3D Culture of Human Ovarian Tissue on Conductive Hybrid Scaffolds with and without Electrical Stimulation

3D Culture of Human Ovarian Tissue on Conductive Hybrid Scaffolds with and without Electrical Stimulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article 3D Culture of Human Ovarian Tissue on Conductive Hybrid Scaffolds with and without Electrical Stimulation Negin chavoshinezhad, Behrooz niknafs This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7638995/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The survival, development, and hormonal activity of ovarian follicles depend on the ovarian tissue microenvironment, making its preservation critical for effective fertility preservation. To maintain the structural and functional integrity of human ovarian cortical tissue during in vitro culture, we developed a conductive scaffold supplemented with hormones and electrical stimulation. Methods Conductive hybrid scaffolds were fabricated from alginate (Alg), carboxymethyl cellulose (CMC), and polypyrrole (PPy) using in situ polymerization of pyrrole monomer followed by ionic cross-linking. These scaffolds supported three-dimensional culture of human ovarian cortical tissue. Three formulations (PPy to Alg-CMC ratios of 2:1, 5:1, and 10:1) were evaluated for structural stability. Human ovarian cortical fragments (1 mm × 1 mm × 500 μm) were cultured on the scaffolds for 15 days; one group received daily electrical stimulation (100 mV for 1 h), while the control group did not. Stromal and follicular responses were assessed via immunohistochemical staining for α-smooth muscle actin (α-SMA), Ki-67, and p53 to evaluate ECM remodeling, proliferation, and stress/apoptosis. Results The 2:1 PPy/Alg-CMC scaffold exhibited superior performance, with biocompatibility, conductivity, and no cytotoxicity. Under electrical stimulation, stromal, granulosa, and endothelial cells displayed strong activation, including sustained α-SMA expression (indicating a myofibroblast-like phenotype and vascular endothelial growth), elevated Ki-67 levels (indicating enhanced proliferation), and absent p53 expression (confirming no cellular stress or apoptosis). Conclusion This study demonstrates that a conductive hybrid scaffold (2:1 PPy to Alg-CMC) combined with daily electrical stimulation promotes stromal proliferation, preserves stromal integrity and tissue structure, and supports primordial follicle progression to the preantral and antral stage, suggesting effective maintenance of the follicular niche. Biological sciences/Biotechnology Biological sciences/Cell biology Physical sciences/Materials science Health sciences/Medical research 3D ovarian tissue culture In-situ follicle culture Electrical stimulation Conductive hybrid scaffold Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction In mammalian ovaries, folliculogenesis is a tightly regulated process that provides a continuous supply of developmentally competent oocytes throughout a woman's reproductive lifespan( 1 ). In vitro follicle growth (IVG) offers transformative potential for assisted reproductive technologies (ART), enabling the generation of mature, fertilizable oocytes from immature follicles. This approach is especially valuable for fertility preservation in prepubertal girls and women undergoing gonadotoxic cancer treatments, where traditional options like oocyte or embryo cryopreservation are not viable ( 2 ). Despite milestones such as the first live births from in vitro-grown oocytes ( 3 ), early in vitro culture methods relied on two-dimensional (2D) platforms that failed to preserve the native three-dimensional architecture of ovarian follicles. In 2D systems, the loss of spatial structure disrupts essential bidirectional communication between the oocyte and surrounding granulosa cells, ultimately impairing oocyte maturation and developmental competence( 4 ). To overcome these limitations, current strategies have shifted toward three-dimensional (3D) culture systems that more closely mimic the natural ovarian microenvironment. Among these, culturing intact ovarian cortical fragments stands out as a promising method, as it maintains stromal context, extracellular matrix (ECM) integrity, and natural tissue architecture—all critical for follicle survival, activation, and coordinated development. Preserving this structural and biochemical niche is essential for maintaining the follicular unit and facilitating physiological signaling during in vitro culture ( 5 , 6 ). Despite recent advances, most modern systems still lack key physiological cues present in vivo, such as bioelectrical signals. Emerging evidence indicates that endogenous electric fields play vital roles in tissue morphogenesis, cell proliferation, and extracellular matrix remodeling ( 7 ). In regenerative medicine, exogenous electrical stimulation has shown therapeutic promise by enhancing cardiac tissue function, neural regeneration, and wound healing ( 8 ). Conductive biomaterials, such as polypyrrole-reinforced hydrogels, offer a powerful way to integrate bioelectrical cues into 3D culture platforms. When combined with natural polymers like alginate and carboxymethyl cellulose, these materials form hybrid scaffolds that are biocompatible, mechanically tunable, highly porous, and electrically conductive ( 9 , 10 ). In this study, we introduce a novel conductive 3D culture system designed to preserve and enhance ovarian tissue structure during in vitro culture. By integrating a PPy/Alg-CMC hybrid scaffold with daily electrical stimulation ( 11 ), our goal is to support the in-situ growth of human ovarian follicles within intact cortical fragments. This approach not only maintains the ovarian tissue's natural 3D architecture but also leverages bioelectrical cues to promote granulosa and stromal cell proliferation ( 12 ). Our central hypothesis is that preserving ovarian tissue structure through the use of a conductive hybrid scaffold and electrical stimulation creates a biomimetic niche that supports follicle survival, growth, and progression to later developmental stages. This integrated strategy represents a significant advancement in ovarian tissue engineering and offers a promising platform for next-generation fertility preservation, particularly for patients at risk of premature ovarian insufficiency due to cancer or other pathologies. 2. Materials and Methods This study was designed as a controlled in vitro experiment to assess the effects of electrical stimulation on ovarian tissue integrity and follicular development during 3D culture. All methods in this study were carried out in accordance with relevant guidelines and regulations. Experimental protocols were reviewed and approved by the Ethics Committee of Tabriz University of Medical Sciences (Approval ID: IR.TBZMED.REC.1402.200). 2.1 Preparation of the Conductive (PPy/ Alg-CMC) Scaffold Conductive hybrid hydrogel scaffolds were fabricated from alginate, carboxymethyl cellulose, and polypyrrole using in situ oxidative polymerization and ionic cross-linking. All reagents were analytical grade (Sigma-Aldrich) and used without further purification ( 9 , 13 ). Briefly, a 2% (w/v) Alg solution was prepared by dissolving alginate in deionized water with constant magnetic stirring at room temperature for 6 hours until fully homogeneous. CMC was dissolved in a 0.5% (v/v) citric acid–acetic acid buffer (pH ≈ 4.5) to yield a 2% (w/v) solution, which was stirred for 24 hours at room temperature to form a clear, viscous mixture. Equal volumes of the Alg and CMC solutions were blended thoroughly for 1 hour at 60–70°C to create a homogeneous Alg-CMC blend ( 13 ). To impart electrical conductivity, pyrrole monomer was incorporated via in situ polymerization. A 0.2 M pyrrole solution in 1 N HCl was added dropwise to the Alg-CMC matrix at three volume ratios: 2:10, 5:10, and 10:10 (PPy: Alg-CMC) to evaluate the effects of polypyrrole content on scaffold properties. The mixture was stirred at 90°C for 30 minutes to ensure uniform dispersion. Polymerization was initiated by slowly adding 0.2 M ferric chloride (FeCl₃) in 1 N HCl as an oxidant. The reaction proceeded under constant stirring at 90°C for 5 hours, during which the solution turned from brown to deep black, indicating successful PPy formation and integration ( 9 , 14 ). To form scaffolds, the conductive PPy/Alg-CMC solution was dispensed into sterile 6-well plates (∼1 mL per well), creating circular scaffolds approximately 4 mm in diameter and 2 mm thick. The droplets were immediately immersed in 0.1 M calcium chloride (CaCl₂) to induce ionic cross-linking via the "egg-box" model, where Ca²⁺ ions cross-link alginate's guluronic acid blocks, leading to rapid gelation and stable 3D porous structures. Cross-linking occurred for 60 minutes at room temperature ( 14 ). After cross-linking, the CaCl₂ solution was discarded, and scaffolds were rinsed three times with sterile phosphate-buffered saline (PBS) to remove residual ions. For sterilization, scaffolds were immersed in 70% ethanol for 30 minutes, followed by UV irradiation (15 minutes per side) in a biosafety cabinet. They were then washed thoroughly with sterile PBS to remove ethanol residues and equilibrated in culture medium before tissue seeding( 9 ). 2.2. Morphological Analysis of the Scaffold The surface morphology, pore structure, and 3D architecture of the conductive PPy/Alg-CMC hybrid scaffolds were examined using scanning electron microscopy (SEM). Pure alginate scaffolds were prepared and analyzed under identical conditions as non-conductive controls for comparison. Scaffold samples were frozen at − 80°C for 4 hours and lyophilized for 24 hours to preserve the porous microstructure. Dried samples were mounted on aluminum stubs and sputter-coated with a 10–15 nm layer of gold/palladium (Au/Pd) for conductivity ( 15 ). SEM imaging was conducted with a scanning electron microscope (e.g., Hitachi SU5000) at an accelerating voltage of 5–15 kV. Images were acquired at various magnifications to evaluate pore size, interconnectivity, wall morphology, and overall architecture ( 16 ). Pore diameter was quantified using ImageJ software, measuring at least 50 pores randomly from multiple fields per sample. Results were averaged across three independent scaffolds per formulation( 16 ). This comparison highlighted how incorporating polypyrrole and carboxymethyl cellulose affected the microstructural properties of the alginate matrix. 