Lokesh Saravanan

Development and Evaluation of a Bioactive Electrospun PVA/Hydroxyapatite/Eugenol Composite Biomaterial for Dental Pulp Regeneration Lokesh Saravanan1†, Saranya Srinivasan1†, Ashwathi Vijayalekha1, Suresh Kumar Anandasadagopan2, Ashok Kumar Pandurangan1* 1School of Life Sciences, B. S. Abdur Rahman Crescent Institute of Science and Technology Vandalur, Chennai, 600048, India 2Biochemistry and Biotechnology Laboratory, CSIR-Central Leather Research Institute Adyar, Chennai, 600020, India Corresponding author* Mail ID:- Abstract Regenerative endodontics aims to restore the vitality and function of dental pulp through tissue engineering approaches, offering a biological alternative to conventional root canal treatment. This study presents the fabrication and evaluation of electrospun nanofibrous scaffolds composed of Polyvinyl alcohol (PVA) and Hydroxyapatite (HAp), incorporated with Eugenol, a phytochemical known for its antimicrobial and anti-inflammatory properties. Three scaffold variants were developed: a control (PVA/HAp) and two test groups containing Eugenol at 100 µg/mL and 200 µg/mL concentrations. The scaffolds were subjected to physicochemical and biological characterization. FTIR and XRD confirmed the successful integration of components and crystallinity, while SEM revealed improved fiber morphology and uniformity in Eugenol-loaded scaffolds. Thermal stability was enhanced as demonstrated by TGA. Antimicrobial activity assays showed moderate inhibition of Streptococcus mutans and Enterococcus faecalis, with the higher Eugenol concentration showing greater efficacy. In silico studies further supported Eugenol’s therapeutic relevance; network pharmacology identified 10 key genes linked to pulp pathology, and molecular docking demonstrated strong binding affinities to targets such as ACE, PLAU, and MME. These results suggest that the PVA/HAp/Eugenol scaffold not only provides structural support but also exhibits bioactivity conducive to inflammation modulation and pulp tissue regeneration, making it a promising candidate for regenerative endodontic applications. Keywords: Dental pulp regeneration, Electrospinning, Eugenol, PVA/HAp scaffold, Anti-microbial activity, Regenerative endodontics. Abbreviations PVA – Polyvinyl alcohol HAp – Hydroxyapatite FTIR – Fourier Transformed Infrared Spectroscopy XRD – X-ray diffraction SEM – Scanning Electron Microscopy TGA – Thermogravimetric Analysis RCT – Root Canal Therapy ECM – Extracellular matrix PCL – Polycaprolactone nHAp – Nano Hydroxyapatite IDPSCs – Inflamed Dental Pulp Stem Cells DSPP – Dentin sialophosphoprotein DMP-1 – Dentin matrix protein 1 BMPs – Bone morphogenetic proteins NGF – Neural growth factor iPSCs – Induced pluripotent stem cells PPI – Protein-protein interaction 1. Introduction The dental pulp is a richly vascularized and innervated tissue enclosed within the hard dentinal walls. It plays multiple roles, including responding to external stimuli, supplying nutrients, and reducing neuronal sensitivity by promoting pulp repair through mineralization. Worldwide, approximately 3.5 billion people are affected by dental problems as stated in the Global Burden of Disease Study 2019, which considers dental caries of the permanent teeth to be the most widespread ailment. Pulpitis along with pulp necrosis is one of the dental caries terms and is quite popular for causing major trouble, often needs to be treated with Root Canal Therapy (RCT) [1]. While RCT is a standard procedure aimed at relieving pain and removing infected pulp tissue, there is complete loss of the dental pulp leading to a non-vital or a devitalized tooth post-procedure. Dental pulp is significant for the teeth as it keeps them alive as well as involving in the formation of dentin (dentinogenesis), supplying blood, and resists bacterial infections [2]. Vital pulp can be defined as being able to react towards stimuli and take part in modulating healing processes when it is damaged but in case of post-RCT procedure, these abilities are rendered inactive, and unparalleled vital pulp tissue becomes incapable of responding to stimuli [3].The focus on regeneration of dental pulp is revolutionizing and represents a transformative approach in modern endodontics, which aims at not only to treat the infected or defected tooth but also to biologically restore the functions of the pulp-dentin complex. The unparalleled activities of the dental pulp underscore the importance of advanced strategies that can enable proper tissue regeneration within the pulp space by addressing the limitations of current treatment modalities. One of the most promising approaches in this field is the use of bioactive scaffolds, a cornerstone of tissue