Dental pulp-derived mesenchymal stem cells reduce lesion progression in a rat model of endometriosis

Endometriosis is a chronic, estrogen -dependent inflammatory disease sustained by aberrant angiogenesis and progressive fibrosis. We evaluated the therapeutic efficacy of human dental pulp – derived mesenchymal stem cells (DP-MSCs) in a surgically induced rat endometriosis model. Twenty- eight adult female Wistar rats were randomized (n = 7/group) to sham group (G1), untreated endometriosis-group (G2), single-dose DP-MSCs-group (G3) (2×106 cells intraperitoneally on day 28) and double-dose DP-MSCs-group (G4) (2×106 cells on days 28 and 35). Endometriosis was induced by autologous uterine tissue implantation onto the peritoneal wall and allowed to establish for 28 days; treatment effects were assessed 7 days after the final DP-MSCs dose. Serum and peritoneal TNF-α, IL- 6, VEGF, and CA-125 were quantified; lesions were evaluated by semi-quantitative histopathology and fibrosis grading and by immunohistochemistry for CA-125, VEGF, type I collagen (Col1), and TNF-α. Untreated endometriosis showed increased systemic TNF -α (p = 0.0207) and IL -6 (p = 0.0003) and marked peritoneal elevations versus sham (all p < 0.0001). DP -MSCs treatment significantly reduced peritoneal TNF-α and IL-6 in both regimens (each p < 0.0001 vs untreated) and decreased peritoneal VEGF, with greater suppression after double dosing (p = 0.0100 between regimens). Double dosing produced stronger systemic TNF-α suppression (p = 0.0027 vs untreated). Histopathology and fibrosis improved, most prominently with double dosing (both p < 0.0001), accompanied by reduced CA -125, VEGF, Col1 and TNF-α immunoreactivity (CA-125 and TNF-α, p < 0.0001). DP-MSCs effectively resolve the hallmark pathological features of endometriosis in a dose -dependent manner. By synergistically targeting inflammatory, angiogenic, and fibrotic pathways, this cell -based strategy offers a potent, disease-modifying approach for clinical management.

Biomarkers; Cytokines; Dental Pulp; Endometriosis; Histopathology; Mesenchymal Stem Cells. ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS

Endometriosis is a chronic, estrogen-dependent inflammatory disorder defined by ectopic endometrial- like glands and stroma, most commonly on the pelvic peritoneum, and it remains a major cause of pelvic pain and infertility. Disease persistence is linked to a permissive peritoneal milieu with immune dysregulation, sustained inflammation, angiogenic activation, and progressive e xtracellular matrix remodeling that supports lesion survival, invasion, and adhesion formation [1]. High symptom burden, recurrence, and the limitations of current medical and surgical options highlight the need for fertility - sparing, mechanism-directed approaches that target inflammatory, angiog enic, and fibrotic pathways [2]. Mesenchymal stem cells (MSCs) are increasingly studied as mechanism -oriented therapeutics, with effects largely attributed to paracrine signaling, immunomodulation, and regulation of tissue repair. Several international consensus statements have proposed minimal criteria and surface -marker panels to standardize MSC definition and improve comparability across studies [3]. Among available sources, dental tissue–derived MSCs are attractive due to practical procurement and relevance to inflammatory and regenerative indications. Dental pulp–derived MSCs (DP-MSCs) show strong expansion capacity, relatively low immunogenicity, and translational feasibility, including tissue -engineering applications [4]. Endometriosis progression depends on angiogenesis and stromal remodeling, domains that DP-MSC may influence through microenvironmental modulation. In addition, emerging work indicates that DP- MSC function and heterogeneity are shaped by epigenetic programs, including DNA methylation, which can affect lineage potential and immunobiology and may influence therapeutic consistency and potency. Despite growing knowledge of dental stem cell biology, direct preclinical testing of DP-MSCs in endometriosis remains limited, supporting the need for well-controlled in vivo studies that address dose regimen and timing [5]. In this context, the present study evaluates the therapeutic potential of human DP-MSCs administration in a surgically induced rat model of endometriosis. Key disease axes—local and systemic inflammation, angiogenesis, fibrotic remodeling, and lesion activity —were evaluated using complementary biochemical (TNF -α, IL -6, VEGF, CA -125), histochemical (H&E and Masson’s trichrome), and immunohistochemical (CA-125, Col1, VEGF, TNF-α) readouts. Single and double-dose intraperitoneal DP-MSCs regimens were compared to assess regimen dependence and to align delivery with peritoneal targeting approaches used to modulate abdominal inflammatory milieus in experimental m odels [6], thereby strengthening the translational rationale for peritoneal adm inistration in endometriosis and supporting the development of fertility-sparing treatment strategies. ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS

Ethical Approval and Experimental Design The human DP-MSCs used in this study were isolated from third molars extracted for clinical reasons at the Ege University Faculty of Dentistry. All experimental protocols and the collection of human samples were approved by the Ege University Medical Research Ethics Committee under the approval number 23-1.1T/37 (Initial approval) and updated/confirmed with the decision 23-9.1T/51. All methods were performed in accordance with the relevant guidelines and regulations (Declaration of Helsinki). Informed consent was obtained from all participants and/or their legal guardians prior to the col lection of dental tissues. All experimental procedures were conducted at the Ege University Experimental Animals Application and Research Center following approval from the Ege University Animal Experiments Local Ethics Committee (decision date: March 27, 2024; Approval number: 2023-072). All procedures complied with institutional and national guidelines for laboratory animal care and the ARRIVE guidelines. Twenty- eight female Wistar albino rats aged 10 - 12 weeks and weighing 180 to 260 g were included. Female rats were housed in single cages in a room with a temperature of 24 ± 1 °C and a 12-h light–dark cycle (lights on at 07.00 a.m.) and were fed a standard laboratory diet and water and food were provided ad libitum. All efforts were made to minimize animal suffering and to reduce the number of animals used. At the end of the study, all rats were euthanized to ensure minimum pain and distress. Euthanasia was performed by an intraperitoneal overdose of a combination of 80 mg/kg ketamine (VetaKetam, Vet - Agro, Lublin, Poland) and 10 mg/kg xylazine (VetaXyl, Vet-Agro, Lublin, Poland). DP-MSC Isolation, Culture, and Characterization Human DP-MSCs were obtained from impacted third molars using commonly applied dental pulp stem cell isolati on procedures [7] . Teeth were transported in sterile Falcon tubes and processed under a biosafety cabinet. Dental pulp tissue was removed under aseptic conditions and processed separately for each donor. The pulp was digested with 3 mg/mL type I collagenase at 37 °C for 45 minutes. Enzymatic digestion was stopped by adding complete culture medium to a final volume of 10 mL (α -MEM supplemented with 10% fetal bovine serum, 1% gentamicin, 1% penicillin/streptomycin, and 0.8% amphotericin B). The suspension was centrifuged at 1 600 rpm for 5 minutes at 4 °C. The supernatant was discarded, the pellet was resuspended in fresh medium (final volume 5 mL), and cells were seeded into T-25 cm² flasks. Cultures were monitored on days 3 and 5 for attachment and early growth. After day 5, the medium was replaced every three days. At ≥80% confluency, cells were detached with trypsin- EDTA, counted using a hemocytometer, and expanded as required. Cells were cryopreserved in freezing medium containing 90% fetal bovine serum and 10% dimethyl sul foxide, following standard MSC ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS handling procedures [8]. After one month, cells were thawed for experiments and recounted, yielding a post-thaw concentration of approximately 9 ×105 cells/mL per vial. Representative images of cultured cells are shown in Figure 1. Flow cytometric immunophenotyping of DP-MSCs DP-MSCs were characterized by flow cytometry (FACS) at the STEMBIO A.Ş. laboratory (TÜBİTAK Marmara Technopark R&D and Innovation Center, Gebze, Kocaeli, Türkiye). Cells were expanded to passage 1 to lim it passage -related changes in MSC propert ies [9]. At ~80% confluency, cells were detached, washed, and resuspended in Dulbecco’s phosphate -buffered saline (DPBS) at ≥5 × 10^6 cells/mL. Immunostaining was performed using the BD Stemflow™ Human MSC Analysis Kit (BD Biosciences, San Jose, CA, USA). The panel included MSC -positive markers (CD73, CD90, CD105) and negative lineage markers (CD45, CD34, CD19, CD11b, HLA-DR), consistent with commonly used MSC phenotyping approaches for DP- MSCs [10]. After staining, cells were washed, resuspended in FACSflow solution, and analyzed on a Navios EX flow cytometer using Navios EX tetra software. DP- MSCs showed high expression of CD90 (99.81%), CD73 (99.7%), CD105 (98.3%), and CD44 (95.6%); CD44 is often reported as an MSC -associated marker [11]. Cells showed low expression of hematopoietic/immune markers (combined panel positivity 1.56%). Overall, 98.35% of analyzed cells matched the expected DP-MSCs immunophenotype (Figure 2). ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS Figure 1. Morphological and immunophenotypic characterization of DP -MSCs. Representative inverted phase-contrast micrographs illustrate the adherent, spindle-shaped, fibroblast-like morphology of DP-MSCs. The upper images display the cells during the early expansion phase (Initial isolation, P1- P2); the middle images represent the confluent culture prior to cryopreservation; and the lower images show the cells after thawing and subsequent re-culture. The consistent morphology observed across all stages indicates that the cryoprese rvation and thawing processes do not adversely affect the typical MSC-like appearance or the plastic-adherence capacity of the cells. ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS Figure 2. Immunophenotypic characterization of DP -MSCs. Representative flow cytometry dot plots showing the gating strategy based on forward (FSC) and side scatter (SSC) ( Gate A, 93.83% ). Analysis of MSC markers: DP-MSCs show high expression levels for CD90 (99.91%), CD44 (95.64%), CD105 (98.32%) , and CD73 (99.74%) . Exclusion of hematopoietic lineages: cells show negat ive expression for CD34 and CD45 (Gate G, 1.56%), confirming their non-hematopoietic origin. Data tables provide event counts, percentage of gated cells (%Gated), and mean fluorescence intensity (MFI) for each marker. Animal Model of Experimental Endometriosis and Study Design Twenty-eight adult female Wistar albino rats (10 –12 weeks old; 180–260 g) were randomly allocated to four experimental groups (n = 7/group). Experimental endometriosis was induced using a validated autologous uterine tissue implantati on method, as previously described [12]. Briefly, under aseptic conditions, a full -thickness uterine fragment containing endometrium (approximately 0.5 × 0.5 × 0.1 cm) was excised from the right uterine horn and sutured to the peritoneal wall with 5 -0 Vicryl. All surgical procedures were performed by the same operator to reduce inter-operator variability. A 28-day post-induction interval was defined as the lesion establishment (baseline) time point, consistent with the original description of this model and with the commonly used ~4 -week period required for stable, histologically confirmable ectopic implants [12]. To standardize treatment evaluation across regimens, animals were euthanized seven days after the final DP-MSCs administration in each treatment arm. This interval was selected to capture early MSCs-mediated immunomodulatory and paracrine effects within the peritoneal milieu, consistent with the concept that MSC s efficacy is primarily driven by secreted mediators rather than long -term engraftmen t [13]. Intraperitoneal delivery was chosen because it is widely used to target abdominal/peritoneal inflammation and has been shown to ameliorate experimental inflammatory disease by modulating immune cell activation in the peritoneal compartment [6,14]. Group Allocation and Interventions Group allocation and interventions were defined a priori based on established endometriosis induction methodology and prior intraperitoneal MSCs administration studies [6, 12,14]. Group 1 (Sham control; day 42 endpoint) underwent laparotomy on day 0, followed by peritoneal irrigation with sterile 0.9% NaCl and closure without tissue implantation; animals were euthanized on day 42 to provide a time - matched control for the longest study duration. Group 2 (Endometriosis base line; day 28 endpoint) underwent endometriosis induction on day 0 without further intervention and was euthanized on day 28 to verify lesion establishment and to define baseline model status [12]. Group 3 (Single-dose DP-MSC; day 35 endpoint) underwent endometriosis induction on day 0 and received a single intraperitoneal dose of 2×106 DP-MSCs on day 28; animals were euthanized on day 35 (i.e., 7 days after dosing). Group 4 ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS (Double-dose DP-MSCs; day 42 endpoint) underwent endometriosis induction on day 0 and received intraperitoneal DP-MSCs (2×106 cells) on days 28 and 35; animals were euthanized on day 42 (i.e., 7 days after the second dose) (Figure 3). Figure 3. Macroscopic evaluation and quantitative measurement of endometriotic lesions across experimental groups. Top row (G2, G3, G4) shows the representative in situ macroscopic appearance of the lesions during laparotomy. Bottom row (G2-1, G3 -1, G4 -1) displays the harvested lesions measured with a digital caliper to calculate the volumes. G2: Untreated endometriotic lesion group exhibiting well-vascularized, large cystic structures. G3: Single-dose DP-MSCs therapy group showing a moderate reduction in lesion size. G4: Double-dose DP-MSCs therapy group showing the most significant regression in lesion volume and vascularization. Caliper measurements correspond to the data distribution presented in the volumetric analysis. Biochemical Analyses Serum and peritoneal lavage fluid were collected at euthanasia. All protein concentrations (TNF-α, IL- 6, VEGF, and CA -125) were determined using commercially available ELISA kits according to the manufacturer’s instructions. To ensure methodological rigor, all samples were analyzed in duplicate. The sensitivity of the assays was 0.86 pg/mL for TNF -α, 7.5 pg/mL for IL -6, and 18.75 pg/mL for VEGF. The intra -assay and inter -assay coefficients of variation (CV) were confirmed to be less than 10% and 12%, respec tively, ensuring high reproducibility and minimal experimental variability. ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS Absorbance was read at 450 nm using a microplate reader (Thermo Scientific Multiskan Go). All assays were performed under the same conditions to allow valid comparisons between groups [15]. Histochemical and Immunohistochemical Analyses Excised endometriotic implants were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned at 5 µm [16]. For histochemical assessment, hematoxylin and eosin staining was performed to evaluate glandular architecture, stromal organization, and inflammatory cell infiltration, and a histopathological score was calculated [17]. Fibrosis was evaluated using Masson’s trichrome staining with aniline blue (BioOptica Milano S.p.A., Milan, Italy , 04 -010802), and a fibrosis score was calculated based on collagen deposition [18]. For immunohistochemistry, sections were incubated with primary antibodies against CA-125 (Raybiotech, 144-61671-100), collagen I (Col 1; Bioss Antibodies, bs-10423R), VEGF (Bioss Antibodies, bs-0279R) and TNF -α ( Elabscience, E-AB-40015) at 1:100 dilution; Col 1 immunostaining is commonly used as a fibrosis-associated marker in endometriosis tissue studies [19], and TNF -α/VEGF are widely used section -level inflammatory/an giogenic readouts [20] Immunoreactivity was visualized using DAB chromogen and counterstained with Mayer’s hematoxylin. Two histologists, blinded to group allocation, scored staining using a semi-quantitative scale (0, absent; 1+, weak; 2+, moderate; 3+, s trong) [21]. Two investigators independently scored all histological outcomes, and any