment with 1 ml of trypsin/EDTA (0.05%) in an incubator. After 3-4 minutes, 3 ml of complete culture medium containing FBS was added to neutralize the trypsin. Cells were collected and centrifuged at 1200 rpm for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in 3 ml of complete culture medium and divided equally into three T75 culture flasks containing 14 ml of the same medium. The flasks were incubated under the same conditions. This process was repeated until passage three. DP-MSC Characterization Cells were cultured in 6-well plates until they reached 70-80% confluence. Two wells were used for each of the following groups: control, osteogenic induction, and adipogenic induction. Cells in each group were treated with a specific culture medium. The control group was maintained in basic culture medium consisting of DMEM, FBS, antibiotics, and non-essential amino acids. Osteogenic induction medium was supplemented with ascorbic acid (50 µM), dexamethasone (100 nM), and glycerol 3-phosphate (100 mM). Adipogenic induction medium was supplemented with ascorbic acid (100 µM), dexamethasone (100 nM), and indomethacin (200 µM). The culture media for all groups were replaced every two days. After 20 days of induction, the culture media were removed, and the cells were washed with PBS. Cells were fixed with 4% formaldehyde (0.5 ml) for 15 minutes, washed several times with dH2O, and stained with Alizarin Red or Oil Red O for the osteogenic and adipogenic groups, respectively. Flow cytometry was performed on passage three cells for further characterization. Cells were detached using trypsin/EDTA, and counted with a hemocytometer. Approximately 100,000 cells were incubated with anti-human-CD34-FITC, anti-human-CD45-FITC, anti-human-CD90-PerCp, and anti-human-CD73-PE antibodies. The cell suspension was washed with 2% PBS, centrifuged at 400g for 5 minutes, and analyzed using a flow cytometer (BD Biosciences, USA). FlowJo software was used for data analysis. Exosome Isolation from DP-MSCs DP-MSCs at passage 3 were cultured in 10 T75 flasks. Upon reaching 50-60% confluence, the culture medium was replaced with DMEM. After 72 hours, the conditioned medium was collected, and exosomes were extracted using the Exocib kit (Zist Fanavaran, Iran) according to the manufacturer’s instructions. The extracted exosomes were stored at -70°C. Scanning Electron Microscopy (SEM) of Exosomes For SEM analysis, 20 µl of the exosome suspension was fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer (pH 7.4) at 4°C for 2 hours. The samples were then dehydrated through a graded ethanol series (30%, 50%, 70%, 90%, 100%, 100%, 10 minutes each). Following dehydration, the samples were mounted onto silicon chips and sputter-coated with a 5 nm thin layer of gold. Imaging was performed using a TESCAN-Vega3 scanning electron microscope (TESCAN, Czech Republic) at an accelerating voltage of 20 kV, with a secondary electron detector. Images were acquired at 59.6kx magnification or 500 nm. Protein Concentration Measurement The protein concentration of the exosome suspension was determined using the Bradford method. Briefly, 25 µl of the suspension was mixed with 475 µl of Bradford solution (Pars Tous, Iran) and incubated in the dark for 5 minutes. Absorbance was measured at 595 nm. A standard curve was generated using bovine serum albumin (fraction V; concentrations: 1000, 500, 250, 125, 62.5, 31.25, 15.6, 7.6 µg/ml) and distilled water, and used to determine the protein concentration of the exosome sample. Human Gingival Fibroblast (hGF) Cell Culture The hGF cell line was obtained from the Pasteur Institute (Tehran, Iran) as a confluent culture in a T25 flask. Upon receipt, cells were incubated at 37 °C in a humidified atmosphere with 5% CO 2 . After 24 hours, cells were harvested and subcultured into three T25 flasks using DMEM-F12 medium supplemented with 10% FBS. The cells were maintained in the same incubator until used for subsequent experiments. MTT Assay hGF cells were seeded in 96-well plates at a density of 5000 cells per 200 µl per well. After 24 hours of incubation, cells were treated with different concentrations of folic acid, exosome suspension, and H 2 O 2 . After 24 hours, the medium was replaced with 200 µl of basic culture medium containing 20 µl of MTT solution (5 mg/ml in sterile PBS) and incubated for 4 hours. The plate was centrifuged, the medium was removed, and 200 µl of DMSO was added to each well. Absorbance was measured at 570 nm. Induction of Inflammation in hGF Cells hGF cells were seeded in 6-well plates at a density of 50,000 cells per well and incubated for 24 hours. Cells were then treated with 50 µM H 2 O 2 for 1 hour to induce inflammation. A 2 ml scratch was created in the center of the cell culture using a scraper to simulate injury (case group). The control group was treated with DMEM-F12 medium only, without H 2 O 2 or a scratch. Following H2O2 treatment, the culture medium was removed, and