engineering [4]. Tissue engineering is the interdisciplinary field that involves either of the compounds such as biomaterials, cells or growth factors to regenerate tissues by aiming to repair or replace the damaged tissues in the body. Research have been developed to later stages of fabricating biomaterials in combination with bioactive molecules to restore, repair and regain the functionality of the damaged tissue. This have shown an immense promise in overcoming the challenges that is faced in conventional endodontic therapies [5]. At present, numerous studies are being conducted to assess tissue engineering approaches for regenerating dental pulp. Among them scaffolds play a significant role by providing a three-dimensional (3D) framework that mimics the Extracellular Matrix (ECM), thereby supporting cell adhesion, proliferation, and differentiation. From this list, the novel construction of a nanofibrous scaffold has caught researcher interest as a result of its beneficial features with high surface area to volume ratios and enhanced cell adhesion, proliferation, and differentiation [6]. Electrospinning has also proven to be an exceptionally useful and versatile method for the preparation of nanofibrous scaffolds with structural and morphological characteristics strongly comparable to natural ECM. These nanofibers present high surface area-to-volume ratios and well-controlled mechanical and biochemical properties and are thus a suitable choice for regenerative dental use [7]. By the process of elecrospinning, nanometer to micrometer-diameter fibers with very similar properties to ECM can be obtained and are thus suitable for regenerative use. The improvement in the mechanical properties and biocompatibility is always assured by the bio-ceramics and polymers that make up the biomaterials. Hydroxyapatite (HAp), a calcium apatite mineral form that occurs naturally, is the main inorganic tooth and bone material. Due to its high osteoconductivity, good biocompatibility, and chemical resemblance to hard tissues of human beings, HAp is a widely utilized bio-ceramic as a component in scaffold systems to improve bioactivity as well as mineralization potential [8]. To improve the pulp regenerative prospect of the biomaterial, the integration of bioactive molecules/phytochemicals into the scaffolds is a growing trend to impart multifunctional properties [9], [10]. Eugenol, a phenolic product of clove oil, exhibits a broad spectrum of biological activities such as antimicrobial, anti-inflammatory, antioxidant, and analgesic activity. Eugenol is an ideal choice for dental tissue engineering, especially for pulp tissue regeneration where antibacterial activity along with the potential to modulate inflammation are both necessary [11]. Hydroxyapatite is suggested to be blended with polymers like chitosan, hyaluronic acid, polyvinyl alcohol, polylactic acid, polyoxyethylene, and polyacrylamide, among others to form composites that improve the mechanical properties and functionality of nanofibers for use in tissue engineering applications [12], [13]. Among them, Polyvinyl alcohol (PVA) is a hydrophilic biocompatible synthetic polymer widely employed in biomedical applications as it possesses strong film-forming properties, mechanical flexibility, and non-toxicity [14]. Anjaneyulu et al., (2017) have successfully electrospun Ag doped HAp–polyvinyl alcohol (PVA) composite nanofibers and tested their antibacterial action against Staphylococcus aureus and Escherichia coli, hemocompatibility, and in vitro bioactivity in Simulated Body Fluid (SBF) solution to determine their applicability as bone tissue engineering applications. Salim et al., (2021) have developed electrospun PVA/HA nanofibers with chitosan and HAp and observed increased mechanical stability and antimicrobial action. The results hold the promise to be used in bone tissue engineering. da Costa Sousa et al., (2022) have developed resorbable electrospun PVA/chitosan nanofibers incorporated with ciprofloxacin and the host defense peptide IDR-1002. These nanofibers showed antibiofilm activity, immunomodulatory effects, and biocompatibility, promoting pulp-like tissue formation in vivo. Their multifunctional properties highlight the potential for dental pulp regeneration procedures. Villanueva-Lumbreras et al., (2024) have fabricated electrospun polycaprolactone (PCL) nanofibers incorporating nano-hydroxyapatite (nHAp) and Humulus lupulus (hop) extract. The composite showed good biocompatibility and slight antimicrobial activity, indicating potential use in oral bone tissue regeneration and as a delivery system for natural medicinal agents. However, to the best of our knowledge, no comprehensive study has investigated the fabrication and evaluation of electrospun PVA-HAp scaffolds functionalied with varying concentrations of Eugenol to enhance their structural, thermal, antimicrobial properties for a dental pulp regeneration application. Through this multifaceted analysis, this study seeks to establish the potential of PVA/HAp/Eugenol nanofibrous scaffold as a bioactive and functional platform for future applications in dental pulp regeneration. 