2.3. Biodegrading Assessment of The Scaffold To evaluate structural stability, scaffold samples were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) at 37°C with gentle shaking at 100 rpm in a sealed container for 15 days. At predefined intervals (days 0, 3, 5, 10, and 15), scaffolds were removed, gently rinsed, and examined under an optical microscope for surface changes. This assessment was performed on Alg, Alg/CMC, and PPy/Alg-CMC composite variants to compare degradation and durability ( 9 ). 2.4. Evaluation of In Vitro Cytotoxicity of The Scaffolds Cytotoxicity of scaffold extracts was assessed using an MTT assay per ISO 10993-5 standards ( 17 ). Human Foreskin Fibroblasts (HFF cells), were purchased from the Cell Bank of the Immunology Research Center, Tabriz University of Medical Sciences. HFF cells were seeded in 96-well plates at 1 × 10⁴ cells/well in RPMI medium with 10% fetal bovine serum (FBS), serving as the negative control. Cells were exposed to extracts from PPy[2]/Alg + CMC, PPy[5]/Alg + CMC, and PPy[10]/Alg + CMC scaffolds. After incubation at 37°C in a humidified 5% CO₂ atmosphere for specified periods, the medium was removed, and cells were washed three times with PBS. Then, 100 µL of MTT solution (5 mg/mL in PBS) was added per well, followed by 4 hours of incubation for formazan crystal formation. The supernatant was aspirated, and 500 µL of dimethyl sulfoxide (DMSO) was added to dissolve the crystals. Absorbance was measured at 570 nm using a BioTek ELx800 Micro ELISA reader (Shoreline, WA, USA). Cell viability was calculated as a percentage relative to the negative control to determine scaffold biocompatibility( 17 ). 2.5. Electrical Conductivity Measurement of The Scaffold Electrical conductivity of the PPy/Alg-CMC hybrid scaffolds was measured at room temperature (25°C) using a Mettler-Toledo SevenGo Duo SG23 portable conductivity meter (Mettler-Toledo AG, Switzerland), with a range of 0.10 µS/cm to 500 mS/cm. The device included an LC310 conductivity cell, automatic temperature compensation (ATC), and Good Laboratory Practice (GLP) data logging for accuracy and reproducibility ( 18 ). Scaffolds were equilibrated in PBS for 24 hours to mimic physiological conditions and ensure stable probe contact. The conductive surface was placed in direct contact with the probe, and readings were taken after signal stabilization (30–60 seconds). For each formulation (PPy/Alg-CMC ratios of 2:10, 5:10, and 10:10), three independent scaffolds were tested in triplicate. Average conductivity was reported in microsiemens per centimeter (µS/cm). Pure Alg and CMC scaffolds (without PPy) served as non-conductive controls to quantify PPy's contribution to conductivity( 18 , 19 ). 2.6. Electrical Stimulation (ES) Setup To examine the effects of electrical stimulation on ovarian tissue constructs, a custom system was used. Silver wire electrodes (Ag) were positioned in each well of a 6-well plate on opposite sides of the scaffold, with an inter-electrode distance of ∼4–6 mm. Electrodes were connected via insulated wires and alligator clips to a programmable pulse generator delivering unipolar square-wave pulses at 100 mV amplitude, 1 Hz frequency, and 1 ms pulse width. Stimulation was applied daily for 1 hour over 15 days, creating an electric field of ∼17–25 mV/mm across the scaffold. This low-intensity regimen was chosen to influence cell behavior without causing electrochemical artifacts ( 20 , 21 ). 2.7. Study Groups The experimental groups were as follows: Group 1 (Control): Ovarian tissue cultured on conductive scaffold without electrical stimulation. Group 2 (Electrical Stimulation): Ovarian tissue cultured on conductive scaffold with daily electrical stimulation. 2.8. Ovarian Tissue Collection and Processing Human ovarian cortical tissue was collected from five women (mean age 33.6 ± 4.8 years, range 20–35) undergoing laparoscopy for benign gynecological conditions at Al-Zahra Hospital, Tabriz University of Medical Sciences, with prior written informed consent. All procedures were conducted in accordance with approved protocols and the guidelines of the Ethics Committee (Approval ID: IR.TBZMED.REC.1402.200). Immediately post-surgery, tissue fragments were transported on ice in sterile PBS supplemented with antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin). In the laboratory, tissues were rinsed three times with cold PBS to remove blood, fat, and debris ( 22 ).Under sterile conditions, the cortex was dissected using fine forceps and microscissors. To maximize follicle exposure to nutrients and signals while promoting primordial follicle activation and growth, tissues were cut into uniform 1–2 mm³ pieces with a sterile scalpel. These fragments were then placed on conductive scaffolds, ensuring good contact for 3D culture and potential electrical interactions ( 6 , 22 ). 2.9. Culture Conditions and Medium Preparation The base medium was alpha-Minimal Essential Medium (α-MEM) supplemented with 10% FBS, 2 mM sodium pyruvate, 50 µg/mL ascorbic acid, 50 mg/L streptomycin sulfate, and 75 mg/L penicillin. To promote follicular development, 75 mIU/mL follicle-stimulating hormone (FSH) and 75 mIU/mL luteinizing hormone (LH) were added( 6 , 23 ). A volume of 2 mL of the complete culture medium was added to each scaffold to ensure complete coverage and even distribution over the tissue fragments. The medium was replaced every other day to maintain nutrient availability and remove metabolic waste. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂ for a total duration of 15 days( 24 ). 2.10. Histological Evaluation of Stroma and Follicular Architecture After 15 days, tissue constructs were gently removed from scaffolds and fixed in 4% (w/v) paraformaldehyde at 4°C for 12–16 hours to preserve cellular and ECM morphology. Samples were dehydrated through graded ethanol (50%, 70%, 80%, 95%, 100%), cleared in xylene, and embedded in paraffin using standard protocols. Serial 5 µm sections were cut on a rotary microtome and mounted on charged slides ( 25 ). Sections were deparaffinized in xylene, rehydrated through descending ethanol grades, stained with hematoxylin for 5–7 minutes to visualize nuclei, and differentiated in acid alcohol. Cytoplasm and ECM were counterstained with eosin for 2–3 minutes. After dehydration and clearing, slides were coverslipped with permanent mounting medium( 25 ). Stained sections were examined under a brightfield microscope equipped with a digital camera (e.g., Olympus BX53 or Zeiss Axio Imager A2). High-resolution images were captured at 100× and 400× magnifications under consistent illumination. Stromal structure and follicle development were qualitatively assessed. 2.12. Immunohistochemical analysis of Ki-67, P53, and α-SMA Expression Immunohistochemistry was used to assess cellular proliferation (Ki-67), apoptosis (p53), and stromal activation/vascular stabilization (α-SMA). Paraffin- or OCT-embedded sections (5 µm) from paraformaldehyde-fixed samples underwent antigen retrieval in citrate buffer (pH 6.0) with heat. Non-specific binding was blocked with 5% BSA or normal serum. Sections were incubated overnight at 4°C with primary antibodies: Ki-67 (1:100–1:200), p53 (1:100), or α-SMA (1:200). Detection involved HRP-conjugated secondary antibodies with DAB for chromogenic visualization or fluorescent secondaries with DAPI counterstaining for fluorescence. Staining was evaluated microscopically and scored semi-quantitatively by intensity and positive cell percentage: negative (–) for no staining, weak (+) for faint staining in 70% of cells ( 26 – 28 ). Scores were assigned for each marker in relevant compartments (granulosa cells, stroma), with discrepancies resolved by consensus. 3. Results The porosity, biodegradability, cytocompatibility, and electrical conductivity of alginate-based scaffolds incorporating carboxymethyl cellulose and polypyrrole were systematically evaluated to confirm their suitability for 3D ovarian tissue culture. 3.1. Porosity of The Scaffold SEM analysis revealed that control alginate scaffolds exhibited a highly porous, interconnected 3D network with pore diameters of 50–150 µm, ideal for cell infiltration, nutrient diffusion, and oxygen transport in 3D cultures (Fig. 2 .a). Incorporating PPy and CMC slightly reduced overall porosity but preserved interconnectivity, which is crucial for uniform nutrient and oxygen distribution. Pore sizes became more uniform and smaller (25–50 µm), attributable to partial pore occlusion during PPy polymerization, increased polymer density, and CMC-induced chain entanglement (Fig. 2 .b). This uniformity likely enhances mechanical stability and provides a consistent environment for follicular encapsulation and development. Notably, pore size and porosity did not vary significantly across PPy concentrations ( 2 , 5 , and 10 ), indicating that PPy loading and polymerization do not substantially alter scaffold microarchitecture. Thus, PPy content can be adjusted to optimize conductivity without compromising porosity. Overall, these scaffolds maintain sufficient porosity for mass transfer and cell penetration while offering improved stability for long-term ovarian tissue or follicle culture (Figs. 2 .a–c). 3.2. Biodegradation of The Scaffolds Degradation was concentration-dependent upon PPy incorporation into the Alg-CMC matrix: PPy[2]-Alg/CMC [1] scaffolds showed excellent structural integrity, with no cracks, minimal erosion, and uniform morphology throughout the culture period, reflecting optimal compatibility and controlled degradation. PPy[5]-Alg/CMC [1] scaffolds displayed moderate changes, such as slight surface roughness and early matrix weakening, suggesting emerging phase inhomogeneity. PPy[10]-Alg/CMC [1] scaffolds underwent severe degradation, including visible cracks, brittleness, and PPy aggregation, likely due to poor interfacial adhesion between hydrophobic PPy and the hydrophilic matrix, accelerating breakdown. Overall, CMC enhances alginate stability, but high PPy compromises integrity. The PPy[2]-Alg/CMC [1] formulation achieves the best balance of durability and controlled degradation, making it ideal for extended 3D ovarian follicle culture. 