the cells were washed twice with PBS. The treatment groups were as follows: • Control group: Complete culture medium. • Group I: Complete culture medium + 50 µM folic acid. • Group II: Complete culture medium + 20 µg/ml DP-MSC-derived exosomes. • Group III: Complete culture medium + 50 µM folic acid + 20 µg/ml DP-MSC-derived exosomes. Cell morphology was monitored after 72 hours using a Nikon-TS100 inverted microscope (Nikon Corporation, Japan) equipped with a digital camera. Cells were then harvested for gene expression analysis. Measuring Expression of Anti-inflammatory, Antioxidant, Apoptotic, and Osteogenic Genes Total RNA was extracted from hGF cells using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA quantity and quality were assessed with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). First-strand cDNA was synthesized using the AddScript cDNA Synthesis Kit (Vivantis Technologies, Selangor Darul Ehsan, Malaysia) and 500 ng of RNA template.Real-time quantitative polymerase chain reaction (RT-qPCR) was used to measure the expression of specific genes involved in inflammation, oxidative stress, apoptosis, and osteogenesis. The following table lists the primer sequences used for RT-qPCR: h.sod.f CCA TGT TCA TGA GTT TGG AGA TAA T h.sod,r TGC CTC TCT TCA TCC TTT GG h.gpx.f CAT CAG GAG AAC GCC AAG AA h.gpx.r GCA CTT CTC GAA GAG CAT GA h.cat.f CCG AGA GAG AAT TCC TGA GG A h.cat.r CTT TGC CTT GGA GTA TTT GGT AAT G f.tnf.f TGC TGC ACT TTG GAG TGA T f.tnf.r GGG TTC GAG AAG ATG ATC TGA C h.pparg.f CAG ATC CAG TGG TTG CAG ATT A h.pparg.r AGA TGC AGG CTC CAC TTT G h.runx2.f CGC TGC AAC AAG AAC CT h.runx2.r TTA CCC GCC ATG ACA GTA AC h.ripk3.f TTC CGG GCG CAA CAT AG h.ripk3.r TTC GTT ATC CAG ACT TGC CAT Table 1 . List of primer sequences used for real-time quantitative polymerase chain reaction (RT-qPCR) analysis of gene expression. RT-qPCR was performed using the Applied Biosystems StepOne Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The reaction conditions were: initial denaturation at 95°C for 15 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 1 minute. The expression levels of superoxide dismutase ( SOD ), catalase ( CAT ), glutathione peroxidase ( GPX ), tumor necrosis factor-alpha ( TNF-α ), runt-related transcription factor 2 ( RUNX2 ), peroxisome proliferator-activated receptor gamma ( PPARγ ), and receptor-interacting serine/threonine-protein kinase 3 ( RIPK3 ) were normalized to the expression of the housekeeping gene, and relative gene expression was calculated using the 2 −ΔΔCt method. Statistical Analyses Quantitative data, representing gene expression levels and MTT assay results, are presented as the mean ± standard deviation (SD). All experiments were performed in triplicate. Statistical analyses were conducted using SPSS version 11.5 (IBM Corp., Armonk, NY, USA). The normality of data distribution for each experimental group was assessed using the Shapiro-Wilk test. For comparisons between two groups, the unpaired Student’s t-test was used when data were normally distributed. If data violated the assumption of normality, the non-parametric Mann-Whitney U test was used. For comparisons involving more than two groups, a one-way analysis of variance (ANOVA) was performed. Where ANOVA indicated a statistically significant difference, Tukey’s post-hoc test was used to determine specific pairwise comparisons between groups. A two-tailed p-value of less than 0.05 (p < 0.05) was considered to indicate statistical significance. RESULTS DP-MSCs Morphology In this study, mesenchymal stem cells (MSCs) were isolated from the dental pulp of six deciduous teeth extracted from four children aged 4-6 years. In the initial days of culture, the cells exhibited heterogeneous morphologies, including spindle-shaped, flattened, star-shaped, and round. However, the majority of cells adhering to the flask surface were spindle-shaped. Non-adherent cells were removed by replacing the culture medium. Over time, the spindle-shaped cells proliferated, and cell colonies emerged and gradually coalesced until they covered the entire surface area of the culture flask. This was designated as passage 0. Following passaging, the rate of cell proliferation increased, and the flask surface reached confluence with spindle-shaped or fibroblast-like cells within approximately 3-4 days (Fig. 1). Figure 1 . ( Left ) Early-stage culture showing initial cell attachment and colony formation. Note the sparse distribution of cells with varied morphology, including spindle-shaped cells and a small, dense cluster. ( Middle ) Increased cell proliferation and organization at the end of passage 0. Cells exhibit a predominantly spindle-shaped morphology and form a more interconnected network, indicating the early stages of confluence. ( Right ) Passage three culture demonstrating a high degree of confluence with cells aligned in a fibroblast-like arrangement. The cells are elongated and densely packed, covering