2. Materials & Methods Polyvinyl alcohol [(C2H4O)x] was dissolved in a mixture of distilled water and glacial acetic acid; Hydroxyapatite [Ca10(PO4)6(OH)2] was used as a bio-ceramic; Eugenol [C10H12O2] stock was prepared at the concentration of 1mg/10ml using ethanol [C 2H 5OH] as a solvent; Muller Hinton Agar, Agar powder, Nutrient Broth (Sisco Research Laboratories) has been used in Anti-microbial assay. 2.1. In-silico Studies 2.1.1. Network Pharmacology The network pharmacology workflow aimed to identify the top 10 genes associated with both eugenol targets and dental pulp defect pathways. Initially, the SMILES structure of eugenol was retrieved from the PubChem database and utilized to analyze its absorption, distribution, metabolism, and excretion (ADME) properties using the SwissADME database. Subsequently, potential eugenol target proteins were identified through the SuperPred database. To refine these findings, key genes involved in the dental pulp defect pathway were retrieved from the GeneCards database. Common genes associated with both eugenol targets and dental pulp defects were identified using the Interaction Venn database. The Protein-Protein Interaction (PPI) network for these common genes was constructed using the STRING database, considering only high-confidence interactions [19]. Cytoscape 3.10.2 software was used to visualize the protein-protein interaction network, and the CytoHubba plugin was utilized to identify the top 10 hub genes [20]. 2.1.2. Molecular Docking Molecular docking was done to determine the binding interactions between the target proteins found and Eugenol. The 3D structures of the top 10 proteins found, which had previously been determined by network pharmacology analysis, were downloaded in.pdb file format from the Protein Data Bank (PDB) database. The 3D structure of Eugenol, as a ligand, was downloaded in.sdf format from the PubChem database and transformed to.pdb format using Molegro Molecular Viewer. Docking experiments were performed utilizing AutoDock 4.2.6. Protein preparation consisted of removal of the water molecules and other heteroatoms, addition of polar hydrogens, and calculation of the Kollman charges. Ligand minimization was performed in energy by the universal force field (UFF) and then torsion-optimization to define its flexibility. The grid box for all of the proteins was placed at the center over the binding site, and its size was optimized to enclose all of the possible sites of interaction. Docking was done using Lamarckian genetic algorithm (LGA) parameters as default. The docking files generated gave the binding affinities (kcal/mol) and binding conformations. These were further processed for 2D interaction mapping and visualization of docked complexes by Biovia Discovery Studio software, which detected hydrogen bonds, hydrophobic interactions, and other important forces involved in ligand-protein stabilization [21]. 2.2. Electrospun Fabrication Electrospun nanofibrous scaffolds were prepared with PVA, HAp, and Eugenol. The starting solution was prepared by mixing acetic acid and distilled water in the ratio of 8:2, to which PVA was dissolved and standardized to a concentration of 80 mg/mL. They were well mixed to prepare it as homogeneous. HAp was added to a concentration of 10 mg/mL and stirred well until it got dispersed. An individual stock solution of Eugenol was also prepared by dissolving 10 mg of Eugenol in 100 mL of distilled water. Three scaffolds were then prepared: a control scaffold made of PVA and HAp only, and two test samples with Eugenol concentrations of 100 µg/mL and 200 µg/mL, respectively. The prepared suspensions were loaded into a syringe with a 9-mm metallic needle, and electrospinning was performed using optimized parameters—26 kV electrical voltage, a flow rate of 0.8 mL/hour, and a 15 cm distance between the needle tip and the collector. The nanofibers were collected on an aluminum foil-wrapped collector as shown in figure 1 and dried at room temperature overnight. The fabricated scaffolds were stored in a desiccator and subjected to investigate its influence on the structural integrity, thermal stability, surface morphology and the antimicrobial efficacy of the nanofibrous scaffolds. 