3.3. Scaffolds' Cytotoxicity and Biocompatibility To confirm safety and suitability for ovarian tissue culture, cytotoxicity and biocompatibility of Alg-CMC-PPy scaffolds were evaluated. HFF cell viability and metabolic activity were measured via MTT assay after 72 hours at 37°C in 5% CO₂. PPy [2]-Alg/CMC [1] scaffolds (Fig. a), exhibited excellent biocompatibility, with viability > 100% of control, indicating robust proliferation and favorable cell-scaffold interactions. Viability was ∼97% for PPy [5]-Alg/CMC [1], (Fig. b) and ∼95% for PPy [10]-Alg/CMC [1] (Fig. c), showing a slight decline with increasing PPy. This minor reduction at higher PPy levels may stem from polymer aggregation, reduced hydrophilicity, or trace reactive species release during PPy synthesis. However, all formulations demonstrated strong biocompatibility, confirming that PPy integration into Alg-CMC does not induce significant cytotoxicity, even at higher concentrations. 3.3. Scaffolds' Electrical Conductivity Control scaffolds of alginate or Alg-CMC (without PPy) displayed very low conductivity (< 10 µS/cm or < 1 × 10⁻² S/m), consistent with their insulating nature. While structurally sound and biocompatible, these lack the ability to transmit electrical signals. PPy incorporation dramatically enhanced conductivity in a dose-dependent manner, enabling electroactive 3D environments: PPy[2]-Alg/CMC[1]: 1.33 × 10⁻⁴ S/cm PPy[5]-Alg/CMC[1]: 1.25 × 10⁻³ S/cm PPy[10]-Alg/CMC[1]: Higher values, reflecting over 1,300-fold increase vs. controls, due to PPy forming efficient conductive networks and percolation pathways. Although PPy[10] offered superior conductivity, PPy[2]/Alg-CMC[1] was selected as optimal, balancing conductivity with structural stability, uniform morphology, and controlled degradation (as per biodegradation results in Section 3.2 ). Higher PPy formulations showed phase separation, cracking, and rapid degradation, potentially unsuitable for long-term follicle culture. 3.4. Analysis of Ovarian Tissue Histologically and Morphologically At day 0, ovarian tissue primarily contained primordial and early primary follicles, featuring a central oocyte surrounded by a single layer of flattened granulosa cells and no theca layer (Fig. 4 .a). After 15 days of 3D culture, marked morphological advancement occurred in the electrical stimulation group. Follicles progressed to preantral and antral stages, with multiple layers of cuboidal to columnar granulosa cells, prominent mitotic figures, and intact oocytes lacking degenerative signs, indicating robust growth and oocyte viability under ES (Fig. 4 .b)( 27 ). The stromal compartment also showed improved organization, with denser, follicle-oriented stromal cells suggesting enhanced cell-matrix interactions and microenvironmental support. Importantly, no histological evidence of damage appeared in the stroma, including vacuolization, pyknotic nuclei, or perinuclear halos—markers of stress, degeneration, or apoptosis. This absence supports the scaffold's biocompatibility, structural integrity, and ES safety (Fig. 4 .b). 3.5. Evaluation of α-SMA, p53, and Ki-67 Expression in Response to Electrical Stimulation 3.5.1 Expression of the Cell Proliferation Marker, Ki-67 Control Group : Without electrical stimulation, Ki-67 expression was minimal, with few positive cells in granulosa and oocyte nucleus, indicating limited mitotic activity during baseline 3D culture (Fig. 5 .a). Electrical Stimulation Group: By day 15, the ES group exhibited a marked increase in Ki-67-positive cells (+++), particularly in granulosa cells and theca layers (Fig. 5 .b) and surrounding stroma (Fig. 5 .c), with enhanced nuclear staining reflecting heightened proliferative activity compared to controls. 3.5.2. P53 Expression (Apoptosis-Associated Marker) P53 expression was negligible or absent under baseline conditions, suggesting minimal cellular stress or apoptotic activation initially (Fig. 6.a). After 15 days of ES, no significant increase in p53 occurred compared to controls. Staining remained low-intensity and sparse, with no nuclear accumulation in granulosa, theca, or stromal cells (Fig. 6.a,6. b). . 3.5.3 α-SMA Expression (Vascular Stability and Stromal Integrity Marker) α-SMA expression was low in stroma, mainly in sparse fibroblasts and perivascular cells around follicles, consistent with baseline ovarian homeostasis and physiological vascular support (Fig. 7 .a). By day 15, α-SMA expression increased substantially (+++), especially in stroma and perivascular cells surrounding follicles, with more organized staining and cellular alignment (Fig. 7 .b). 4. Discussion Our study developed a novel conductive 3D scaffold integrated with electrical stimulation to enhance stromal organization, vascular stabilization, and in situ follicular development. Histological analyses confirm that 3D culture with ES and hormonal supplementation promotes key biological processes, preserving ovarian tissue architecture and creating an optimal niche for follicle growth without inducing stromal damage or cellular stress. In reproductive bioengineering, 3D culture systems provide a more physiologically relevant environment for in vitro follicle growth than traditional 2D methods( 4 , 29 , 30 ). A common approach involves encapsulating isolated secondary follicles in natural or synthetic hydrogels, particularly alginate, which maintains 3D structure and supports bidirectional oocyte-granulosa cell communication, enabling progression to the antral stage( 4 , 31 – 33 ). Xu et al. demonstrated the potential of this method, showing that murine secondary follicles (150–180 µm) cultured in alginate for 8 days yielded oocytes with high developmental competence: 71% reached metaphase II post-IVM, 68% fertilized, and produced live offspring ( 33 , 34 ). Similarly, Telfer et al. ( 35 ) and Amorim et al. ( 36 ), reported successful in vitro maturation of human and animal follicles in 3D systems, highlighting their promise for fertility preservation. However, isolating individual follicles disrupts the native ovarian environment, eliminating essential stromal interactions, vascular cues, and paracrine signaling critical for folliculogenesis ( 37 ). To address this, 3D culture of ovarian cortical fragments has gained traction, as it preserves tissue architecture, ECM integrity, and inter-follicular communication, leading to improved follicle survival and activation while mimicking the in vivo niche( 5 , 38 ). For example, alginate-encapsulated human ovarian tissue maintained > 50% structural integrity and supported multilayer follicle formation( 39 , 40 ). Porous alginate scaffolds also enhanced follicle viability and reduced apoptosis, underscoring the value of engineered hydrogels( 41 ). Nevertheless, conventional hydrogels like fully cross-linked alginate pose challenges due to high stiffness, which can restrict stromal proliferation and nutrient diffusion, potentially hindering follicle development( 42 , 43 ). Pais et al. emphasized the importance of mechanical compliance, showing that softer gels (0.25–0.7% w/v) promote diffusion, steroidogenesis, and follicular expansion, while rigid matrices suppress stromal proliferation( 42 ). To mitigate rigidity without losing support, strategies include blending alginate with biocompatible polymers like collagen, fibrin, or carboxymethyl cellulose to improve flexibility, porosity, and cell-matrix interactions ( 44 ),or adjusting alginate concentration and cross-linking density to better match native ovarian tissue softness ( 44 , 45 ). While these hybrid scaffolds improve mechanics, they remain passive and unable to deliver dynamic cues like bioelectrical signals, increasingly recognized as key regulators of cellular behavior, tissue organization, and regeneration. Native ovarian tissue, like other biological tissues, exhibits intrinsic electrical activity influencing proliferation, differentiation, and communication. Traditional hydrogels lack electroactive properties, limiting their ability to replicate the in vivo environment fully.Interest in conductive polymers (CPs) has grown since their discovery in the late 1970s( 46 ). These materials combine semiconductor-like electrical and optical properties with polymer-like mechanical and processing advantages. Among CPs, polypyrrole (PPy) stands out for its high conductivity and electrochemical stability( 47 ). We engineered a hybrid conductive scaffold by embedding PPy in an Alg-CMC matrix( 14 , 19 , 48 ), to provide daily bioelectrical stimulation (100 mV, 1 h/day) during culture. This voltage (100–200 mV), validated in cardiac, skeletal, and neural systems, safely enhances proliferation, differentiation, regeneration, and matrix synthesis( 49 – 51 ). For instance, PPy-mediated 100 mV stimulation promotes fibroblast-to-myofibroblast transition via the TGF-β1/ERK/NF-κB pathway, sustaining an activated phenotype for wound healing( 52 ). Falling within physiological ranges, this low voltage supplies dynamic cues while avoiding damage from pH changes, electrode corrosion, or toxic byproducts ( 53 ), making it suitable for ovarian tissue. Mechanistically, ES likely activates parallel pathways like PI3K/Akt, ERK/MAPK, Ca²⁺/CaMKII, and TGF-β/SMAD, regulating survival, proliferation, cytoskeletal organization, and ECM synthesis( 52 ). ES may also enhance localized ECM deposition and cross-linking—possibly via enzymes like lysyl oxidase—modulating scaffold biomechanics to support follicle development ( 54 , 55 ). Pulsed electric fields, for example, double collagen and elastin production in fibroblasts without elevating matrix metalloproteinase (MMP) activity, indicating balanced ECM remodeling( 56 , 57 ). Our findings align, showing increased fibroblast deposition without fibrosis, suggesting constructive remodeling( 58 ). Although not directly assessed here, these mechanisms may underlie the enhanced intercellular communication in our cultures. We observed that ES significantly boosted Ki-67 expression—a marker of active cell cycle phases (G1, S, G2, M)—in follicular and stromal compartments ( 59 ). This pro-proliferative effect, likely mediated by pathways like ERK1/2 (MAPK) driving G1/S transition ( 52 , 60 ). ), suggests ES promotes cell cycle entry, aiding stromal regeneration and follicular growth. Beyond proliferation, ES guides differentiation and patterning, acting as morphogenic signals for coordinated growth and organization ( 61 , 62 ). In our system, ES elevated α-SMA expression, indicating stromal fibroblast proliferation and myofibroblast differentiation, which supports constructive remodeling ( 63 , 64 ). These α-SMA⁺ cells likely aid vascular stabilization, follicular integrity, and ECM organization—key for a functional perifollicular niche. Similarly, pulsed electrical stimulation (PES) upregulated α-SMA in human dermal fibroblasts via Smad2/3 phosphorylation, confirming ES activates the TGF-β/Smad axis to drive myofibroblast conversion and contractility ( 63 , 65 ). Histological data further show that ES reinforces stromal health and regenerative capacity through mature microvasculature establishment. Stromal activation aligns with preantral follicle development, maintaining architecture without apoptosis or fibrosis. Thus, ES-driven remodeling is supportive, fostering a physiologically active, mechanically robust microenvironment for follicle growth and function. 