Near Field Melt-Electrowriting of Bioglass loaded Ultrathin Fibrous 3D-Hierarchy as Tissue Engineering Template – A Practical Approach

Near Field Melt-Electrowriting of Bioglass loaded Ultrathin Fibrous 3D-Hierarchy as Tissue Engineering Template – A Practical Approach | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Near Field Melt-Electrowriting of Bioglass loaded Ultrathin Fibrous 3D-Hierarchy as Tissue Engineering Template – A Practical Approach Samir Das, Nitish Das Kashyap, Nantu Dogra, Santanu Dhara This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7860311/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 Personalized Three-dimensional (3D) printed scaffolds represent an advancement in tissue engineering and regenerative medicine, offering customizable implants that closely match the unique anatomical and pathological needs of individual patients. 3D printing of polymeric scaffolds enables the precise fabrication of complex structures with customizable porosity and mechanical properties. Despite the potential of 3D printing, achieving interconnected fibrous hierarchy that closely mimic the natural extracellular matrix remains a challenge. In this context, melt electrospinning and its utilization in near field electrowriting could be an emerging technique in the field of tissue engineering for tailorable architecture and mechanical properties that meet the specific requirements of various tissue types. But the challenges remain in optimizing processing parameters to ensure reproducibility and functionality of the scaffolds in cost effective way. Hence, we aim to refine this knowledge by developing a specialized 3D printing system that reduces polymer viscosity through controlled heating while enhancing electrical conductivity. Bioactive cues were integrated into fibrous scaffolds to improve biological activity and maintain mechanical strength for native tissue models. Process parameters were systematically optimized, and scaffold properties were characterized through physicochemical analyses and cellular assays, demonstrating effective cell–matrix interactions and anisotropy comparable to native tissue, outperforming conventional electrospun scaffolds. Melt electrowriting Computer-aided design Biofunctionalization In-vitro study Anatomical model Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Biological tissues exhibit an anisotropic and hierarchical fibrous architecture of native extracellular matrix (ECM), highlighting the need for fibrous scaffold design in tissue engineering applications. Conventional electrospinning yields continuous fibers with diameters in the nanomicrometer range, as reported by Ragvi et. al., 2023. These processes typically produce randomly oriented fibers that form an interconnected porous network, with pore sizes in few hundred nanometers range (Filippe et. al., 2023). However, these techniques are time-consuming, suffer from design customization, mechanical strength for sufficient cell infiltration, vascularization, and nutrient transport in complex tissue engineering application (Greiner et. al., 2010). Thus, researchers have explored alternative methods such as co-electrospinning (Bazilevsky et. al., 2007), salt-leaching (Hou et. al., 2003), and multi-layering (Kidoaki et. al., 2005) to improve cell infiltration and nutrient diffusion (Huang et. al., 2003), but uneven fiber distribution, lack of 3-dimensional hierarchy with nano-level pore sizes, insufficient mechanical strength, and residual contaminants limit their application in large-scale production (Pham QP et. al., 2006). On the other hand, near-field electrospinning is an advanced technique that enables the precise fabrication of 3D fibrous scaffolds with microscale architectures (Brown et. al., 2011). Unlike conventional electrospinning, MEW utilizes a molten polymer jet that solidifies upon deposition, allowing controlled fiber alignments with spatial arrangements (Rnjak-Kovacina J et. al., 2011). This process is particularly advantageous for fabricating tailorable scaffold shape and hierarchy for patient-specific applications with tunable mechanical properties, making it ideal for tissue engineering applications (Stankus JJ et. al., 2008). Nonetheless, the requirement for heat treatment to maintain ink viscosity, combined with the application of high voltage, increases the overall system cost. Additionally, the potential interaction between electrical heating systems and high-voltage components could lead to short-circuit hazards (Wang et. al., 2022). In this context, advanced research is focused on enhancing the scalability of MEW for commercial