2.3. Characterization of Bioactive Nanofibrous Scaffolds 2.3.1. Fourier Transformed Infrared Spectroscopy (FTIR) FTIR analysis has been done for the exploration of chemical interaction and functional group of scaffolds being synthesized. In the study used in this case, FTIR analysis involved Jasco Ins FT/IR-6300 model spectrometer. Spectra of wavenumber range from 4000 cm⁻¹ to 400 cm⁻¹ of 4 cm⁻¹ resolution and at the scan rate of 2 mm/sec average was taken using four scans/spectrum. The test specimens PVA/HAp and two of concentrations of 100 µg/mL and 200 µg/mL of Eugenol, respectively, were the target scaffolds. The study centered on determining the loading of Eugenol and the occurrence of significant functional groups specific to PVA, HAp, and Eugenol and any likely molecular interactions between the compounds. 2.3.2. X-ray diffraction (XRD) The structural and crystalline phases of the electrospun nanofibrous scaffolds were evaluated through X-ray diffraction measurement. The measurements were obtained using a Bruker XRD machine possessing 40 kV accelerating voltage, 30 mA current using Cu Kα as an X-ray source. Diffraction patterns were acquired from 2θ range of 5° to 90° with scan speed of 0.1820°/min. The gathered data was then analyzed using BmltoV4Converter software (6.5.0.0). Later the gathered patterns were compared with reference data of International Centre for Diffraction Data (ICDD) database. The analysis made possible to detect HAp phase existence and structural modification upon Eugenol incorporation into the PVA/HAp matrix. 2.3.3. Scanning Electron Microscopy (SEM) The traits of surface morphology and fiber arrangement of the electrospun scaffolds were studied through SEM analysis with an Apreo S field emission scanning electron microscope (FE-SEM). Each sample was assessed individually with the control sample PVA/HAp compared with Eugenol-loaded scaffolds (Test Sample I: 100 µg/mL and Test Sample II: 200 µg/mL) to evaluate the properties. SEM analysis was executed on different magnification levels to assess the fiber arrangement, surface texture, uniformity and electrospun morphology. The images attained from SEM are essential in analyzing the impact of integrating eugenol on fiber morphology, porosity, and potential changes in nanofiber arrangement, which are important for supporting cellular interactions on dental pulp regeneration applications. 2.3.4. Thermogravimetric Analysis (TGA) Thermal and decomposition characteristics of the prepared electrospun scaffolds were characterized by TGA using Perkin Elmer STA 6000 equipment. Samples were first heated between 25°C and 300°C with a heating rate of 50°C/min, and subsequently from 200°C to 800°C with a heating rate of 20°C/min under nitrogen atmosphere. The flow rate of the nitrogen was maintained at 50 mL/min during the analysis for keeping the atmosphere inert. The gauge was standardized by an indium standard for excellent thermal response. 2.4. Antimicrobial Activity Assessment Antimicrobial activity of the synthesized scaffolds was screened against Enterococcus faecalis and Streptococcus mutans, the oral pathogenic bacterial strains; by the agar disc diffusion method. Mueller-Hinton Agar (MHA) was prepared, autoclaved and poured into sterile petri dishes under aseptic conditions. After solidified, each plate was inoculated with the individual bacterial strain using a sterile L-rod to assure lawn culture everywhere on each plate. Each inoculated plate was divided into four equal sections, and the following samples were placed in the respective quadrants: C (Control): PVA/HAp scaffold; T-I (Test I): PVA/HAp/Eugenol (100 µg/mL) scaffold; T-II (Test II): PVA/HAp/Eugenol (200 µg/mL) scaffold; Ab: Standard antibiotic disc (Ampicillin) as positive control. An additional sterile control was kept as a negative control. Then the plates are incubated at 37°C for 24h and each plate was then observed and measured for its zones of inhibition (in mm) around the different samples to evaluate their antibacterial efficiency. 3. Results and Discussion 3.1. In-silico Studies 3.1.1. Network Pharmacology The network pharmacology analysis showed the top ten hub genes linked to Eugenol targets and dental pulp defects: NFKB1, ACE, MME, CCR1, SLC2A1, TACR1, CTSD, PLAU, C5AR1, and MAOA. These genes were identified using Cytoscape (CytoHubba plugin) after combining data from the GeneCards, PubChem, SuperPred, and STRING databases. Venn analysis revealed common genes, demonstrating the relationship between eugenol targets and dental pulp problem pathways. Each identified gene plays an important part in dental pulp defects or regeneration pathways, as well as being a target for Eugenol. Hub Genes Role in Dental Pulp Defect Contributing actions of Eugenol References NFKB1 (Nuclear