5. Conclusion Our findings demonstrate that a conductive PPy/Alg-CMC scaffold combined with electrical stimulation elicits a regulated, regenerative stromal response mirroring key aspect of the native ovarian microenvironment. By enhancing stromal organization, vascularization, and perifollicular support without inducing fibrosis or stress, this approach creates a biomimetic niche that advances follicular development to the preantral stage. These results highlight the promise of electroactive biomaterials in ovarian tissue engineering, offering a robust strategy to improve in vitro follicle growth and fertility preservation solutions. Abbreviations Assisted reproductive technology (ART), In vitro fertilization (IVF), Automatic temperature compensation (ATC), Good Laboratory Practice (GLP), Two-dimensional culture (2D), Three-dimensional culture (3D), Extracellular matrix (ECM), Matrix metalloproteinase (MMP), Electrical stimulation (ES), Conductive polymers (CPs), Polypyrrole (PPy), Carboxymethyl cellulose (CMC), Alginate (Alg), alpha-Minimal Essential Medium (α-MEM), Dulbecco’s Modified Eagle’s Medium (DMEM), Dimethyl sulfoxide (DMSO), Fetal bovine serum (FBS), Phosphate-buffered saline (PBS), Human Foreskin Fibroblasts (HFF cells), In vitro activation (IVA), In vitro Growth (IVG), In vitro maturation (IVM), Luteinizing hormone (LH), Follicle-stimulating hormone (FSH), Transforming growth factor-beta (TGF-β), Extracellular Signal-Regulated Kinase (ERK), Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B cells (NF-κB), α-smooth muscle actin (α-SMA), Ferric chloride (FeCl₃), Hydrochloric Acid (HCl), Calcium chloride (CaCl₂), Micro siemens per centimeter (µS/cm), Scanning electron microscopy (SEM). Declarations 6.1. Ethical statement Not applicable. 6.2. Consent for publication Written informed consent was obtained from all participating individuals. 6.3. Competing interests The authors declare that they have no competing interests. 6.4. Availability of data and materials The datasets used during the current study are available from the corresponding author upon reasonable request. 6.5. Funding Statement This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors . 6.6. Acknowledgements This work is part of the Ph.D. thesis of Negin Chavoshinezhad at Tabriz University of Medical Sciences. 6.7. Author contribution statement N.CH. and B.NI. conceptualized the study and contributed to writing the original draft, review, and editing. N.CH. prepared the data. B.NI. supervised the study, reviewed and edited the manuscript, and finalized the version for submission. All authors have read and approved the final manuscript. 6.8. Authors' information Negin Chavoshinezhad, Ph.D. Student, Department of Anatomy, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. Email: [email protected] | Tel: +98 918 981 9671 Behrooz Niknafs, Full Professor, Department of Anatomical Sciences, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. Email: [email protected] | Tel: +98 914 316 7148 References Abdi, S. I., Ng, S. M. & Lim, J. O. An enzyme-modulated oxygen-producing micro-system for regenerative therapeutics. Int. J. Pharm. 409 (1–2), 203–205 (2011). Marin, L. et al. Fertility preservation in girls and women: State of art and future possibilities. Clin. Exp. Obstet. Gynecol. 49 (9), 206 (2022). Eppig, J. J. & Schroeder, A. C. Capacity of mouse oocytes from preantral follicles to undergo embryogenesis and development to live young after growth, maturation, and fertilization in vitro. Biol. Reprod. 41 (2), 268–276 (1989). Khunmanee, S. & Park, H. 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14:24:19","extension":"xml","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":134207,"visible":true,"origin":"","legend":"","description":"","filename":"6c62ba28c3f04ead82a3e555f75cad4f1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7638995/v1/5ce7a80d80f4dd2e61a25fc7.xml"},{"id":92873931,"identity":"e315280c-19c8-41b1-9f7e-d19594552f3d","added_by":"auto","created_at":"2025-10-06 14:24:16","extension":"html","order_by":40,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":151383,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7638995/v1/e40a9f002aa564c8d95f8a3d.html"},{"id":92873967,"identity":"52c900c0-ebd2-48ea-a55b-835ff09bf5ba","added_by":"auto","created_at":"2025-10-06 14:24:19","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":158947,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental setup for electrical stimulation of ovarian tissue constructs.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7638995/v1/9f2ca7442707454ad5aaccdc.jpg"},{"id":92873957,"identity":"5e042ba0-b18b-41c3-aad0-e0788fd90ada","added_by":"auto","created_at":"2025-10-06 14:24:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":541742,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM image of control and final Scaffolds,\u003c/strong\u003e \u003cstrong\u003eMagnification of the Fig 2.a: 250x, Fig 2.b: 500x, and Fig 2.c: 2,50kx.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7638995/v1/eb2ccf2f87a3daaeb666c4d6.jpg"},{"id":92873943,"identity":"6d402875-8dab-414c-b22a-30e845bd7aa4","added_by":"auto","created_at":"2025-10-06 14:24:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":132427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytotoxity of HFF cells seeded on PPy[2]/Alg-CMC(Fig.a), PPy[5]/Alg-CMC(Fig.b),and PPy[10]/Alg-CMC(Fig.c) scaffolds after 24, 48 and 72 h by MTT assay.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figue3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7638995/v1/cdcc1577dda411489d5a5882.jpg"},{"id":92875034,"identity":"169b16e2-5b9b-491b-8b15-82741de7ecfa","added_by":"auto","created_at":"2025-10-06 14:32:20","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1267616,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOvarian Tissue Morphological Assessment.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7638995/v1/b74a109891c5ea9efcb218b2.jpg"},{"id":92873971,"identity":"b52a4da1-924e-4498-9a2c-3a845228667f","added_by":"auto","created_at":"2025-10-06 14:24:20","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":697465,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of Ki-67 in control and ES groups.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7638995/v1/f9a344f097289a0bdc5bfe1c.jpg"},{"id":92873927,"identity":"4d2de6c6-38e2-44b2-8ecb-fefc9d133192","added_by":"auto","created_at":"2025-10-06 14:24:14","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":536654,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of P53 in control and experimental groups.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7638995/v1/a298346282cd67a2d3c1d00c.jpg"},{"id":92873963,"identity":"6356b94e-c9c1-4fae-8352-d85b2a7f4529","added_by":"auto","created_at":"2025-10-06 14:24:19","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":781817,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of α-SMA in control and experimental groups.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7638995/v1/39e28e8a72b1d615f166eedf.jpg"},{"id":100357585,"identity":"2c3426f1-f563-4875-ab8b-ce2efa305e8d","added_by":"auto","created_at":"2026-01-16 07:20:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5477644,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7638995/v1/a61f40f9-96a0-4a4c-bf9f-646f4282c3aa.pdf"},{"id":92873956,"identity":"901c951f-4561-4635-a3ea-8b7d89d4d0c8","added_by":"auto","created_at":"2025-10-06 14:24:18","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":14247,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7638995/v1/0f5e71b2e45e72327ad04ca9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"3D Culture of Human Ovarian Tissue on Conductive Hybrid Scaffolds with and without Electrical Stimulation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn mammalian ovaries, folliculogenesis is a tightly regulated process that provides a continuous supply of developmentally competent oocytes throughout a woman's reproductive lifespan(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). In vitro follicle growth (IVG) offers transformative potential for assisted reproductive technologies (ART), enabling the generation of mature, fertilizable oocytes from immature follicles. This approach is especially valuable for fertility preservation in prepubertal girls and women undergoing gonadotoxic cancer treatments, where traditional options like oocyte or embryo cryopreservation are not viable (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite milestones such as the first live births from in vitro-grown oocytes (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), early in vitro culture methods relied on two-dimensional (2D) platforms that failed to preserve the native three-dimensional architecture of ovarian follicles. In 2D systems, the loss of spatial structure disrupts essential bidirectional communication between the oocyte and surrounding granulosa cells, ultimately impairing oocyte maturation and developmental competence(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). To overcome these limitations, current strategies have shifted toward three-dimensional (3D) culture systems that more closely mimic the natural ovarian microenvironment. Among these, culturing intact ovarian cortical fragments stands out as a promising method, as it maintains stromal