use by optimizing processing parameters and integrating bioactive elements into 3D fibrous architectures (Lifson et. al., 2019). The present study introduces a facile system for fabricating bioactive cue-incorporated, mechanically robust melt-spun fibers through 3D patterning. The newly developed nozzle design enables the controlled melting of low-temperature biocompatible thermoplastic polymers, such as polycaprolactone (PCL). By circulating non-electroconductive, high–heat-capacity fluids (e.g., oil or silicone gel) around the nozzle, localized heating is maintained while minimizing heat loss and reducing the risk of electrical hazards. This approach facilitates the fabrication of anatomically precise scaffolds capable of supporting tissue regeneration. Optimization of the process parameters further demonstrates the potential of these scaffolds for on-site wound healing applications and highlights their promise for future clinical translation in healthcare. 2. Materials and Methods 2.1. Experimental setup The elevated temperature electrowriting system was developed by integrating a high-voltage power supply with the 3D printer to fabricate bioactive ultrathin fibrous scaffolds. A customized nozzle with a fluid-based 3D printer provided precise control for layer-by-layer fiber deposition, ensuring accurate scaffold architecture, as mentioned in the patent entitled “A facile system and method for melt-electro-writing of ultrathin fibrous 3D scaffold and tubular graft” by our group. The engineered metal nozzle is designed to accommodate the thermoplastic polymer and is equipped with a heating jacket to ensure efficient melting within an optimal processing time. An emulsion pump-based hot fluid circulation system was utilized to maintain a consistent temperature around the nozzle periphery, effectively preventing polymer solidification during extrusion. This approach also eliminated short-circuit risks typically encountered with electrically heated platens. This system maintains the polymer in a molten state, enabling precise fiber deposition under high voltage. The CAD model of the nozzle is shown in Fig. 1 a, while a visual representation of the assembled nozzle is presented in Fig. 1 b. 2.2. Viscosity measurement An engineered syringe with an internal diameter of 14.5 mm was employed under controlled conditions as a capillary viscometer, hereafter referred to as a syringe capillary rheometer, following the method reported by Teng et. al. (1997). This approach was chosen to closely replicate direct ink-based printing, in which extrusion is driven by a screw mechanism through a blunt capillary nozzle (22G × 15 mm; outer diameter 0.7 mm, inner diameter 0.4 mm). The shear rate and shear stress were calculated using Equations (1) and (2), respectively, across extrusion velocities (V) ranging from 10 to 120 mm/s at temperatures between 80°C and 240°C. The apparent viscosity was subsequently determined using Eq. (3), mentioned in Fig. 2c. γ= \(\:\frac{4AV}{\pi\:{r}^{3}}\) …………………..( 1 ) τ= \(\:\frac{Fr}{{\pi\:}{R}^{2}\text{L}}\) ………………………( 2 ) η= \(\:\frac{{\tau\:}}{{\gamma\:}}\) ……………….……( 3 ) Where A is the cross-sectional area of the nozzle, V is the velocity of extrusion, r is the radius of the capillary, F is the plunging force, L is the length of the nozzle, and R is the radius nozzle. 2.3. Physico-Chemical Characterisation of the Scaffold The surface morphology and elemental analysis of the samples were conducted using scanning electron microscopy (SEM) (Sigma 300 VP-FESEM, Zeiss, Germany) coupled with energy dispersive X-ray spectroscopy (EDS) with an acceleration voltage of 10 kV in both backscattering and secondary electron imaging modes. Furthermore, the microstructure of the samples was observed through stereo-zoom microscopy (Leica Microsystems, Germany). The confirmation of functional groups was further investigated by X-ray photoelectron spectroscopy (XPS) (PHI 5000 VERSA PROBE III, ULVAC PHI, USA). 