factor NF- kappa-B p105 subunit) Regulates inflammation and immune responses critical for pulp repair. Modulates inflammatory pathways, enhancing regeneration. [22], [23] ACE (Angiotensin-converting enzyme) Associated with vascular remodeling and angiogenesis, vital for pulp regeneration. Influences vascular processes, improving angiogenesis. [24], [25] MME (Neprilysin) Plays a role in matrix remodeling, essential for pulp healing. Supports structural matrix modifications during regeneration. [26] CCR1 (C-C chemokine receptor type1) Regulates chemotaxis and cellular recruitment during inflammatory responses. Enhances tissue repair by regulating cell migration. [27] SLC2A1 (Glucose transporter) Encodes GLUT1, a transporter involved in glucose uptake, providing energy for regeneration. Supports cellular energy metabolism, boosting regenerative processes. [28], [29] TACR1 (Neurokinin 1 receptor) Associated with nociception and inflammatory regulation. Reduces pain and inflammation through pathway modulation. [30], [31] CTSD (Cathepsin D) A lysosomal enzyme linked to tissue homeostasis and regeneration. Contributes to maintaining tissue balance and repair. [32], [32] PLAU (Urokinase-type plasminogen activator) Implicated in fibrinolysis, aiding tissue repair and remodeling. Facilitates fibrinolysis, accelerating remodeling during healing. [33] C5AR1 (C5a anaphylatoxin chemotactic receptor) Plays a role in immune regulation and inflammation, critical for healing. Improves immune modulation and inflammation management. [34] MAOA (Monoamine oxidase A) Related to cellular oxidative stress management. Reduces oxidative stress, enhancing tissue survival and repair. [35], [36] Table 1 provides a concise summary about the role of the identified genes and their interaction with Eugenol to enhance dental pulp regeneration. Figure 2: (A) The top 10 genes that is associated with eugenol targets and dental pulp defects analyzed with the help of the Cytoscape software (cytohubba plugin) (B) The protein-protein interactions of the top 10 genes obtained using string database Protein-protein interaction (PPI) research with STRING revealed substantial connections between these genes, supporting their interconnected roles in regulating cellular and molecular processes critical for dental pulp repair (figure 2). This functional insight supports Eugenol's potential as a therapeutic drug for these important pathways. 3.1.2. Molecular Docking Molecular docking revealed Eugenol's interactions with key proteins related with dental pulp abnormalities. The findings highlight Eugenol's potential as a bioactive chemical that targets various regeneration-related pathways. The key highlights of our findings with the help of molecular docking study indicates that ACE (-7.97 kcal/mol) exhibited the highest binding affinity, suggesting strong interactions that might support vascular remodeling and angiogenesis. MME (-6.46 kcal/mol) and PLAU (-6.47 kcal/mol) showed strong binding interactions, indicating roles in matrix remodeling and fibrinolysis. Moderate binding was observed with NFKB1 (-5.5 kcal/mol) and TACR1 (-5.91 kcal/mol), highlighting their potential involvement in inflammation and pain modulation. The different kinds of interactions found in the 2 D formation of the docked components were listed in the table 2. Protein (PDB ID) Binding Affinity (kcal/mol) Interaction Highlights ACE (1O8A) -7.97 Hydrogen bonding, alkyl interactions, Pi-sulfur interaction, amide-Pi stacking PLAU (1C5W) -6.47 Salt bridge, sulfur-X, hydrogen bonding and alkyl interactions MME (1R1H) -6.46 Hydrogen bonding, van der Waals forces, Pi-sigma and alkyl interactions TACR1 (6HLP) -5.91 Pi-Anion, Pi-Sigma and alkyl interactions CTSD (6QCB) -5.83 Hydrogen bonding and alkyl interactions NFKB1 (1U36) -5.5 Pi-Pi stacking, hydrogen bonding MAOA (6EZZ) -5.32 Hydrogen bonding, Pi-Sigma and alkyl interaction C5AR1 (6C1R) -5.11 Van der waals interaction, hydrogen bonding and alkyl interactions SLC2A1 (6THA) -4.73 Hydrogen bonding, alkyl interaction and Pi-Pi T-shaped interaction CCR1 (7VL8) -4.7 Hydrogen bonding and alkyl interactions Table 2, provides the binding affinity values and highlights the types of interactions between the ligand and the receptor. Figure 3: The 2D & 3D interaction of Eugenol with the receptors as follows: (A) ACE (B) PLAU (C) MME (D) TACR1 (E) CTSD (F) NFKB1 (G) MAOA (H) C5AR1 (I) SLC2A1 (J) CCR1 These docking results demonstrate Eugenol's capacity to bind efficiently to key proteins, impacting processes such as inflammation, tissue remodeling, and cellular metabolism, all of which are essential for dental pulp regeneration [23]. 