context, extracellular matrix (ECM) integrity, and natural tissue architecture\u0026mdash;all critical for follicle survival, activation, and coordinated development. Preserving this structural and biochemical niche is essential for maintaining the follicular unit and facilitating physiological signaling during in vitro culture (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite recent advances, most modern systems still lack key physiological cues present in vivo, such as bioelectrical signals. Emerging evidence indicates that endogenous electric fields play vital roles in tissue morphogenesis, cell proliferation, and extracellular matrix remodeling (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). In regenerative medicine, exogenous electrical stimulation has shown therapeutic promise by enhancing cardiac tissue function, neural regeneration, and wound healing (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Conductive biomaterials, such as polypyrrole-reinforced hydrogels, offer a powerful way to integrate bioelectrical cues into 3D culture platforms. When combined with natural polymers like alginate and carboxymethyl cellulose, these materials form hybrid scaffolds that are biocompatible, mechanically tunable, highly porous, and electrically conductive (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study, we introduce a novel conductive 3D culture system designed to preserve and enhance ovarian tissue structure during in vitro culture. By integrating a PPy/Alg-CMC hybrid scaffold with daily electrical stimulation (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), our goal is to support the in-situ growth of human ovarian follicles within intact cortical fragments. This approach not only maintains the ovarian tissue's natural 3D architecture but also leverages bioelectrical cues to promote granulosa and stromal cell proliferation (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur central hypothesis is that preserving ovarian tissue structure through the use of a conductive hybrid scaffold and electrical stimulation creates a biomimetic niche that supports follicle survival, growth, and progression to later developmental stages. This integrated strategy represents a significant advancement in ovarian tissue engineering and offers a promising platform for next-generation fertility preservation, particularly for patients at risk of premature ovarian insufficiency due to cancer or other pathologies.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003eThis study was designed as a controlled in vitro experiment to assess the effects of electrical stimulation on ovarian tissue integrity and follicular development during 3D culture. All methods in this study were carried out in accordance with relevant guidelines and regulations. Experimental protocols were reviewed and approved by the Ethics Committee of Tabriz University of Medical Sciences (Approval ID: IR.TBZMED.REC.1402.200).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Preparation of the Conductive (PPy/ Alg-CMC) Scaffold\u003c/h2\u003e\u003cp\u003eConductive hybrid hydrogel scaffolds were fabricated from alginate, carboxymethyl cellulose, and polypyrrole using in situ oxidative polymerization and ionic cross-linking. All reagents were analytical grade (Sigma-Aldrich) and used without further purification (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBriefly, a 2% (w/v) Alg solution was prepared by dissolving alginate in deionized water with constant magnetic stirring at room temperature for 6 hours until fully homogeneous. CMC was dissolved in a 0.5% (v/v) citric acid\u0026ndash;acetic acid buffer (pH\u0026thinsp;\u0026asymp;\u0026thinsp;4.5) to yield a 2% (w/v) solution, which was stirred for 24 hours at room temperature to form a clear, viscous mixture. Equal volumes of the Alg and CMC solutions were blended thoroughly for 1 hour at 60\u0026ndash;70\u0026deg;C to create a homogeneous Alg-CMC blend (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). To impart electrical conductivity, pyrrole monomer was incorporated via in situ polymerization. A 0.2 M pyrrole solution in 1 N HCl was added dropwise to the Alg-CMC matrix at three volume ratios: 2:10, 5:10, and 10:10 (PPy: Alg-CMC) to evaluate the effects of polypyrrole content on scaffold properties. The mixture was stirred at 90\u0026deg;C for 30 minutes to ensure uniform dispersion. Polymerization was initiated by slowly adding 0.2 M ferric chloride (FeCl₃) in 1 N HCl as an oxidant. The reaction proceeded under constant stirring at 90\u0026deg;C for 5 hours, during which the solution turned from brown to deep black, indicating successful PPy formation and integration (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo form scaffolds, the conductive PPy/Alg-CMC solution was dispensed into sterile 6-well plates (\u0026sim;1 mL per well), creating circular scaffolds approximately 4 mm in diameter and 2 mm thick. The droplets were immediately immersed in 0.1 M calcium chloride (CaCl₂) to induce ionic cross-linking via the \"egg-box\" model, where Ca\u0026sup2;⁺ ions cross-link alginate's guluronic acid blocks, leading to rapid gelation and stable 3D porous structures. Cross-linking occurred for 60 minutes at room temperature (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). After cross-linking, the CaCl₂ solution was discarded, and scaffolds were rinsed three times with sterile phosphate-buffered saline (PBS) to remove residual ions. For sterilization, scaffolds were immersed in 70% ethanol for 30 minutes, followed by UV irradiation (15 minutes per side) in a biosafety cabinet. They were then washed thoroughly with sterile PBS to remove ethanol residues and equilibrated in culture medium before tissue seeding(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Morphological Analysis of the Scaffold\u003c/h2\u003e\u003cp\u003eThe surface morphology, pore structure, and 3D architecture of the conductive PPy/Alg-CMC hybrid scaffolds were examined using scanning electron microscopy (SEM). Pure alginate scaffolds were prepared and analyzed under identical conditions as non-conductive controls for comparison.\u003c/p\u003e\u003cp\u003eScaffold samples were frozen at \u0026minus;\u0026thinsp;80\u0026deg;C for 4 hours and lyophilized for 24 hours to preserve the porous microstructure. Dried samples were mounted on aluminum stubs and sputter-coated with a 10\u0026ndash;15 nm layer of gold/palladium (Au/Pd) for conductivity (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSEM imaging was conducted with a scanning electron microscope (e.g., Hitachi SU5000) at an accelerating voltage of 5\u0026ndash;15 kV. Images were acquired at various magnifications to evaluate pore size, interconnectivity, wall morphology, and overall architecture (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePore diameter was quantified using ImageJ software, measuring at least 50 pores randomly from multiple fields per sample. Results were averaged across three independent scaffolds per formulation(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). This comparison highlighted how incorporating polypyrrole and carboxymethyl cellulose affected the microstructural properties of the alginate matrix.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Biodegrading Assessment of The Scaffold\u003c/h2\u003e\u003cp\u003eTo evaluate structural stability, scaffold samples were incubated in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM) at 37\u0026deg;C with gentle shaking at 100 rpm in a sealed container for 15 days. At predefined intervals (days 0, 3, 5, 10, and 15), scaffolds were removed, gently rinsed, and examined under an optical microscope for surface changes. This assessment was performed on Alg, Alg/CMC, and PPy/Alg-CMC composite variants to compare degradation and durability (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Evaluation of In Vitro Cytotoxicity of The Scaffolds\u003c/h2\u003e\u003cp\u003eCytotoxicity of scaffold extracts was assessed using an MTT assay per ISO 10993-5 standards (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Human Foreskin Fibroblasts (HFF cells), were purchased from the Cell Bank of the Immunology Research Center, Tabriz University of Medical Sciences.\u003c/p\u003e\u003cp\u003eHFF cells were seeded in 96-well plates at 1 \u0026times; 10⁴ cells/well in RPMI medium with 10% fetal bovine serum (FBS), serving as the negative control. Cells were exposed to extracts from PPy[2]/Alg\u0026thinsp;+\u0026thinsp;CMC, PPy[5]/Alg\u0026thinsp;+\u0026thinsp;CMC, and PPy[10]/Alg\u0026thinsp;+\u0026thinsp;CMC scaffolds. After incubation at 37\u0026deg;C in a humidified 5% CO₂ atmosphere for specified periods, the medium was removed, and cells were washed three times with PBS. Then, 100 \u0026micro;L of MTT solution (5 mg/mL in PBS) was added per well, followed by 4 hours of incubation for formazan crystal formation. The supernatant was aspirated, and 500 \u0026micro;L of dimethyl sulfoxide (DMSO) was added to dissolve the crystals. Absorbance was measured at 570 nm using a BioTek ELx800 Micro ELISA reader (Shoreline, WA, USA). Cell viability was calculated as a percentage relative to the negative control to determine scaffold biocompatibility(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Electrical Conductivity Measurement of The Scaffold\u003c/h2\u003e\u003cp\u003eElectrical conductivity of the PPy/Alg-CMC hybrid scaffolds was measured at room temperature (25\u0026deg;C) using a Mettler-Toledo SevenGo Duo SG23 portable conductivity meter (Mettler-Toledo AG, Switzerland), with a range of 0.10 \u0026micro;S/cm to 500 mS/cm. The device included an LC310 conductivity cell, automatic temperature compensation (ATC), and Good Laboratory Practice (GLP) data logging for accuracy and reproducibility (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eScaffolds were equilibrated in PBS for 24 hours to mimic physiological conditions and ensure stable probe contact. The conductive surface was placed in direct contact with the probe, and readings were taken after signal stabilization (30\u0026ndash;60 seconds). For each formulation (PPy/Alg-CMC ratios of 2:10, 5:10, and 10:10), three independent scaffolds were tested in triplicate. Average conductivity was reported in microsiemens per centimeter (\u0026micro;S/cm). Pure Alg and CMC scaffolds (without PPy) served as non-conductive controls to quantify PPy's contribution to conductivity(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Electrical Stimulation (ES) Setup\u003c/h2\u003e\u003cp\u003eTo examine the effects of electrical stimulation on ovarian tissue constructs, a custom system was used. Silver wire electrodes (Ag) were positioned in each well of a 6-well plate on opposite sides of the scaffold, with an inter-electrode distance of \u0026sim;4\u0026ndash;6 mm. Electrodes were connected via insulated wires and alligator clips to a programmable pulse generator delivering unipolar square-wave pulses at 100 mV amplitude, 1 Hz frequency, and 1 ms pulse width. Stimulation was applied daily for 1 hour over 15 days, creating an electric field of \u0026sim;17\u0026ndash;25 mV/mm across the scaffold. This low-intensity regimen was chosen to influence cell behavior without causing electrochemical artifacts (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Study Groups\u003c/h2\u003e\u003cp\u003eThe experimental groups were as follows:\u003c/p\u003e\u003cp\u003eGroup 1 (Control): Ovarian tissue cultured on conductive scaffold without electrical stimulation.