discrepancies were resolved by joint re -evaluation to reach a consensus score for each section [21]. Statistical Analysis Data analyses were performed using IBM SPSS Statistics for Windows, Version 25.0 (IBM Corp., Armonk, NY, USA). The normality of the data distribution was assessed using the Shapiro -Wilk test. Descriptive statistics are presented as mean ± standard deviation (SD) for normally distributed variables and as median (interquartile range [IQR]) for non -normally distributed variables. For intergroup comparisons of normally distributed continuous variables, one-way analysis of variance (ANOVA) was employed, followed by the Bonferroni post -hoc test for multipl e comparisons to maintain the family - wise error rate. For variables that did not conform to a normal distribution (e.g., serum VEGF and lesion volume), the non -parametric Kruskal -Wallis test was used, with Dunn’s post -hoc test (including Bonferroni correction) applied for pairwise comparisons. Homogeneity of variances was verified using Levene’s test. A p-value of < 0.05 was considered statistically significant for all tests. ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS

A. Comparative evaluation of cytokines and the tumor marker in serum and peritoneal fluid Analysis of systemic (serum) and local (peritoneal fluid) samples demonstrated that the untreated endometriosis group exhibited elevated levels of proinflammatory cytokines (TNF -α, IL -6) and the angiogenic factor VEGF. DP-MSCs administration shifted these readouts toward a lower-activity state, with a generally stronger effect observed under the repeated -dose regimen across multiple endpoints. Serum TNF-α levels were significantly increased in the endometriosis group compared with controls (p = 0.0207). Single-dose DP-MSCs treatment did not yield a statistically significant reduction in serum TNF-α. In contrast, the two -dose regimen produced a significant decrease relative to untreated endometriosis (p = 0.0027) and relative to the single-dose group (p = 0.0097), indicating that repeated administration conferred a more robust systemic anti -inflammatory effect. In peritoneal fluid, TNF -α differed substantially across groups: levels were markedly elevated in endometriosis compared with controls (p < 0.0001) and were significantly reduced by both DP -MSCs regimens relative to untreated endometriosis (p < 0.0001 for both comparisons). However, peritoneal TNF -α remained significantly higher in both treated groups than in controls (p < 0 .0001 for control versus endometriosis plus single dose, and for control versus endometriosis plus two doses), supporting partial rather than complete normalization of local inflammatory activity within the observation period (Figure 4). A similar pattern was observed for IL -6. Serum IL -6 was significantly higher in endometriosis than in controls (p = 0.0003) and decreased in both DP -MSCs-treated groups compared with untreated endometriosis (endometriosis versus endometriosis plus single dose, p = 0.0016; e ndometriosis versus endometriosis plus two doses, p = 0.0008), consistent with attenuation of systemic inflammatory signaling. In peritoneal fluid, IL-6 showed a strong separation between controls and endometriosis (p < 0.0001) and was significantly reduced by both DP-MSCs regimens relative to untreated endometriosis (p < 0.0001 for both). Nevertheless, peritoneal IL-6 remained significantly elevated in treated groups compared with controls (control versus endometriosis plus single dose, p = 0.0002; control versus endometriosis plus double dose , p = 0.0044), again indicating incomplete resolution of local inflammation during the follow-up interval (Figure 4). VEGF levels demonstrated a coherent disease -associated increase and treatment-associated decrease, w ith an overall pattern compatible with dose dependence. In serum, VEGF was higher in endometriosis than in controls (p = 0.0278) and was reduced by the double-dose DP-MSCs regimen (p = 0.0345). In peritoneal fluid, VEGF was markedly increased in endometrio sis compared with controls (p < 0.0001) and decreased after DP -MSCs treatment (endometriosis versus endometriosis plus single dose, p = 0.0002; endometriosis versus endometriosis plus double dose, p = 0.0073). In addition, the significant difference betwee n treatment regimens (endometriosis plus single dose versus endometriosis plus double dose, p = 0.0100) supports a stronger local anti-angiogenic effect with repeated dosing (Figure 4). ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS For CA-125, serum measurements showed a significant reduction in the double- dose group compared with the single -dose group (p = 0.0496), consistent with diminished lesion -related biological activity under repeated DP-MSCs exposure. In peritoneal fluid, CA-125 did not exhibit statistically significant separation in post hoc analyses, suggesting either limited sensitivity of local CA -125 to short -term treatment-related changes relative to inflammatory and angiogenic mediators, or the need for greater statistical power or longer follow-up to resolve smaller effect sizes (Figure 4). Figure 4. Impact of DP-MSCs on Serum and Peritoneal Biomarker Profiles. Comparative ELISA quantification of TNF-α (ng/L), IL-6 (ng/L), and CA-125 (U/mL) levels in serum and peritoneal lavage fluid (PF) across experimental groups: Intact control (G1), Endometriosis (G2), Single-dose DP-MSCs (G3) and Double-dose DP-MSCs (G4). The untreated endometriosis group (G2) exhibited significantly elevated inflammatory and disease-associated markers compared to the sham control. In contrast, DP - MSCs treatment led to a dose -dependent reduction in these markers, with the double-dose group (G4) demonstrating the most robust suppression. Data are presented as mean ± standard deviation (SD). Individual p-values for intergroup comparisons are indicated above the brackets. Statistical significance was defined as p<0.05. ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS B. Dose-Dependent Effects of Stem Cell Therapy on Lesion Volume The analysis of lesion volumes across experimental groups revealed a clear downward trend associated with stem cell administration. Compared to the untreated endometriosis group (G2), a reduction in lesion volume was observed in the single -dose group (G3) and further pronounced in the double-dose group (G4). Although the difference between G2 and G4 approached statistical significance (p= 0.071), it did not cross the conventional threshold of p 0.999). Notably, the G4 group exhibited the lowest variance among all groups, suggesting that double -dose stem cell application may provide a more consistent therapeutic response in reducing lesion size (Figure 5). Figure 5. Comparison of Lesion Volumes (mm3) Among Experimental Groups. The distribution of lesion sizes following stem cell applications. G2 (Untreated endometriosis group), G3 (Single-dose i.p 2×106 DP-MSCs group) G4 (Double-dose i.p 2×106 DP-MSCs group). Data are presented as box-and- whisker plots indicating median values and interquartile ranges. While a downward trend in lesion volume is observed with increasing doses, the di fference between G2 and G4 did not reach statistical significance ( p=0.071). No significant difference was observed between the single and double -dose groups (p>0.999). ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS C. Histopathological assessment by histochemical stainings Semi-quantitative grading (0 absent to 3 severe) indicated that untreated endometriosis displayed the highest overall histopathology and fibrosis scores, consistent with active lesion biology accompanied by stromal remodeling. Both DP-MSCs regimens significantly reduced histological severity relative to untreated endometriosis, with improvement observed in the single-dose group (p = 0.0370) and a more pronounced reduction in the double-dose group (p < 0.0001). Fibrosis scores followed a comparable trajectory, with DP-MSCs treatment decreasing fibrotic burden compared with untreated endometriosis (single dose, p = 0.0370; double doses, p < 0.0001). The parallel improvement across overall histopathology and fibrosis supports the interpretation that DP -MSCs therapy mitigated inflammatory tissue injury and collagenous remodeling within endometriotic foci, with greater efficacy after repeated administration (Figure 6). ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS Figure 6. Histochemical evaluation of endometriotic lesions and fibrosis. Representative hematoxylin and eosin (H&E) –stained sectio ns (A) show glandular –stromal architecture and inflammatory cell infiltration in untreated endometriotic lesions, with reduced histological activity after DP-MSCs treatment, most prominently in the double-dose grou p. Masson’s trichrome staining with aniline blue (B) highlights collagen deposition, demonstrating marked stromal fibrosis in untreated lesions and reduced collagen accumulation following DP-MSCs therapy. G2 (Untreated endometriosis group), G3 (Single- dose i.p 2×10 6 DP-MSCs group) G4 ( Double- dose i.p 2×10 6 DP-MSCs group). Scale bar: 100 µm (10×), 50 µm (20×), 20 µm (40×). D. Histopathological assessment by immunohistochemical stainings Immunohistochemical scoring demonstrated strong positivity for CA-125, VEGF, Col 1 and TNF-α in untreated endometriosis, consistent with active epithelial or glandular features, heightened angiogenic signaling, extracellular matrix accumulation, and robust inflammatory pathway activation within lesions. DP-MSCs therapy was associated with reductions across these markers, most consistently and prominently in the double-dose group. CA -125 immunoreactivity was markedly decreased in the double-dose group relati ve to untreated endometriosis (p < 0.0001), supporting regression of lesion - associated biological activity. Col 1 staining was significantly reduced in both DP-MSCs-treated groups compared with untreated endometriosis (single dose, p = 0.0242; double dose, p = 0.0002), indicating an anti -fibrotic effect at the tissue level that was strengthened by repeated dosing. VEGF immunoreactivity was significantly lower in the double-dose group than in untreated endometriosis (p = 0.0002), corroborating the biochemica l evidence for suppression of angiogenic signaling. TNF -α staining was also substantially reduced in the double-dose group compared with untreated endometriosis (p< 0.0001), consistent with inhibition of proinflammatory pathways within the local lesion microenvironment (Figure 7). ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS Figure 7. Immunohistochemical assessment of lesion activity, inflammation, angiogenesis, and fibrosis. Representative immunohistochemical staining for CA-125, Col 1, VEGF and TNF-α in ectopic endometriotic lesions. Untreated endometriosi s shows strong immunoreactivity while DP -MSCs treatment reduces staining intensity and extent, with the most pronounced decrease in the double-dose group, consistent with reduced lesion act ivity, inflammatory signaling, angiogenic drive, and extracellular matrix remodeling. Scale bar: 20 µm (40×).