the majority of the culture area. DP-MSC Characterization To confirm the mesenchymal identity of the proliferated cells at passage 3 (P3), their differentiation potential and mesenchymal marker expression were assessed. Cells cultured in specific induction media successfully differentiated into osteocytes, as visualized by Alizarin Red staining, and adipocytes, identified by Oil Red O staining. Flow cytometry analysis revealed high expression of mesenchymal markers CD73 and CD90, with 90% and 98% of cells positive, respectively. In contrast, expression of hematopoietic markers CD34 and CD45 was low, with only 7% and 8% of cells positive, respectively (Fig. 2). Figure 2 . ( Left ) Evidence of osteogenic differentiation in MSCs cultured in osteogenic induction medium for 20 days, visualized by Alizarin Red staining. The image shows the presence of numerous Alizarin Red-positive deposits, indicative of calcium accumulation and the formation of a mineralized extracellular matrix. ( Right ) Evidence of adipogenic differentiation in MSCs cultured in adipogenic induction medium for 20 days, visualized by Oil Red O staining. The image reveals the intracellular accumulation of Oil Red O-positive lipid droplets, indicating the formation of mature adipocytes. Flowcytometry of the proliferated cells at passage three Flow cytometry analysis of passage 3 cells revealed the expression of specific cell surface markers. The cells showed low expression of hematopoietic markers CD34 and CD45, with only 1.56% and 1.06% of cells testing positive, respectively (Fig. 3, top left and top right). In contrast, the cells exhibited high expression of mesenchymal markers CD90 and CD73, with 98.9% and 96.3% of cells testing positive, respectively (Fig. 3, bottom left and bottom right). Figure 3 . Flow cytometry analysis of DP-MSCs at passage 3. Histograms represent cell counts versus fluorescence intensity for CD34-PE, CD45-FITC, CD90-FITC, and CD73-PerCP. Numbers indicate the percentage of cells positive for each marker, confirming the mesenchymal phenotype of the cells. SEM of the extracted exosomes Scanning electron microscopy (SEM) revealed the morphology of the isolated exosomes. The exosomes exhibited a predominantly spherical or cup-like shape, with sizes ranging from 150 to 300 nm (Fig. 4). The particles were distributed across the substrate, demonstrating a relatively smooth surface. Figure 4 . Representative SEM micrograph of exosomes derived from dental pulp mesenchymal stem cells, demonstrating their spherical or cup-like morphology and size distribution (150-300 nm). Protein Concentration (Bradford Assay) The protein concentration of the exosome sample was determined using the Bradford assay. A standard curve was generated using known concentrations of bovine serum albumin (BSA), and the absorbance values were plotted against protein concentration (Fig. 5). The standard curve exhibited a logarithmic relationship, best described by the equation y = 0.123ln(x) + 0.5059, with a strong correlation coefficient (R² = 0.9592). Based on this curve, the protein concentration of the exosome solution was calculated to be 850 µg/mL. Figure 5. Bradford assay standard curve used to determine the protein concentration of exosome samples. The curve was generated using known concentrations of bovine serum albumin, and the resulting logarithmic equation (y = 0.123ln(x) + 0.5059) and correlation coefficient (R² = 0.9592) are shown. MTT Assay The MTT assay was performed to evaluate the effects of folic acid, exosomes, and H2O2 on hGF cell viability. Treatment with exosomes at concentrations of 2 µg/ml, 10 µg/ml, and 20 µg/ml did not significantly alter cell viability compared to the control group (Fig. 6, middle ). In contrast, treatment with 100 µM folic acid resulted in a statistically significant increase in cell viability (p = 0.0343), while 50 µM and 10 µM folic acid had no significant effect (Fig. 6, left ). H2O2 treatment induced a concentration-dependent decrease in cell viability. Concentrations of 200 µM, 300 µM, and 400 µM H2O2 significantly reduced cell viability (p = 0.0006, p = 0.0001, and p < 0.0001, respectively). Lower concentrations of H2O2 (50 µM and 100 µM) also significantly decreased cell viability (p = 0.0005 and p = 0.0206, respectively), while 5 µM H2O2 showed a slight increase in cell viability (Fig. 6, right ). Based on these results, 50 µM H2O2 was selected for the induction of inflammation, as it induced a significant reduction in cell viability while allowing for a substantial population of cells to remain viable. Furthermore, 50 µM folic acid and 20 µg/ml exosome suspension were chosen for subsequent experiments to assess their potential to counteract the effects of inflammation, as these concentrations did not significantly decrease cell viability. Figure 6: MTT assay results showing the effects of exosomes, folic acid, and H2O2 on hGF cell viability. (A) Cell viability (%) after treatment