\u003c/p\u003e\u003cp\u003eGroup 2 (Electrical Stimulation): Ovarian tissue cultured on conductive scaffold with daily electrical stimulation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Ovarian Tissue Collection and Processing\u003c/h2\u003e\u003cp\u003eHuman ovarian cortical tissue was collected from five women (mean age 33.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8 years, range 20\u0026ndash;35) undergoing laparoscopy for benign gynecological conditions at Al-Zahra Hospital, Tabriz University of Medical Sciences, with prior written informed consent. All procedures were conducted in accordance with approved protocols and the guidelines of the Ethics Committee (Approval ID: IR.TBZMED.REC.1402.200).\u003c/p\u003e\u003cp\u003eImmediately post-surgery, tissue fragments were transported on ice in sterile PBS supplemented with antibiotics (100 U/mL penicillin and 100 \u0026micro;g/mL streptomycin). In the laboratory, tissues were rinsed three times with cold PBS to remove blood, fat, and debris (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).Under sterile conditions, the cortex was dissected using fine forceps and microscissors. To maximize follicle exposure to nutrients and signals while promoting primordial follicle activation and growth, tissues were cut into uniform 1\u0026ndash;2 mm\u0026sup3; pieces with a sterile scalpel. These fragments were then placed on conductive scaffolds, ensuring good contact for 3D culture and potential electrical interactions (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Culture Conditions and Medium Preparation\u003c/h2\u003e\u003cp\u003eThe base medium was alpha-Minimal Essential Medium (α-MEM) supplemented with 10% FBS, 2 mM sodium pyruvate, 50 \u0026micro;g/mL ascorbic acid, 50 mg/L streptomycin sulfate, and 75 mg/L penicillin. To promote follicular development, 75 mIU/mL follicle-stimulating hormone (FSH) and 75 mIU/mL luteinizing hormone (LH) were added(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). A volume of 2 mL of the complete culture medium was added to each scaffold to ensure complete coverage and even distribution over the tissue fragments. The medium was replaced every other day to maintain nutrient availability and remove metabolic waste. Cultures were maintained at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂ for a total duration of 15 days(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Histological Evaluation of Stroma and Follicular Architecture\u003c/h2\u003e\u003cp\u003eAfter 15 days, tissue constructs were gently removed from scaffolds and fixed in 4% (w/v) paraformaldehyde at 4\u0026deg;C for 12\u0026ndash;16 hours to preserve cellular and ECM morphology. Samples were dehydrated through graded ethanol (50%, 70%, 80%, 95%, 100%), cleared in xylene, and embedded in paraffin using standard protocols. Serial 5 \u0026micro;m sections were cut on a rotary microtome and mounted on charged slides (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Sections were deparaffinized in xylene, rehydrated through descending ethanol grades, stained with hematoxylin for 5\u0026ndash;7 minutes to visualize nuclei, and differentiated in acid alcohol. Cytoplasm and ECM were counterstained with eosin for 2\u0026ndash;3 minutes. After dehydration and clearing, slides were coverslipped with permanent mounting medium(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Stained sections were examined under a brightfield microscope equipped with a digital camera (e.g., Olympus BX53 or Zeiss Axio Imager A2). High-resolution images were captured at 100\u0026times; and 400\u0026times; magnifications under consistent illumination. Stromal structure and follicle development were qualitatively assessed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.12. Immunohistochemical analysis of Ki-67, P53, and α-SMA Expression\u003c/h2\u003e\u003cp\u003eImmunohistochemistry was used to assess cellular proliferation (Ki-67), apoptosis (p53), and stromal activation/vascular stabilization (α-SMA). Paraffin- or OCT-embedded sections (5 \u0026micro;m) from paraformaldehyde-fixed samples underwent antigen retrieval in citrate buffer (pH 6.0) with heat. Non-specific binding was blocked with 5% BSA or normal serum. Sections were incubated overnight at 4\u0026deg;C with primary antibodies: Ki-67 (1:100\u0026ndash;1:200), p53 (1:100), or α-SMA (1:200). Detection involved HRP-conjugated secondary antibodies with DAB for chromogenic visualization or fluorescent secondaries with DAPI counterstaining for fluorescence.\u003c/p\u003e\u003cp\u003eStaining was evaluated microscopically and scored semi-quantitatively by intensity and positive cell percentage: negative (\u0026ndash;) for no staining, weak (+) for faint staining in \u0026lt;\u0026thinsp;30% of cells, moderate (++) for visible staining in 30\u0026ndash;70% of cells, and strong (+++) for intense staining in \u0026gt;\u0026thinsp;70% of cells (\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Scores were assigned for each marker in relevant compartments (granulosa cells, stroma), with discrepancies resolved by consensus.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eThe porosity, biodegradability, cytocompatibility, and electrical conductivity of alginate-based scaffolds incorporating carboxymethyl cellulose and polypyrrole were systematically evaluated to confirm their suitability for 3D ovarian tissue culture.\u003c/p\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Porosity of The Scaffold\u003c/h2\u003e\u003cp\u003eSEM analysis revealed that control alginate scaffolds exhibited a highly porous, interconnected 3D network with pore diameters of 50\u0026ndash;150 \u0026micro;m, ideal for cell infiltration, nutrient diffusion, and oxygen transport in 3D cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.a).\u003c/p\u003e\u003cp\u003eIncorporating PPy and CMC slightly reduced overall porosity but preserved interconnectivity, which is crucial for uniform nutrient and oxygen distribution. Pore sizes became more uniform and smaller (25\u0026ndash;50 \u0026micro;m), attributable to partial pore occlusion during PPy polymerization, increased polymer density, and CMC-induced chain entanglement (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.b). This uniformity likely enhances mechanical stability and provides a consistent environment for follicular encapsulation and development.\u003c/p\u003e\u003cp\u003eNotably, pore size and porosity did not vary significantly across PPy concentrations (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, and \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), indicating that PPy loading and polymerization do not substantially alter scaffold microarchitecture. Thus, PPy content can be adjusted to optimize conductivity without compromising porosity. Overall, these scaffolds maintain sufficient porosity for mass transfer and cell penetration while offering improved stability for long-term ovarian tissue or follicle culture (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.a\u0026ndash;c).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Biodegradation of The Scaffolds\u003c/h2\u003e\u003cp\u003eDegradation was concentration-dependent upon PPy incorporation into the Alg-CMC matrix: PPy[2]-Alg/CMC [1] scaffolds showed excellent structural integrity, with no cracks, minimal erosion, and uniform morphology throughout the culture period, reflecting optimal compatibility and controlled degradation.\u003c/p\u003e\u003cp\u003ePPy[5]-Alg/CMC [1] scaffolds displayed moderate changes, such as slight surface roughness and early matrix weakening, suggesting emerging phase inhomogeneity.\u003c/p\u003e\u003cp\u003ePPy[10]-Alg/CMC [1] scaffolds underwent severe degradation, including visible cracks, brittleness, and PPy aggregation, likely due to poor interfacial adhesion between hydrophobic PPy and the hydrophilic matrix, accelerating breakdown.\u003c/p\u003e\u003cp\u003eOverall, CMC enhances alginate stability, but high PPy compromises integrity. The PPy[2]-Alg/CMC [1] formulation achieves the best balance of durability and controlled degradation, making it ideal for extended 3D ovarian follicle culture.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Scaffolds' Cytotoxicity and Biocompatibility\u003c/h2\u003e\u003cp\u003eTo confirm safety and suitability for ovarian tissue culture, cytotoxicity and biocompatibility of Alg-CMC-PPy scaffolds were evaluated. HFF cell viability and metabolic activity were measured via MTT assay after 72 hours at 37\u0026deg;C in 5% CO₂.\u003c/p\u003e\u003cp\u003ePPy [2]-Alg/CMC [1] scaffolds (Fig. a), exhibited excellent biocompatibility, with viability\u0026thinsp;\u0026gt;\u0026thinsp;100% of control, indicating robust proliferation and favorable cell-scaffold interactions. Viability was \u0026sim;97% for PPy [5]-Alg/CMC [1], (Fig. b) and \u0026sim;95% for PPy [10]-Alg/CMC [1] (Fig. c), showing a slight decline with increasing PPy.\u003c/p\u003e\u003cp\u003eThis minor reduction at higher PPy levels may stem from polymer aggregation, reduced hydrophilicity, or trace reactive species release during PPy synthesis. However, all formulations demonstrated strong biocompatibility, confirming that PPy integration into Alg-CMC does not induce significant cytotoxicity, even at higher concentrations.