The present study provides integrated biochemical and histopathological evidence indicating that dental pulp–derived mesenchymal stem c ells (DP -MSCs) can modulate several key biological processes involved in experimental endometriosis. Untreated animals displayed pronounced systemic and peritoneal inflammatory activation, reflected by elevated TNF -α and IL -6 levels, increased VEGF signaling, and higher histopathological and fibrosis scores. Administration of DP-MSCs was associated with marked reductions in inflammatory cytokines and angiogenic mediators, accompanied by improvements in lesion histopathology and extracellular matrix remodeli ng. Notably, several of these effects were more pronounced following repeated dosing, as evidenced by reductions in serum TNF -α (p = 0.0027), serum VEGF (p = 0.0345), and peritoneal VEGF (p = 0.0073). Collectively, these findings suggest that DP-MSC therapy may exert multi-axis regulatory effects on the inflammatory, angiogenic, and fibrotic components of the endometriotic microenvironment [22]. Inflammation is widely recognized as a central driver of endometriosis progression and lesion persistence. In line with this concept, untreated animals in the present study exhibited elevated serum TNF-α levels compared with controls (p = 0.0207). While a single administration of DP-MSCs did not ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS significantly modify systemic TNF-α levels, repeated dosing resulted in a significant reduction relative to untreated endometriosis (p = 0.0027) and relative to the single -dose group (p = 0.0097). These findings suggest that sustained exposure to DP -MSC-derived regulatory signals may be necessary to achieve measurable systemic modulation of inflammatory cytokines. A similar pattern was observed for IL -6, a cytokine strongly implicated in the inflammatory cascade associated with endometriosis. Serum IL -6 levels were significantly increased in untreated disease compared with controls (p = 0.0003) but were markedly reduced following DP-MSC treatment in both intervention groups (single dose, p = 0.0016; double dose, p = 0.0008). Together, these observations support the concept that DP -MSCs attenuate systemic inflammatory signalin g in experimental endometriosis, consistent with the well -established immunomodulatory properties of mesenchymal stromal cells [23]. Inflammatory modulation was even more evident within the peritoneal cavity, which represents the primary inflammatory niche for ectopic lesions. Both peritoneal TNF -α and IL -6 were markedly elevated in untreated disease compared with controls (both p < 0.0001). DP-MSC therapy significantly reduced these cytokines relative to untreated animals (all p < 0.0001), although levels remained higher than those observed in controls (TNF-α: both p < 0.0001; IL-6: p = 0.0002 for single dose and p = 0.0044 for double dose). This pattern suggests that DP -MSC treatment substantially attenuates local inflammatory activity but may not fully no rmalize the peritoneal inflammatory milieu within the relatively short observation period of the present study [24]. Angiogenesis represents another critical process supporting the establishment and survival of ectopic endometrial tissue. Consistent with t his paradigm, serum VEGF levels were elevated in untreated endometriosis compared with controls (p = 0.0278). A significant reduction was observed following repeated DP-MSC administration (p = 0.0345), whereas the single -dose regimen did not produce a statistically significant change. Within the peritoneal cavity, VEGF concentrations were markedly increased in untreated disease relative to controls (p < 0.0001) and were significantly reduced following DP-MSC treatment (single dose, p = 0.0002; double dose, p = 0.0073). The significant difference between treatment regimens (p = 0.0100) suggests that repeated administration may produce a stronger suppression of angiogenic signaling within the lesion microenvironment. Histological findings further supported these biochemical results, demonstrating reduced VEGF immunoreactivity, particularly in the repeated-dose group (p = 0.0002), indicating concordant molecular and tissue-level responses [25]. Histopathological evaluation further supports the possibility that DP-MSC therapy may influence lesion severity and stromal remodeling. Untreated animals exhibited the highest histopathological and fibrosis scores, whereas both treatment regimens significantly reduced these parameters (single dose, p = 0.0370; ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS double dose p < 0.0001). The more pronounced improvement observed following repeated dosing suggests that treatment frequency may influence the magnitude of tissue remodeling and repair. Consistent immunohistochemical patterns were observed across several lesion-associated markers. CA- 125 immunoreactivity was markedly reduced in the repeated-dose group (p < 0.0001), which may reflect decreased epithelial or glandular activity within ectopic lesions. Similarly, collagen type I (C ol 1) staining was significantly reduced following DP-MSC therapy (single dose, p = 0.0242; double dose, p = 0.0002), indicating attenuation of fibrotic remodeling. Reduced TNF -α immunoreactivity in the repeated-dose group (p < 0.0001) further supports the interpretation that DP-MSC treatment modulates inflammatory activity directly within the lesion microenvironment [19]. Interestingly, CA-125 demonstrated compartment -dependent dynamics in the present model. Serum CA-125 levels differed between treatment regimens (double dose vs single dose, p = 0.0496), suggesting reduced systemic biomarker activity following repeated DP-MSC exposure. In contrast, peritoneal CA- 125 did not show significant post hoc differences between groups. This observation aligns with previous studies indicating th at CA -125 may exhibit limited