with different concentrations of folic acid. The asterisk (*) indicates a statistically significant difference compared to the control group (p = 0.0343). (B) Cell viability (%) after treatment with different concentrations of exosomes. (C) Cell viability (%) after treatment with different concentrations of H2O2. Asterisks (, **, ***, ****) indicate statistically significant differences compared to the control group (p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively). Error bars represent standard deviation . Morphology of the hGF cells after treatment Exposure of hGF cells to H2O2 induced a morphological change from a spindle-shaped to a more rounded or retracted morphology (Fig. 7). In this condition, cells also exhibited reduced spreading. Upon removal of H2O2, cells began to recover their spindle-shaped morphology. However, the extent of recovery varied among treatment groups. Cells treated with H2O2 and folic acid showed a slight improvement in morphology compared to cells treated with H22 alone. A more pronounced recovery was observed in cells treated with H2O2 and exosomes. Notably, cells treated concurrently with H2O2, folic acid, and exosomes displayed the most significant restoration of normal spindle-shaped morphology and cell spreading, closely resembling the untreated control cells (Fig. 7). Figure 7. Phase-contrast microscopy images of hGF cells demonstrating the effect of H2O2 on cell morphology and the influence of folic acid and exosome treatment. The panels show (from left to right): untreated control cells, cells treated with H2O2, cells treated with H2O2 and folic acid, cells treated with H2O2 and exosomes, and cells treated with H2O2, folic acid, and exosomes. Scale bar = 5.0 mm. Gene expression analyses The expression of genes related to osteogenesis (RUNX2), apoptosis (Bax/Bcl2, RIPK3), antioxidant activity (CATA, GPx, SOD), and inflammation (IL-6, TGFB, TNF-α) was measured in hGF cells treated with H2O2, followed by exosome, folic acid, or combined exosome and folic acid treatment (Fig. 8). RUNX2 expression was not significantly altered by any treatment (Fig. 8, top left). H2O2 treatment significantly increased the expression of RIPK3, SOD, CATA, GPx, IL-6, and TGFB, as well as the Bax/Bcl2 ratio (p < 0.01 to p < 0.0001) (Fig. 8, top middle, top right, middle left, middle right, bottom left, and bottom middle). H2O2 treatment also significantly increased TNF-α expression (p < 0.05) (Fig. 8, bottom right). Exosome treatment alone, folic acid treatment alone, and the combined treatment significantly reduced the H2O2-induced increase in RIPK3, SOD, CATA, GPx, and TGFB expression, and the Bax/Bcl2 ratio (p < 0.0001 for all comparisons) (Fig. 8, top middle, top right, middle left, middle right, and bottom middle). No significant differences were observed between exosome, folic acid, and combined treatments for these genes. IL-6 expression was significantly reduced by exosome treatment (p < 0.01), folic acid treatment (p < 0.001), and the combined treatment (p < 0.0001) compared to the H2O2-treated group (Fig. 8, bottom left). Again, no significant differences were found between exosome, folic acid, and combined treatments. TNF-α expression was significantly reduced only by folic acid treatment (p < 0.05) compared to the H2O2-treated group (Fig. 8, bottom right). Exosome treatment alone and the combined treatment did not significantly reduce TNF-α expression. These results indicate that H2O2 induced significant changes in the expression of genes associated with osteogenesis, apoptosis, antioxidant activity, and inflammation, while exosome and folic acid treatments effectively mitigated many of these changes. However, none of the treatments affected the expression of RUNX2, an osteogenic marker. Furthermore, folic acid treatment was uniquely effective in reducing H2O2-induced TNF-α expression. Figure 8 : Relative gene expression in hGF cells following treatment with H2O2 and subsequent treatment with exosome, folic acid, or a combination of both. Graphs represent the fold change in gene expression relative to the control group for the following genes: RUNX2, RIPK3, SOD, CATA, Bax/Bcl2, GPx, IL-6, TGFB, and TNF-α. Error bars indicate standard deviation. Asterisks indicate statistical significance: **p < 0.01, ***p < 0.001, ****p < 0.0001; ns = not significant . DISCUSSION Although MSC treatments have shown promise, they are hindered by operational and logistical difficulties in handling and storing the cells in a way that preserves their viability and vitality for transplantation (20). These difficulties include the time-consuming and expensive nature of cell handling, as well as the requirement for specialized facilities. Interestingly, exosomes (25), which are lipid bi-layered membrane-bound extracellular vesicles of nanometer size, are released by stem cells as part of their paracrine actions. Furthermore, stem cells use exosomes to communicate with their peers