3.2 Electrospinned mat of PVA/HAp/Eugenol combination Figure 1: Shows the fabricated electrospun with eugenol as a bioactive compound with the composition of (A) PVA/HAp (B) PVA/HAp/Eugenol 100 µg/mL (C) PVA/HAp/Eugenol 200 µg/mL. 3.3. Physico-chemical Characterizations of the Biomaterials 3.3.1. FTIR Analysis Fourier Transform Infrared Spectroscopy was performed to investigate the presence of functional groups and to assess the molecular interactions between PVA, HAp, and Eugenol in the fabricated nanofibrous scaffold. The FTIR spectra of the control (PVA/HAp) and test groups (PVA/HAp/Eugenol 100 µg/mL and 200 µg/mL) revealed a combination of characteristic peaks corresponding to each of the components, confirming the successful incorporation of Eugenol and HAp within the PVA matrix. The broad absorption band observed around- cm⁻¹ in all three samples corresponds to the O–H stretching vibrations, which can be attributed to the hydroxyl groups present in both PVA and HAp . Notably, a shift and broadening of this peak in the Eugenol-loaded scaffolds (Test 1 and Test 2) indicate the possibility of hydrogen bonding interactions between Eugenol and the polymeric matrix. A distinct peak at ~2910 cm⁻¹ was attributed to C–H stretching vibrations from the methylene groups in the PVA backbone. The C–O stretching vibrations appeared in the region of- cm⁻¹, indicating the presence of alcohol functional groups, which are characteristic of PVA. The presence of Eugenol in the test scaffolds was further supported by a moderate peak observed at ~1642 cm⁻¹, which corresponds to aromatic C=C stretching associated with the phenyl ring of Eugenol. This peak was more elevated in the Test 2 scaffold, steady with the higher Eugenol concentration. Additionally, the phosphate-related vibrational bands of HAp were evident at ~1022 cm⁻¹ (P–O stretching) and 560 cm⁻¹ (O–P–O bending), confirming the presence and structural integrity of HAp in all scaffold types (figure 4) [15]. The tandem spectral signatures prove that all three constituents, i.e., PVA, HAp and Eugenol were successfully incorporated into the matrix of scaffolds without showing any detectable chemical degradation [37]. Peaks shifts in terms of positions and intensities as seen in scaffolds loaded with Eugenol indicate intermolecular interaction as hydrogen bonding whose effect may find expression in terms of mechanical as well as biological behavior of scaffolds. Figure 4: The FTIR spectra for the Raw PVA, HAp and the fabricated biomaterials were analyzed using Origin software. 3.3.2. XRD X-ray diffraction analysis was performed to evaluate the crystalline structure and phase distribution of the electrospun scaffolds and to confirm the successful incorporation of HAp into the PVA matrix. The diffraction patterns of the control (PVA/HAp) and test scaffolds (Eugenol 100 µg/mL and 200 µg/mL) were compared with standard data from the International Centre for Diffraction Data (ICDD) to identify characteristic phases. The control scaffold (PVA/HAp) displayed sharp diffraction peaks at 2θ = 25.9°, 31.8°, 32.9°, and 34.1°, which correspond to the (002), (211), (112), and (300) crystal planes of hexagonal hydroxyapatite, in agreement with JCPDS card no. 09-0432 (figure 5). These peaks confirm the successful incorporation of crystalline HAp within the polymer matrix. The decrease in peak intensity and broadening of diffraction peaks were observed in all the fabricated scaffolds. This suggests a crystalline nature of the fabricated bioactive electrospun scaffolds. The aromatic and phenolic structures of Eugenol may interfere with the ordered arrangement of HAp crystals, leading to a more amorphous scaffold structure. This partial amorphization can enhance flexibility and influence degradation rates—desirable features in scaffolds for soft tissue regeneration such as dental pulp. No new peaks were observed in the test scaffolds, indicating that the incorporation of Eugenol did not result in the formation of any unwanted crystalline byproducts. The overall XRD profiles confirm the preservation of the HAp phase and the structural compatibility of Eugenol with the PVA/HAp system [15], [37]. The amorphous nature may also influence the biological performance of the scaffolds, potentially enhancing biodegradability and cellular infiltration, which are beneficial for tissue engineering applications. Figure 5: The X-ray diffraction pattern of the Control (PVA/HAp): PVAC and the two test groups (PVA/HAp/Eugenol 100 µg/mL): PVAT1, (PVA/HAp/Eugenol 200 µg/mL): PVAT2 of electrospun composites were analysed using Origin software. 