sensitivity in experimental rodent models of endometriosis, particularly over relatively short observation intervals [26]. The multi -axis modulation of inflammation, angiogenesis, and fibrosis observed in this study is consistent with the broader biological profile of mesenchymal stromal cells. Increasing evidence indicates that many MSC -mediated therapeutic effects are primarily driven by paracrine and immunomodulatory signaling rather than long -term cellular engraftment [27]. Several xenogeneic studies have demonstrated measurable biological activity despite transient MSC survival [6,23,28]. Within this framework, the therapeutic effects observed here may reflect transient reprogramming of the lesion microenvironment mediated by DP-MSC-derived secretory factors. Consistent with this interpretation, lesion volume analysis demonstrated a trend toward dose-dependent reduction following DP -MSC administration, with the lowest mean lesion volume observed in the repeated-dose group (G4). Previous experimental studies have reported that MSC -based therapies can suppress the progression of endometriotic lesions through anti -inflammatory and immunomodulatory mechanisms [29,30]. Similarly, Sun et al. [29] and Abbas et al. [31] reported that increasing MSC dosage may enhance cytokine regulation and angiogenesis suppression. In the present study, the difference between the untreated endometriosis group (G2) and the repeated-dose group (G4) approached statistical significance (p = 0.071). Although this did not reach the conventional p < 0.05 threshold—likely due to the limited sample size—the observed reduction may still be biologically meaningful. The absence of a significant difference between the single- and repeated-dose groups (G3 vs G4, p > 0.999) may indicate that therapeutic responses plateau beyond a certain exposure threshold or that dosing intervals require further optimization. ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS Several limitations of the present study should be acknowledged. First, the surgically induced rat model may not fully replicate the complex heterogeneity and hormonal cyclicity characteristic of human endometriosis. Second, the study employed a fixed sacrifice time point, preventing evaluation of long- term therapeutic durability, disease recurrence, or fertilit y outcomes. Third, lesion burden was not assessed using detailed volumetric or stereological approaches. In addition, the biodistribution, persistence, and homing behavior of DP -MSCs were not directly tracked. Mechanistic investigations, including immune c ell phenotyping, extracellular vesicle analysis, or transcriptomic profiling of the lesion microenvironment, were also beyond the scope of the present study. An additional methodological consideration relates to the xenogeneic transplantation of human-derived cells into immunocompetent hosts. Host immune recognition typically limits long -term cellular engraftment; however, current evidence suggests that MSC therapeutic effects are largely mediated through transient paracrine and immunomodulatory signaling r ather than permanent cellular replacement [27]. This “hit -and-run” mechanism has been supported in multiple xenogeneic models demonstrating significant biological effects despite limited MSC survival [6,23,28]. Finally, the absence of significant alteratio ns in peritoneal CA -125 levels requires cautious interpretation. Although CA-125 is widely used as a clinical biomarker, its translational value in rodent models of endometriosis remains limited because correlations with lesion burden or therapeutic response are inconsistent [32 –34]. Model-specific variability may arise from immune responses and surgical induction techniques [1,35]. Therefore, stable CA -125 levels should not necessarily be interpreted as evidence of therapeutic inefficacy but rather highlight the importance of evaluating treatment responses using a multi-parameter framework that integrates inflammatory cytokines, angiogenic mediators, and histopathological outcomes. In conclusion, DP-MSC administration significantly modulated the pathologica l microenvironment of experimental endometriosis by attenuating inflammatory cytokines, suppressing angiogenic signaling, and reducing fibrotic remodeling within lesions. Repeated dosing produced the most consistent improvements across both biochemical and histological parameters. These findings support the potential of DP -MSCs as a promising cell -based therapeutic strategy for modulating the endometriotic microenvironment while underscoring the need for further mechanistic and translational studies to optimize treatment protocols and clarify the underlying biological mechanisms [36]. ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS

In summary, human dental pulp -derived MSCs (DP -MSCs) effectively mitigated the hallmark pathological features of experimental endometriosis, including pro-inflammatory activation, angiogenic signaling, and fibrotic remodeling. Systematic administration significantly reduced systemic and local concentrations of TNF-α, IL-6, and VEGF , with the most pronounced therapeutic efficacy observed under a repeated dosing regimen . These biochemical improvements were corroborated by histopathological recovery, characterized by reduced fibrosis scores and diminished lesional immunoreactivity for Col1, VEGF, and TNF-α. While peritoneal inflammatory mediators did not ret urn to baseline control levels within the study period, the concordant improvements across biochemical, histochemical, and immunohistochemical parameters underscore the potential of DP -MSCs as a potent disease-modifying, cell-based strategy. Our findings highlight a clear regimen-dependent therapeutic profile, suggesting that optimized dosing is critical for clinical efficacy. Future investigations incorporating extended follow -up, quantitative lesion burden analysis, and functional endpoints are warranted to further define the durability of these effects and facilitate the clinical translation of DP-MSCs therapy for endometriosis. Ackowledgement The authors express their gratitude to the Scientific and Technological Research Council of Türkiye (TÜBİTAK), which played a major role in conducting the research. Funding Information This study (Project number: SBAG 224S142) received both scientific and financial support from The Scientific and Technological Research Council of Turkey (TÜBİTAK). Data Availability Statement The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Conflict of Interest The authors have no conflicts of interest to declare that are relevant to the content of this article. ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS

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Adipose tissue-derived mesenchymal stem cells reduce endometriosis cellular proliferation through their anti -inflammatory effects. Clin. Exp. Reprod. Med. 48, 322–336 (2021). ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS Figure Legends Figure 1. Morphological and immunophenotypic characterization of DP -MSCs. Representative inverted phase-contrast micrographs illustrate the adherent, spindle-shaped, fibroblast-like morphology of DP-MSCs. The upper images display the cells during the early expansion phase (Initial isolation, P1-P2); the middle images represent the confluent culture prior to cryopreservation; and the lower images show the cells after thawing and subsequent re -culture. The consistent morphology observed across all stages indicates that the cryopreservation and thawing processes do not adversely affect the typical MSC-like appearance or the plastic-adherence capacity of the cells. Figure 2. Immunophenotypic characterizati on of DP -MSCs. Representative flow cytometry dot plots showing the gating strategy based on forward (FSC) and side scatter (SSC) ( Gate A, 93.83% ). Analysis of MSC markers: DP-MSCs show high expression levels for CD90 (99.91%), CD44 (95.64%), CD105 (98.32%) , and CD73 (99.74%) . Exclusion of hematopoietic lineages: cells show negative expression for CD34 and CD45 (Gate G, 1.56%), confirming their non-hematopoietic origin. Data tables provide event counts, percentage of gated cells (%Gated), and mean fluorescen ce intensity (MFI) for each marker. Figure 3. Macroscopic evaluation and quantitative measurement of endometriotic lesions across experimental groups. Top row (G2, G3, G4) shows the representative in situ macroscopic appearance of the lesions during laparo tomy. Bottom row (G2-1, G3 -1, G4 -1) displays the harvested lesions measured with a digital caliper to calculate the volumes. G2: Untreated endometriotic lesion group exhibiting well-vascularized, large cystic structures. G3: Single-dose DP-MSCs therapy group showing a moderate reduction in lesion size. G4: Double-dose DP -MSCs therapy group showing the most significant regression in lesion volume and vascularization. Caliper measurements correspond to the data distribution presented in the volumetric analysis. Figure 4. Impact of DP-MSCs on Serum and Peritoneal Biomarker Profiles . Comparative ELISA quantification of TNF-α (ng/L), IL-6 (ng/L), and CA-125 (U/mL) levels in serum and peritoneal lavage fluid (PF) across experimental groups: Intact control (G1), Endometriosis (G2), Single-dose DP-MSCs (G3) and Double-dose DP-MSCs (G4). The untreated endometriosis group (G2) exhibited significantly elevated inflammatory and disease-associated markers compared to the sham control. In contrast, DP - MSCs treatment led to a dose-dependent reduction in these markers, with the double -dose group (G4) demonstrating the most robust suppression. Data are presented as mean ± standard deviation (SD). Individual p-values for intergroup comparisons are indicated above the brackets. Statistical significance was defined as p<0.05. ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS Figure 5. Comparison of Lesion Volumes (mm3) Among Experimental Groups. The distribution of lesion sizes following stem cell applications. G2 (Untreated endometriosis group), G3 (Single-dose i.p 2×106 DP-MSCs group) G4 (Double-dose i.p 2×106 DP-MSCs group). Data are presented as box-and- whisker plots indicating median values and interquartile ranges. While a downward trend in lesion volume is observed with increasing doses, the difference between G2 an d G4 did not reach statistical significance ( p=0.071). No significant difference was observed between the single and double -dose groups (p>0.999). Figure 6. Histochemical evaluation of endometriotic lesions and fibrosis. Representative hematoxylin and eos in (H&E) –stained sections ( A) show glandular –stromal architecture and inflammatory cell infiltration in untreated endometriotic lesions, with reduced histological activity after DP-MSCs treatment, most prominently in the double -dose group. Masson’s trichro me staining with aniline blue ( B) highlights collagen deposition, demonstrating marked stromal fibrosis in untreated lesions and reduced collagen accumulation following DP-MSCs therapy. G2 (Untreated endometriosis group), G3 (Single - dose i.p 2×10 6 DP-MSCs group) G4 (Double - dose i.p 2×10 6 DP-MSCs group). Scale bar: 100 µm (10×), 50 µm (20×), 20 µm (40×). Figure 7. Immunohistochemical assessment of lesion activity, inflammation, angiogenesis, and fibrosis. Representative immunohistochemical staining for CA-125, Col 1, VEGF and TNF-α in ectopic endometriotic lesions. Untreated endometriosis shows strong immunoreactivity while DP -MSCs treatment reduces staining intensity and extent, with the most pronounced decrease in the double -dose group, consisten t with reduced lesion activity, inflammatory signaling, angiogenic drive, and extracellular matrix remodeling. Scale bar: 20 µm (40×). ACCEPTED MANUSCRIPT ARTICLE IN PRESSARTICLE IN PRESS