who are not stem cells and/or with each other, just like other cells in the body (26). Because exosomes produce distinct sets of soluble secretomes, they may be thought of as miniature versions of their donor cells. Consequently, exosomes derived from stem cells inherit their parent cell’s therapeutic anti-inflammatory and tissue-regeneration properties (27). Initially used as biomarkers for inflammatory disease diagnosis, exosomes are now being explored as therapeutic cargo delivery vehicles, leveraging their biological properties and therapeutic potential. Further optimization through engineering design may enhance their efficacy in treating chronic inflammatory diseases (24). Compared to stem cells, exosomes offer advantages such as the absence of ethical concerns, reduced immunogenicity and tumorigenicity, versatile delivery methods (28), low toxicity and immunogenicity (29), good biocompatibility, and the ability to traverse biological barriers (14). Previous studies have demonstrated the therapeutic potential of MSC exosomes in various contexts. One study examining the effect of MSC exosomes on chondrocyte biological activity revealed a significant increase in chondrocyte numbers via a synergistic combination of enhanced recruitment, decreased apoptosis, and increased proliferation while promoting matrix synthesis. Exosome-mediated increases in the expression of genes linked to anti-apoptosis (Survivin and Bcl-2) and proliferation (PCNA and FGF-2) occur concurrently with this synergistic combination (30). It appears that MSC exosomes must have elicited this response within minutes of contacting the cells, and that receptor-mediated signal transduction via phosphorylation of survival kinases like AKT and ERK represented the most likely path for this rapid elicitation of cellular proliferation. The observation that exosome-induced cellular proliferation was detected as early as 24 hours is noteworthy, given that the average mammalian cell cycle is also approximately 24 hours (31). This suggests that exosomes can rapidly influence cell cycle progression. Indeed, MSC exosomes have been shown to induce rapid phosphorylation of AKT and ERK in chondrocytes, occurring in less than an hour. This early phosphorylation is crucial for triggering downstream cellular responses, including migration and proliferation, as demonstrated by the marked decrease in these processes upon inhibition of AKT and ERK phosphorylation with wortmannin and U0126, respectively. Interestingly, this inhibition did not affect s-GAG synthesis. The dependence of exosome-mediated tissue repair and regeneration on AKT and/or ERK pro-survival signaling has been demonstrated in various contexts, including wound healing (32), bone repair (33), and myocardial regeneration following ischemic injury (34). Prior proteomic analysis of MSC exosomes revealed that the cargo is extremely complex and includes multiple components, including growth differentiation factor (GDF)-5, platelet-derived growth factor (PDGF), and transforming growth factor (TGF)-b, which may also activate AKT and/or ERK signaling (35-39). Major pathways (such as the PTEN/PI3K/Akt/mTOR, NF-κB, TGF-β, HIF-1α, Wnt, MAPK, JAK-STAT, Hippo, and Notch signaling cascades) and minor pathways are examples of the diverse signaling cascades that are activated and regulated by exosomes. These signaling pathways do, in fact, have a great deal of crosstalk (40). In THP-1 monocytes, MSC exosomes may increase the expression of anti-inflammatory IL-10 and TGF-b1 while decreasing the expression of proinflammatory IL-1b, IL-6, TNF-a, and IL-12P40 (30). Our study also observed modulation of TGF-β expression, suggesting that the exosome-mediated effects we observed may involve these signaling pathways. Given the role of exosomes in modulating inflammation and tissue repair, their potential for periodontal regeneration is of great interest. Exosomes can also be released by periodontal ligament (PDL) cells. By stopping NF-κB from translocating into the nucleus, these exosomes can inhibit the expression of NLRP3 and pro-IL-1β, which in turn suppresses NRLP3 activation and IL-1β secretion from LPS-primed and nigericin-treated macrophages (41). TLR4, NF-κB, NLRP3, caspase-1, and IL-1β levels in the spinal cords of mice with encephalomyelitis can be reduced by a combination of exosomes and microvesicles released from human periodontal ligament stem cells isolated from relapsing-remitting multiple sclerosis patients, according to another study (42). One intriguing study demonstrated that, in an adult immunocompetent rat model (6), collagen sponge loaded with human MSC exosomes improved periodontal regeneration without causing any negative side effects (10, 43, 44). In animal experiments, periodontal ligament (PDL) stem cell and bone marrow MSC transplantation, or their conditioned medium in collagen sponges, has been shown to stimulate periodontal regeneration (9, 45). It was clear that MSC