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Scaffolds' Electrical Conductivity\u003c/h2\u003e\u003cp\u003eControl scaffolds of alginate or Alg-CMC (without PPy) displayed very low conductivity (\u0026lt;\u0026thinsp;10 \u0026micro;S/cm or \u0026lt;\u0026thinsp;1 \u0026times; 10⁻\u0026sup2; S/m), consistent with their insulating nature. While structurally sound and biocompatible, these lack the ability to transmit electrical signals.\u003c/p\u003e\u003cp\u003ePPy incorporation dramatically enhanced conductivity in a dose-dependent manner, enabling electroactive 3D environments:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003ePPy[2]-Alg/CMC[1]: 1.33 \u0026times; 10⁻⁴ S/cm\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003ePPy[5]-Alg/CMC[1]: 1.25 \u0026times; 10⁻\u0026sup3; S/cm\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003ePPy[10]-Alg/CMC[1]: Higher values, reflecting over 1,300-fold increase vs. controls, due to PPy forming efficient conductive networks and percolation pathways.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eAlthough PPy[10] offered superior conductivity, PPy[2]/Alg-CMC[1] was selected as optimal, balancing conductivity with structural stability, uniform morphology, and controlled degradation (as per biodegradation results in Section \u003cspan refid=\"Sec16\" class=\"InternalRef\"\u003e3.2\u003c/span\u003e). Higher PPy formulations showed phase separation, cracking, and rapid degradation, potentially unsuitable for long-term follicle culture.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Analysis of Ovarian Tissue Histologically and Morphologically\u003c/h2\u003e\u003cp\u003eAt day 0, ovarian tissue primarily contained primordial and early primary follicles, featuring a central oocyte surrounded by a single layer of flattened granulosa cells and no theca layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.a).\u003c/p\u003e\u003cp\u003eAfter 15 days of 3D culture, marked morphological advancement occurred in the electrical stimulation group. Follicles progressed to preantral and antral stages, with multiple layers of cuboidal to columnar granulosa cells, prominent mitotic figures, and intact oocytes lacking degenerative signs, indicating robust growth and oocyte viability under ES (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.b)(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe stromal compartment also showed improved organization, with denser, follicle-oriented stromal cells suggesting enhanced cell-matrix interactions and microenvironmental support. Importantly, no histological evidence of damage appeared in the stroma, including vacuolization, pyknotic nuclei, or perinuclear halos\u0026mdash;markers of stress, degeneration, or apoptosis. This absence supports the scaffold's biocompatibility, structural integrity, and ES safety (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.b).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Evaluation of α-SMA, p53, and Ki-67 Expression in Response to Electrical Stimulation\u003c/h2\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e3.5.1 Expression of the Cell Proliferation Marker, Ki-67\u003c/h2\u003e\u003cp\u003e\u003cb\u003eControl Group\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eWithout electrical stimulation, Ki-67 expression was minimal, with few positive cells in granulosa and oocyte nucleus, indicating limited mitotic activity during baseline 3D culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.a).\u003c/p\u003e\u003cp\u003eElectrical Stimulation Group: By day 15, the ES group exhibited a marked increase in Ki-67-positive cells (+++), particularly in granulosa cells and theca layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.b) and surrounding stroma (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.c), with enhanced nuclear staining reflecting heightened proliferative activity compared to controls.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\u003ch2\u003e3.5.2. P53 Expression (Apoptosis-Associated Marker)\u003c/h2\u003e\u003cp\u003eP53 expression was negligible or absent under baseline conditions, suggesting minimal cellular stress or apoptotic activation initially (Fig.\u0026nbsp;6.a). After 15 days of ES, no significant increase in p53 occurred compared to controls. Staining remained low-intensity and sparse, with no nuclear accumulation in granulosa, theca, or stromal cells (Fig.\u0026nbsp;6.a,6. b).\u003c/p\u003e\u003cp\u003e. \u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003e3.5.3 α-SMA Expression (Vascular Stability and Stromal Integrity Marker)\u003c/h2\u003e\u003cp\u003eα-SMA expression was low in stroma, mainly in sparse fibroblasts and perivascular cells around follicles, consistent with baseline ovarian homeostasis and physiological vascular support (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e.a).\u003c/p\u003e\u003cp\u003eBy day 15, α-SMA expression increased substantially (+++), especially in stroma and perivascular cells surrounding follicles, with more organized staining and cellular alignment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e.b).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur study developed a novel conductive 3D scaffold integrated with electrical stimulation to enhance stromal organization, vascular stabilization, and in situ follicular development. Histological analyses confirm that 3D culture with ES and hormonal supplementation promotes key biological processes, preserving ovarian tissue architecture and creating an optimal niche for follicle growth without inducing stromal damage or cellular stress. In reproductive bioengineering, 3D culture systems provide a more physiologically relevant environment for in vitro follicle growth than traditional 2D methods(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). A common approach involves encapsulating isolated secondary follicles in natural or synthetic hydrogels, particularly alginate, which maintains 3D structure and supports bidirectional oocyte-granulosa cell communication, enabling progression to the antral stage(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eXu et al. demonstrated the potential of this method, showing that murine secondary follicles (150\u0026ndash;180 \u0026micro;m) cultured in alginate for 8 days yielded oocytes with high developmental competence: 71% reached metaphase II post-IVM, 68% fertilized, and produced live offspring (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Similarly, Telfer et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) and Amorim et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), reported successful in vitro maturation of human and animal follicles in 3D systems, highlighting their promise for fertility preservation.\u003c/p\u003e\u003cp\u003eHowever, isolating individual follicles disrupts the native ovarian environment, eliminating essential stromal interactions, vascular cues, and paracrine signaling critical for folliculogenesis (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). To address this, 3D culture of ovarian cortical fragments has gained traction, as it preserves tissue architecture, ECM integrity, and inter-follicular communication, leading to improved follicle survival and activation while mimicking the in vivo niche(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). For example, alginate-encapsulated human ovarian tissue maintained\u0026thinsp;\u0026gt;\u0026thinsp;50% structural integrity and supported multilayer follicle formation(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Porous alginate scaffolds also enhanced follicle viability and reduced apoptosis, underscoring the value of engineered hydrogels(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eNevertheless, conventional hydrogels like fully cross-linked alginate pose challenges due to high stiffness, which can restrict stromal proliferation and nutrient diffusion, potentially hindering follicle development(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Pais et al. emphasized the importance of mechanical compliance, showing that softer gels (0.25\u0026ndash;0.7% w/v) promote diffusion, steroidogenesis, and follicular expansion, while rigid matrices suppress stromal proliferation(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). To mitigate rigidity without losing support, strategies include blending alginate with biocompatible polymers like collagen, fibrin, or carboxymethyl cellulose to improve flexibility, porosity, and cell-matrix interactions (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e),or adjusting alginate concentration and cross-linking density to better match native ovarian tissue softness (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWhile these hybrid scaffolds improve mechanics, they remain passive and unable to deliver dynamic cues like bioelectrical signals, increasingly recognized as key regulators of cellular behavior, tissue organization, and regeneration. Native ovarian tissue, like other biological tissues, exhibits intrinsic electrical activity influencing proliferation, differentiation, and communication. Traditional hydrogels lack electroactive properties, limiting their ability to replicate the in vivo environment fully.Interest in conductive polymers (CPs) has grown since their discovery in the late 1970s(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). These materials combine semiconductor-like electrical and optical properties with polymer-like mechanical and processing advantages. Among CPs, polypyrrole (PPy) stands out for its high conductivity and electrochemical stability(\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). We engineered a hybrid conductive scaffold by embedding PPy in an Alg-CMC matrix(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e), to provide daily bioelectrical stimulation (100 mV, 1 h/day) during culture. This voltage (100\u0026ndash;200 mV), validated in cardiac, skeletal, and neural systems, safely enhances proliferation, differentiation, regeneration, and matrix synthesis(\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). For instance, PPy-mediated 100 mV stimulation promotes fibroblast-to-myofibroblast transition via the TGF-β1/ERK/NF-κB pathway, sustaining an activated phenotype for wound healing(\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Falling within physiological ranges, this low voltage supplies dynamic cues while avoiding damage from pH changes, electrode corrosion, or toxic byproducts (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e), making it suitable for ovarian tissue.