2.4. Hemocompatibility and In-vitro protein adsorption Assay Samples of identical size and weight were placed in a 24-well plate for evaluation. The hemocompatibility study was carried out to assess the blood compatibility of MWE samples as per ASTM standard F756 guidelines. Blood was collected in ethylenediaminetetraacetic acid (EDTA)-coated vials to prevent coagulation. Red blood cells (RBCs) were isolated by centrifugation at 4000 rpm for 10 minutes at 37°C, followed by washing with phosphate-buffered saline (PBS, pH 7.4). The assay evaluated hemolytic activity of the scaffold samples in comparison to positive (Triton X-100) and negative (PBS) controls, and the optical density of the supernatant was measured at 540 nm. The percentage of hemolysis was calculated using the following formula (Sperling et. al., 2023): Hemolysis ratio (%) = \(\:\frac{(\text{s}\text{u}\text{s}\text{p}\text{e}\text{n}\text{s}\text{i}\text{o}\text{n}-\text{n}\text{e}\text{g}\text{a}\text{t}\text{i}\text{v}\text{e}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}{(\text{p}\text{o}\text{s}\text{i}\text{t}\text{i}\text{v}\text{e}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}-\text{n}\text{e}\text{g}\text{a}\text{t}\text{i}\text{v}\text{e}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l})}\) x100% For the protein adsorption study, each well was filled with 300 µL of a 0.1% bovine serum albumin (BSA, fraction V, Merck) solution prepared in PBS (pH 7.4). Following incubation at 37°C, samples were rinsed with PBS to remove unbound proteins. Adsorbed proteins were then desorbed using 300 µL of a 2% sodium dodecyl sulfate (SDS) solution. Protein concentration was quantified using the bicinchoninic acid (BCA) protein assay kit (Sigma-Aldrich), which consists of BCA reagent and a 4% copper sulfate pentahydrate solution mixed in a 50:1 ratio. The absorbance of the resulting purple-blue complex was measured at 562 nm using a microplate reader (BIO-RAD, iMark) (Smith et. al., 1985). 2.5. In vitro cytocompatibility Assessment using L929 cell lines The scaffold was sterilized using ethanol, followed by sterile PBS wash. After sterilization, the samples were put into a 24-well plate filled with complete DMEM low-glucose media (Gibco, Life Technologies), and placed in an incubator at an operating temperature of 37°C within 5% CO 2 atmosphere. Each well was seeded with 5 × 10 4 cells to incubate for 1, 3, and 5 days, with the media refreshed every 24 h. 2.5.1. Cell viability and morphological Analysis Cytotoxicity and cellular viability of the materials were evaluated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The samples were incubated with L929 cells obtained from NCCS, Pune, in complete DMEM high glucose cell culture medium (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic. Following incubation, MTT reagent (0.5 mg/mL) was added, and optical density was measured at 570 nm using a Thermo Scientific Multiskan GO plate reader. Cell proliferation over different time intervals was assessed using a live/dead assay Kit (Life Technologies). In order to assess the cellular morphology over scaffold surface, the cells cultured samples were fixed with 4% paraformaldehyde, and permeabilized using 0.1% Triton X-100, and rinsed with PBS (pH 7.4). Cytoskeletal and nuclear staining was performed using Rhodamine/DAPI, staining as per Invitrogen protocol. Fluorescence microscopy (Axio Observer Z1, Carl Zeiss) was used to visualize cell attachment and distribution. Subsequently, the cell-laden scaffolds were dehydrated and imaged using SEM to further evaluate cell adherence and morphological characteristics. 2.6. Design and fabrication of scaffold temptress following anatomy The Digital Imaging and Communications in Medicine (DICOM) files of anonymized patient-specific defective phalanx data were collected from Datta Meghe Institute of Medical Sciences, Wardha, after the informed consent of the patient and with permission of the Ethics Committee of the institute. Reconstruct of the customized phalanx into 3D CAD model was performed using Materialise Mimics Research version 19. The whole phalanx bone is divided into two phalanx bones, i.e., distal and intermediate, in a sagittal section, which is more prone to damage. After the removal of defects form MP phalanx model, refining of the curvature of the 3D reconstruction was done by mashing. Next, the reconstructed phalanx model was saved in. stl file format and imported into the open-source slicing software CURA (v15.04.5, Ultimaker, the Netherlands), to generate the GCODE using optimized printing parameters. 2.7. Statistical Analysis The statistical analysis was performed to assess significant disparities between the experimental and control groups using a one-way analysis of variance (ANOVA) followed by t-test in GraphPad Prism software (version 5.02) with statistical significance at *p < 0.05 and ***p < 0.001. 