exosomes replicated the therapeutic efficacy of MSCs and conditioned medium in promoting periodontal regeneration in a rat model, as the therapeutic effect of the MSC conditioned medium had been increasingly attributed to its exosome content (6). Even, a single implantation of exosome-loaded collagen sponge in a surgically created periodontal defect model promoted periodontal regeneration with enhanced bone growth and increased functional PDL length at week 4. This observation suggests that despite the rapid release and decay of exosomes from the collagen sponges within the first 48h, the effect of exosome on periodontal regeneration appeared to persist and propagate for at least 4 weeks as evident by the significant improvements in bone regeneration and functionally oriented PDL formation in the exosome-treated animals over a period of 4 weeks (6). These findings in animal models support our in vitro observations that exosomes can promote periodontal regeneration. Through a synergistic combination of improved cell viability, migration, proliferation, matrix synthesis, and differentiation to form new bone and PDL attachment, exosomes promoted periodontal regeneration (6). Consequently, the collagen sponge’s comparatively brief 48-hour exposure to MSC exosomes may be enough to encourage periodontal regeneration. Our findings that MSC exosomes were quickly absorbed by PDL cells in vitro in a matter of minutes, that they triggered signal transduction in 15 minutes, and that they altered gene expression and cellular functions like migration, viability, and proliferation in 48 hours further corroborate this. The effects of exosomes on these cellular activities may extend beyond 48 hours, specifically through the exosome-mediated upregulation of genes linked to cell migration (IGF-1, FGF-2), survival and anti-apoptosis (IGF-1, Survivin, and Bcl-2), and proliferation (IGF-1, FGF-2, and PCNA) (46-49). Exosome-mediated upregulation of PDL matrix-associated genes, including extracellular matrix proteins POSTN and COL1A1, as well as related growth factors for matrix synthesis, TGF-b1 and IGF-1, was also noted (47, 50). Exosomes have been used in more than 300 preclinical investigations. While the therapeutic potential of exosomes extends to various diseases, including Crohn’s disease (52), these applications fall into three general categories: drug delivery methods, therapeutic applications, and diagnostic analyses (51). For example, a phase 2 study assessed the safety and effectiveness of exosomes derived from human placental BMSCs in treating anal fistula in Crohn’s disease patients (52). However, stem cell-derived exosome therapy still has certain drawbacks and difficulties, which can be broadly divided into three areas: low yield, difficult extraction, and long-term effects (40). In this sense, various tactics are used to get past these. Isolating exosomes from vast amounts of easily accessible biological materials, for example, is the most likely method for increasing production (24). To sum up, investigating and comprehending the context-specific molecular mechanism of SC-Exo therapy aids in directing and advancing additional preclinical research and clinical trials. In conclusion, findings of the present study show that exosomes derived from dental pulp stem cells have improving impact on periodontal injury. Compared to conventional nanomaterial delivery methods, the exosome-based ”without cells” delivery strategy offers several benefits, such as being ready-to-use and more amenable to reformulation to support various routes of administration. Additionally, superior exosome targeting promotes cargo accumulation at disease sites and lessens harm to healthy cells and the body. Our study demonstrates that exosomes derived from dental pulp stem cells can modulate the expression of key genes involved in inflammation and apoptosis in an in vitro model of periodontal injury. These findings suggest that exosome-based therapies hold promise for periodontal regeneration, warranting further investigation into their clinical potential. REFERENCES 1. Kassebaum NJ, Smith AG, Bernabé E, Fleming TD, Reynolds AE, Vos T, et al. Global, regional, and national prevalence, incidence, and disability-adjusted life years for oral conditions for 195 countries, 1990–2015: a systematic analysis for the global burden of diseases, injuries, and risk factors. 2017;96(4):380-7.2. Kassebaum N, Bernabé E, Dahiya M, Bhandari B, Murray C, Marcenes WJJodr. Global burden of severe periodontitis in 1990-2010: a systematic review and meta-regression. 2014;93(11):1045-53.3. Heitz‐Mayfield LJ, Lang NPJP. Surgical and nonsurgical periodontal therapy. Learned and unlearned concepts. 2013;62(1):218-31.4. Hoang DM, Pham PT, Bach TQ, Ngo AT, Nguyen QT, Phan TT, et al. Stem cell-based therapy for human diseases. 2022;7(1):272.5. Zakrzewski W, Dobrzyński M, Szymonowicz M, Rybak ZJScr, therapy. Stem cells: past, present, and future. 