\u003c/p\u003e\u003cp\u003eMechanistically, ES likely activates parallel pathways like PI3K/Akt, ERK/MAPK, Ca\u0026sup2;⁺/CaMKII, and TGF-β/SMAD, regulating survival, proliferation, cytoskeletal organization, and ECM synthesis(\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). ES may also enhance localized ECM deposition and cross-linking\u0026mdash;possibly via enzymes like lysyl oxidase\u0026mdash;modulating scaffold biomechanics to support follicle development (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePulsed electric fields, for example, double collagen and elastin production in fibroblasts without elevating matrix metalloproteinase (MMP) activity, indicating balanced ECM remodeling(\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). Our findings align, showing increased fibroblast deposition without fibrosis, suggesting constructive remodeling(\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Although not directly assessed here, these mechanisms may underlie the enhanced intercellular communication in our cultures. We observed that ES significantly boosted Ki-67 expression\u0026mdash;a marker of active cell cycle phases (G1, S, G2, M)\u0026mdash;in follicular and stromal compartments (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). This pro-proliferative effect, likely mediated by pathways like ERK1/2 (MAPK) driving G1/S transition (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). ), suggests ES promotes cell cycle entry, aiding stromal regeneration and follicular growth. Beyond proliferation, ES guides differentiation and patterning, acting as morphogenic signals for coordinated growth and organization (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). In our system, ES elevated α-SMA expression, indicating stromal fibroblast proliferation and myofibroblast differentiation, which supports constructive remodeling (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e). These α-SMA⁺ cells likely aid vascular stabilization, follicular integrity, and ECM organization\u0026mdash;key for a functional perifollicular niche. Similarly, pulsed electrical stimulation (PES) upregulated α-SMA in human dermal fibroblasts via Smad2/3 phosphorylation, confirming ES activates the TGF-β/Smad axis to drive myofibroblast conversion and contractility (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHistological data further show that ES reinforces stromal health and regenerative capacity through mature microvasculature establishment. Stromal activation aligns with preantral follicle development, maintaining architecture without apoptosis or fibrosis. Thus, ES-driven remodeling is supportive, fostering a physiologically active, mechanically robust microenvironment for follicle growth and function.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eOur findings demonstrate that a conductive PPy/Alg-CMC scaffold combined with electrical stimulation elicits a regulated, regenerative stromal response mirroring key aspect of the native ovarian microenvironment. By enhancing stromal organization, vascularization, and perifollicular support without inducing fibrosis or stress, this approach creates a biomimetic niche that advances follicular development to the preantral stage. These results highlight the promise of electroactive biomaterials in ovarian tissue engineering, offering a robust strategy to improve in vitro follicle growth and fertility preservation solutions.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAssisted reproductive technology (ART), In vitro fertilization (IVF), Automatic temperature compensation (ATC), Good Laboratory Practice (GLP), Two-dimensional culture (2D), Three-dimensional culture (3D), Extracellular matrix (ECM), Matrix metalloproteinase (MMP), Electrical stimulation (ES), Conductive polymers (CPs), Polypyrrole (PPy), Carboxymethyl cellulose (CMC), Alginate (Alg), alpha-Minimal Essential Medium (α-MEM), Dulbecco’s Modified Eagle’s Medium (DMEM), Dimethyl sulfoxide (DMSO), Fetal bovine serum (FBS), Phosphate-buffered saline (PBS), Human Foreskin Fibroblasts (HFF cells), In vitro activation (IVA), In vitro Growth (IVG), In vitro maturation (IVM), Luteinizing hormone (LH), Follicle-stimulating hormone (FSH), Transforming growth factor-beta (TGF-β), Extracellular Signal-Regulated Kinase (ERK), Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B cells (NF-κB), α-smooth muscle actin (α-SMA), Ferric chloride (FeCl₃), Hydrochloric Acid (HCl), Calcium chloride (CaCl₂), Micro siemens per centimeter (µS/cm), Scanning electron microscopy (SEM).\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e6.1. Ethical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.2. Consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from all participating individuals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.3. Competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.4. Availability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.5. Funding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.6. Acknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is part of the Ph.D. thesis of Negin Chavoshinezhad at Tabriz University of Medical Sciences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.7. Author contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN.CH. and B.NI. conceptualized the study and contributed to writing the original draft, review, and editing. N.CH. prepared the data. B.NI. supervised the study, reviewed and edited the manuscript, and finalized the version for submission. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.8. Authors' information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNegin Chavoshinezhad, Ph.D. Student, Department of Anatomy, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.\u003c/p\u003e\n\u003cp\u003eEmail: [email protected] | Tel: +98 918 981 9671\u003c/p\u003e\n\u003cp\u003eBehrooz Niknafs, Full Professor, Department of Anatomical Sciences, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.\u003c/p\u003e\n\u003cp\u003eEmail: [email protected] | Tel: +98 914 316 7148\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdi, S. I., Ng, S. M. \u0026amp; Lim, J. O. An enzyme-modulated oxygen-producing micro-system for regenerative therapeutics. \u003cem\u003eInt. J. Pharm.\u003c/em\u003e \u003cb\u003e409\u003c/b\u003e (1\u0026ndash;2), 203\u0026ndash;205 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarin, L. et al. 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Med.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e (4), 1110\u0026ndash;1121 (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"3D ovarian tissue culture, In-situ follicle culture, Electrical stimulation, Conductive hybrid scaffold","lastPublishedDoi":"10.21203/rs.3.rs-7638995/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7638995/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe survival, development, and hormonal activity of ovarian follicles depend on the ovarian tissue microenvironment, making its preservation critical for effective fertility preservation. To maintain the structural and functional integrity of human ovarian cortical tissue during in vitro culture, we developed a conductive scaffold supplemented with hormones and electrical stimulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConductive hybrid scaffolds were fabricated from alginate (Alg), carboxymethyl cellulose (CMC), and polypyrrole (PPy) using in situ polymerization of pyrrole monomer followed by ionic cross-linking. These scaffolds supported three-dimensional culture of human ovarian cortical tissue. Three formulations (PPy to Alg-CMC ratios of 2:1, 5:1, and 10:1) were evaluated for structural stability. Human ovarian cortical fragments (1 mm × 1 mm × 500 μm) were cultured on the scaffolds for 15 days; one group received daily electrical stimulation (100 mV for 1 h), while the control group did not. Stromal and follicular responses were assessed via immunohistochemical staining for α-smooth muscle actin (α-SMA), Ki-67, and p53 to evaluate ECM remodeling, proliferation, and stress/apoptosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 2:1 PPy/Alg-CMC scaffold exhibited superior performance, with biocompatibility, conductivity, and no cytotoxicity. Under electrical stimulation, stromal, granulosa, and endothelial cells displayed strong activation, including sustained α-SMA expression (indicating a myofibroblast-like phenotype and vascular endothelial growth), elevated Ki-67 levels (indicating enhanced proliferation), and absent p53 expression (confirming no cellular stress or apoptosis).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study demonstrates that a conductive hybrid scaffold (2:1 PPy to Alg-CMC) combined with daily electrical stimulation promotes stromal proliferation, preserves stromal integrity and tissue structure, and supports primordial follicle progression to the preantral and antral stage, suggesting effective maintenance of the follicular niche.\u003c/p\u003e","manuscriptTitle":"3D Culture of Human Ovarian Tissue on Conductive Hybrid Scaffolds with and without Electrical Stimulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-06 14:23:52","doi":"10.21203/rs.3.rs-7638995/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4d9f57a8-5d8e-4587-91ca-9324461755d8","owner":[],"postedDate":"October 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55729982,"name":"Biological sciences/Biotechnology"},{"id":55729983,"name":"Biological sciences/Cell biology"},{"id":55729984,"name":"Physical sciences/Materials science"},{"id":55729985,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-01-09T06:09:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-06 14:23:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7638995","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7638995","identity":"rs-7638995","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}