3.3.3. SEM The surface morphology of the electrospun scaffolds was assessed at 50 µm, 100 µm, and 200 µm magnifications for all three groups: Control (A1–A3), TS I (B1–B3), and TS II(C1–C3) as shown in Figure 6. In the all the three groups at each magnification i.e. 50 µm, 100 µm, and 200 µm, we can able to observe a smooth plane formation which indicates the well interconnected network without the formation of any HAp beads this shows the uniform dispersion and the homogenization of HAp into the polymer matrix. While the TS I and TS II nanofibers displays smoother surface and mild fiber fusion suggests improved viscosity and intermolecular interactions due to Eugenol incorporation, enhancing scaffold integrity also showed continuous fibers. Overall, the incorporation of Eugenol improved fiber morphology, with TS I and TS II showing optimal characteristics for tissue engineering applications due to balanced fiber uniformity.(figure 6). Figure 6: The images depict the morphological character of the fabricated electrospun nanofibers: Control (A1–A3), TS I (B1–B3), and TS II(C1–C3). 3.3.4. TGA Thermogravimetric analysis was conducted to assess the thermal stability and decomposition profile of the electrospun scaffolds. The TGA curves for the control (PVA/HAp) and test groups (PVA/HAp/Eugenol at 100 µg/mL and 200 µg/mL) revealed a multi-stage degradation pattern, reflecting the sequential decomposition of water, organic polymer (PVA and Eugenol), and the inorganic HAp component. The initial weight loss, observed below 150°C, was attributed to the evaporation of physically adsorbed moisture and residual solvents. This phase was common across all samples, indicating the hydrophilic nature of PVA and the hygroscopic behavior of the scaffolds. The major weight loss occurred in the temperature range of 250°C to 500°C, corresponding to the thermal decomposition of the PVA polymer matrix and the breakdown of organic components including Eugenol. The control scaffold (PVA/HAp) exhibited a sharp degradation event in this region, while the Eugenol-loaded scaffolds showed a broader and slightly shifted degradation profile, suggesting that the presence of Eugenol may have altered the thermal behavior of the matrix. Interestingly, TS I (100 µg/mL Eugenol) displayed a slightly delayed onset of major decomposition, indicating improved thermal stability compared to the control and where the TS II (200 µg/mL Eugenol) showed a most delayed decomposition due to the presence of higher concentration of eugenol as shown in Figure 7. This enhancement could be attributed to intermolecular interactions such as hydrogen bonding between Eugenol and PVA, which may increase the energy required to break down the scaffold matrix. However, at the higher Eugenol concentration (TS II), a slightly earlier degradation onset was noted, possibly due to the plasticizing effect of excess Eugenol reducing the cohesive forces within the polymer network [38]. Beyond 600°C, a small amount of residual mass remained, corresponding to the inorganic HAp component, which is thermally stable and does not decompose within the tested range. The residual weight was consistent across all samples, confirming that the HAp phase was retained during thermal processing and reinforcing its role as a stable scaffold constituent [39]. Overall, the TGA analysis confirms the thermal integrity and compositional behavior of the fabricated scaffolds. The incorporation of Eugenol at optimized concentrations not only adds biofunctionality but may also subtly influence the thermal stability and degradation kinetics, which are critical parameters for biomedical applications, particularly in temperature-sensitive environments such as the oral cavity. Figure 7: The thermal stability and the stage wise decomposition of the fabricated nanofibrous scaffold analysed using Thermogravimetry and plotted using Graph Pad Prism software. 3.4. Antimicrobial Activity The antimicrobial potential of the fabricated scaffolds was evaluated against Enterococcus faecalis and Streptococcus mutans by measuring the zone of inhibition as shown in figure 8. Against E. faecalis, the Control (C), TS I and TS II scaffolds exhibited zones of 2 mm, 1 mm, and 1 mm, respectively, compared to 8 mm for the standard antibiotic disc ampicillin. In contrast, for S. mutans, the zones were 3 mm (C), 3 mm (TS I), and 4 mm (TS II), while the antibiotic showed 9 mm inhibition. Although all scaffolds showed lower antibacterial activity than the standard antibiotic, Test 2 displayed slightly enhanced inhibition against S. mutans, potentially due to the higher concentration of Eugenol, known for its antimicrobial properties. These results suggest that Eugenol-incorporated scaffolds, especially at higher concentrations, may offer moderate antibacterial effects, particularly against S. mutans, which is significant in dental applications. Figure 8: Shows that Disc diffusion method was employed to assess the antimicrobial property of the fabricated scaffold. Where (A) Represents the Negative control – a plate without any microbial strain, (B) Represents the Antimicrobial activity assessment against S.mutans and (C) Represents the Antimicrobial activity assessment against E.faecalis. 