2019;10:1-22.6. Chew JRJ, Chuah SJ, Teo KYW, Zhang S, Lai RC, Fu JH, et al. Mesenchymal stem cell exosomes enhance periodontal ligament cell functions and promote periodontal regeneration. 2019;89:252-64.7. da Silva Meirelles L, Fontes AM, Covas DT, Caplan AIJC, reviews gf. Mechanisms involved in the therapeutic properties of mesenchymal stem cells. 2009;20(5-6):419-27.8. Nagata M, Iwasaki K, Akazawa K, Komaki M, Yokoyama N, Izumi Y, et al. Conditioned medium from periodontal ligament stem cells enhances periodontal regeneration. 2017;23(9-10):367-77.9. Kawai T, Katagiri W, Osugi M, Sugimura Y, Hibi H, Ueda MJC. Secretomes from bone marrow–derived mesenchymal stromal cells enhance periodontal tissue regeneration. 2015;17(4):369-81.10. Lai RC, Arslan F, Lee MM, Sze NSK, Choo A, Chen TS, et al. Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury. 2010;4(3):214-22.11. Huňáková K, Hluchý M, Kuricová M, Ševčík K, Rosocha J, Ledecký VJFV. Role of mesenchymal stem cells—derived exosomes in osteoarthritis treatment. 2018;62(4):19-23.12. Hu G-w, Li Q, Niu X, Hu B, Liu J, Zhou S-m, et al. Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells attenuate limb ischemia by promoting angiogenesis in mice. 2015;6:1-15.13. Lee C, Mitsialis SA, Aslam M, Vitali SH, Vergadi E, Konstantinou G, et al. Exosomes mediate the cytoprotective action of mesenchymal stromal cells on hypoxia-induced pulmonary hypertension. 2012;126(22):2601-11.14. Sil S, Dagur RS, Liao K, Peeples ES, Hu G, Periyasamy P, et al. Strategies for the use of extracellular vesicles for the delivery of therapeutics. 2020;15:422-42.15. Noonin C, Thongboonkerd VJT. Exosome-inflammasome crosstalk and their roles in inflammatory responses. 2021;11(9):4436.16. Zhang H, Wang L, Li C, Yu Y, Yi Y, Wang J, et al. Exosome-induced regulation in inflammatory bowel disease. 2019;10:1464.17. Chang T-H, Wu C-S, Chiou S-H, Chang C-H, Liao H-JJB. Adipose-derived stem cell exosomes as a novel anti-inflammatory agent and the current therapeutic targets for rheumatoid arthritis. 2022;10(7):1725.18. Yang L, Wang T, Zhang X, Zhang H, Yan N, Zhang G, et al. Exosomes derived from human placental mesenchymal stem cells ameliorate myocardial infarction via anti-inflammation and restoring gut dysbiosis. 2022;22(1):61.19. Lai RC, Yeo RWY, Lim SK, editors. Mesenchymal stem cell exosomes. Seminars in cell & developmental biology; 2015: Elsevier.20. Toh WS, Lai RC, Hui JHP, Lim SK, editors. MSC exosome as a cell-free MSC therapy for cartilage regeneration: implications for osteoarthritis treatment. Seminars in cell & developmental biology; 2017: Elsevier.21. Liu W, Rong Y, Wang J, Zhou Z, Ge X, Ji C, et al. Exosome-shuttled miR-216a-5p from hypoxic preconditioned mesenchymal stem cells repair traumatic spinal cord injury by shifting microglial M1/M2 polarization. 2020;17:1-22.22. Nakazaki M, Morita T, Lankford KL, Askenase PW, Kocsis JDJJoev. Small extracellular vesicles released by infused mesenchymal stromal cells target M2 macrophages and promote TGF‐β upregulation, microvascular stabilization and functional recovery in a rodent model of severe spinal cord injury. 2021;10(11):e12137.23. Huang J-H, Fu C-H, Xu Y, Yin X-M, Cao Y, Lin F-YJNr. Extracellular vesicles derived from epidural fat-mesenchymal stem cells attenuate NLRP3 inflammasome activation and improve functional recovery after spinal cord injury. 2020;45:760-71.24. Wang C, Xu M, Fan Q, Li C, Zhou XJAjops. Therapeutic potential of exosome‐based personalized delivery platform in chronic inflammatory diseases. 2023;18(1):100772.25. Vizoso FJ, Eiro N, Cid S, Schneider J, Perez-Fernandez RJIjoms. Mesenchymal stem cell secretome: toward cell-free therapeutic strategies in regenerative medicine. 2017;18(9):1852.26. Zhang Y, Bi J, Huang J, Tang Y, Du S, Li PJIjon. Exosome: a review of its classification, isolation techniques, storage, diagnostic and targeted therapy applications. 2020:6917-34.27. Ren KJO. Exosomes in perspective: a potential surrogate for stem cell therapy. 2019;107(3):271-84.28. Hastuti S, Idroes R, Imran I, Ramli Y, Abas AH, Tallei TEJP. hUMSC vs. hUMSC–Exosome: Which One Is Better for Epilepsy? 2022;15(10):1247.29. Hwang HS, Kim H, Han G, Lee JW, Kim K, Kwon IC, et al. Extracellular vesicles as potential therapeutics for inflammatory diseases. 2021;22(11):5487.30. Zhang S, Chuah SJ, Lai RC, Hui JHP, Lim SK, Toh WSJB. MSC exosomes mediate cartilage repair by enhancing proliferation, attenuating apoptosis and modulating immune reactivity. 2018;156:16-27.31. Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell JJNCfBI, Bookshelf. Molecular cell biology 4th edition. 2000;9.32. Zhang B, Wang M, Gong A, Zhang X, Wu X, Zhu Y, et al. HucMSC-exosome mediated-Wnt4 signaling is required for cutaneous wound healing. 2015;33(7):2158-68.33. Zhang J, Liu X, Li H, Chen C, Hu B, Niu X, et al. Exosomes/tricalcium phosphate combination scaffolds can enhance bone regeneration by activating the PI3K/Akt signaling pathway. 2016;7:1-14.34. Arslan F, Lai RC, Smeets MB, Akeroyd L, Choo A, Aguor EN, et al. Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury. 2013;10(3):301-12.35. Lai RC, Tan SS, Teh BJ, Sze SK, Arslan F, De Kleijn DP, et al. Proteolytic potential of the MSC exosome proteome: Implications for an exosome‐mediated delivery of therapeutic proteasome. 2012;2012(1):971907.36. Qureshi HY, Ahmad R, Sylvester J, Zafarullah MJCs. Requirement of phosphatidylinositol 3-kinase/Akt signaling pathway for regulation of tissue inhibitor of metalloproteinases-3 gene expression by TGF-β in human chondrocytes. 2007;19(8):1643-51.37. Qureshi HY, Sylvester J, Mabrouk ME, Zafarullah MJJocp. TGF‐β‐induced expression of tissue inhibitor of metalloproteinases‐3 gene in chondrocytes is mediated by extracellular signal‐regulated kinase pathway and Sp1 transcription factor. 2005;203(2):345-52.38. Gharibi B, Ghuman MS, Hughes FJJJoc, medicine m. A kt‐and E rk‐mediated regulation of proliferation and differentiation during PDGFR β‐induced MSC self‐renewal. 2012;16(11):2789-801.39. Nakamura K, Shirai T, Morishita S, Uchida S, Saeki-Miura K, Makishima FJEcr. p38 mitogen-activated protein kinase functionally contributes to chondrogenesis induced by growth/differentiation factor-5 in ATDC5 cells. 1999;250(2):351-63.40. Li D, Li D, Wang Z, Li J, Shahzad KA, Wang Y, et al. Signaling pathways activated and regulated by stem cell-derived exosome therapy. 2024;14(1):105.41. Wang Z, Maruyama K, Sakisaka Y, Suzuki S, Tada H, Suto M, et al. Cyclic stretch force induces periodontal ligament cells to secrete exosomes that suppress IL-1β production through the inhibition of the NF-κB signaling pathway in macrophages. 2019;10:1310.42. Soundara Rajan T, Giacoppo S, Diomede F, Bramanti P, Trubiani O, Mazzon EJIjoi, et al. Human periodontal ligament stem cells secretome from multiple sclerosis patients suppresses NALP3 inflammasome activation in experimental autoimmune encephalomyelitis. 2017;30(3):238-52.43. Tan CY, Lai RC, Wong W, Dan YY, Lim S-K, Ho HKJScr, et al. Mesenchymal stem cell-derived exosomes promote hepatic regeneration in drug-induced liver injury models. 2014;5:1-14.44. Zhang S, Chu W, Lai R, Lim S, Hui J, Toh WJO, et al. Exosomes derived from human embryonic mesenchymal stem cells promote osteochondral regeneration. 2016;24(12):2135-40.45. Sakaguchi K, Katagiri W, Osugi M, Kawai T, Sugimura-Wakayama Y, Hibi HJB, et al. Periodontal tissue regeneration using the cytokine cocktail mimicking secretomes in the conditioned media from human mesenchymal stem cells. 2017;484(1):100-6.46. Han X, Amar SJJoDR. IGF-1 signaling enhances cell survival in periodontal ligament fibroblasts vs. gingival fibroblasts. 2003;82(6):454-9.47. Yu Y, Mu J, Fan Z, Lei G, Yan M, Wang S, et al. Insulin-like growth factor 1 enhances the proliferation and osteogenic differentiation of human periodontal ligament stem cells via ERK and JNK MAPK pathways. 2012;137:513-25.48. Sant’Ana AC, Marques MM, Barroso EC, Passanezi E, de Rezende MLRJJop. Effects of TGF‐β1, PDGF‐BB, and IGF‐1 on the rate of proliferation and adhesion of a periodontal ligament cell lineage in vitro. 2007;78(10):2007-17.49. Shimabukuro Y, Terashima H, Takedachi M, Maeda K, Nakamura T, Sawada K, et al. Fibroblast growth factor‐2 stimulates directed migration of periodontal ligament cells via PI3K/AKT signaling and CD44/hyaluronan interaction. 2011;226(3):809-21.50. Fujii S, Maeda H, Tomokiyo A, Monnouchi S, Hori K, Wada N, et al. Effects of TGF-β1 on the proliferation and differentiation of human periodontal ligament cells and a human periodontal ligament stem/progenitor cell line. 2010;342:233-42.51. Rezaie J, Feghhi M, Etemadi TJCC, Signaling. A review on exosomes application in clinical trials: perspective, questions, and challenges. 2022;20(1):145.52. Califf RM, Zarin DA, Kramer JM, Sherman RE, Aberle LH, Tasneem AJJ. Characteristics of clinical trials registered in ClinicalTrials. gov, 2007-2010. 2012;307(17):1838-47. Information & Authors Information Version history V1 Version 1 08 May 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Natural Sciences Authors Affiliations Saghar Zarei Islamic Azad University Shiraz View all articles by this author Mostafa Montazeri 0000-0001-6364-2658 [email protected] University of Michigan School of Dentistry View all articles by this author Nader Tanideh Shiraz University of Medical Sciences View all articles by this author Shahrokh Zare Shiraz University of Medical Sciences View all articles by this author Mahintaj Dara Shiraz University of Medical Sciences View all articles by this author Metrics & Citations Metrics Article Usage 300 views 124 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Saghar Zarei, Mostafa Montazeri, Nader Tanideh, et al. Synergistic Effects of Folic Acid and Dental Pulp Stem Cell-Derived Exosomes on Gene Expression in a Periodontal Injury Model. Authorea . 08 May 2025. 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