4. Discussion The objective of this study was to fabricate and evaluate the properties of scaffolds for dental pulp tissue engineering applications by combining Polyvinyl Alcohol (PVA), Hydroxyapatite (HAp), and Eugenol into a bioactive electrospun nanofibrous structure. For these scaffolds which were intended for the regeneration of dental pulp, the design was based on evaluating the physicochemical, thermal, morphological, and antimicrobial traits of the scaffolds. The in-silico studies provided crucial insights into Eugenol’s therapeutic potential. Network pharmacology identified 10 key hub genes associated with pulp pathology, while molecular docking revealed strong binding affinities of Eugenol, particularly with ACE and MME. These interactions suggest that Eugenol may influence inflammation, tissue remodeling, and healing processes, complementing the structural and biological functions of the scaffold. The incorporation of HAp and Eugenol into the PVA matrix was evidenced by FTIR spectra. The test scaffolds TS I and TS II showed the presence of O–H, C–H, and phosphate peaks with shifting bands. These shifts indicate possible hydrogen bonding and intermolecular interactions among scaffold components. Such features are essential for the stability of the scaffold and controlled drug release systems. Largely, XRD patterns showed several peaks that confirmed the existence of the crystalline phase of HAp and the amorphous nature of the overall scaffold. There was a noticeable drop of peak intensity and slight broadening observed in all the samples, which suggested a decline in crystallinity. The enhancement of scaffold flexibility along with structure integrity led to increased biodegradability making it ideal for use in pulp tissue regeneration. TGA results showed that there was multi-step degradation for Eugenol loaded scaffolds which also exhibited delayed thermal decomposition compared to the control sample. This increase in thermal stability is indicative of Eugenol acting as a stabilizer within the scaffold matrix due to the interaction with the PVA chains. SEM imaging captured the enhancement of the morphology of the nanofibers on Eugenol addition. TS I and TS II displayed the smoothest, beadless, and best aligned fibers with well interconnected network, which is vital for cellular attachment and nutrient transport, also it maintained structural integrity which is ideal for tissue applications. TS II outperformed TS I in having greater antimicrobial activity in both test groups, being the more inhibited towards Streptococcus mutans, while all scaffold types displayed moderate activity with Enterococcus faecalis. Although the inhibition zone observed with Eugenol was comparatively lesser than that of standard antibiotics, its incorporation still highlights Eugenol’s potential role in providing antimicrobial protection, which is significant in endodontic procedures. All of these are contributory evidence reinforcing that the multifunctionality hypothesis of fabricated PVA/HAp/Eugenol scaffolds holds true. This serves as a promising candidate for regenerative endodontics, considering it combines structural support, thermal and morphological integrity, antimicrobial activity, and molecular bioactive potential. The scaffold’s clinical utility, however, requires further investigation into in vitro cytocompatibility and in vivo regenerative efficacy. 5. Conclusion The present study reports the fabrication and evaluation of electrospun nanofibrous scaffolds comprising Polyvinyl Alcohol (PVA), Hydroxyapatite (HAp), and Eugenol for dental pulp regeneration. Structural analyses using FTIR and XRD confirmed the successful integration of all components, while SEM revealed improved fiber uniformity and morphology in Eugenol-loaded samples. TGA findings indicates enhanced thermal stability and antimicrobial assays showed mild inhibition zones in all samples, with the TS II group performing better against S.mutans. Complementing the experimental work, in-silico analyses provided insight into the biological relevance of Eugenol. Network pharmacology identified ten target genes linked to dental pulp defects, including ACE, PLAU and MME. Molecular docking further supported strong binding affinities between Eugenol and several key proteins, suggesting its role in regulating inflammatory and regenerative pathways. 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