3. Results and Discussion The facile system for developing ultrathin, fibrous scaffolds was established using optimized printing parameters, as mentioned in the following section: 3.1. Fabrication of Scaffold template The rheological characterisation of the polymer ink revealed shear-thinning behaviour, as shown in Fig. 2a. The apparent viscosity decreased consistently with increasing extrusion velocity and temperature, indicating suitability of the formulation for direct ink-based 3D printing. This trend suggests that higher shear rates reduce flow resistance, thereby facilitating controlled fiber extrusion. Based on optimized viscosity of 112 Pa-s, as reported by Ding et. al., 2018, the temperature and velocity of printing were determined to 134 o C and 90 mm/sec, respectively. The CAD-based design and corresponding G-code generation (Fig. 2b) enabled accurate translation of the scaffold template into a full-scale 3D printed construct. Further, the modified system is connected to a high-voltage power supply (Glassman, US), generating an electric field that draws out polymer fibers onto the grounded collector as shown in Fig. 2c. The nozzle-to-collector distance is carefully controlled to achieve the desired fiber diameter and morphology. Under the influence of the electric field, the polymer solution forms ultrathin fibers, typically under 10 microns in diameter, making them suitable for biomedical scaffold pplications. Figure 2d shows the uniform grid pattern with consistent spacing and structural integrity for a full-scale 3D fibrous scaffold template. The magnified optical images evidence the fiber morphology of unit cell within the gridded scaffold templates. A highly ordered porous architecture with well-defined, continuous fibers and strong interconnections at the cross-over points is clearly observed. This structural organization is essential for maintaining mechanical integrity while simultaneously supporting nutrient diffusion and facilitating cell migration under biological conditions, as reported by previous studies on fiber-based tissue scaffolds (Tamayol et. al., 2013). Further, the viscosity was measured using capillary rheometer techniques as mention in Eq. 3 to determine the optimized flow condition during melt-electrowriting. 3.2. Effect of Nozzle to Collector Distance on fibrous extrusion The distance between the nozzle tip and collector, commonly known as the tip-to-collector distance, is a crucial parameter in the MWE process, which significantly influences fiber formation and overall scaffold architecture, as depicted in Fig. 3 . Variations in this distance affect the electrostatic forces acting on the polymer jet, thereby impacting fiber diameter, morphology, and uniformity (Fig. 3 a). At longer distances (e.g., 4 cm and 3.5 cm), the electrostatic field weakens, leading to a reduced concentration of charged polymer molecules at the collector. This results in loosely connected, tangled fibers with poor definition and lower structural uniformity. In contrast, reducing the tip-to-collector distance strengthens the electric field even at lower voltages, enhancing fiber alignment and uniformity as shown under Stereo-zoom microscope (Fig. 3 b). Specifically, a reduced tip-to-collector distance can decrease the voltage required for fiber extrusion and shorten the processing time. Such optimized conditions are also favorable for achieving precise, fibrous, and pattern-based live cell printing with high cell viability, as reported by He et. al., 2017. Overall, the results confirm that reducing the tip-to-collector distance enhances fiber control, leading to structurally defined scaffolds with desirable mechanical properties for biological performance. 3.3. Physico-chemical Characterization of fibrous Scaffold templates The unit cell of the scaffold was observed using a stereo zoom microscope (Leica, Germany), as depicted in Fig. 4 a. Melt-spun fiber diameters were measured in the range of 25 µm to 40 µm, as shown in Fig. 4 b. The distribution of bioglass particles within the core of the melt-spun fibers is evident in Fig. 4 c. The incorporation of bioactive glass into the fiber core is particularly significant, as it enhances cell migration and supports mineralization, thereby improving the scaffold potential for site-specific tissue regeneration (Gentile et. al., 2012).The surface morphology and elemental composition of the 3D-printed fibrous scaffold made of PCL (Fig. 4 d) and PCL/Bioglass (PCL/BG) composite were analysed using SEM. SEM images confirmed uniform fiber formation across the scaffold in both types of samples, with well-defined porosity. The presence of bioactive glass particles embedded within the fiber matrix was visually confirmed and validated through elemental composition via EDX, as shown in Fig. 4 e. Furthermore, the XPS analysis reveals significant differences in surface composition between PCL and PCL/Bioglass printed scaffold templates. The PCL spectrum (Fig. 4 f) shows prominent peaks corresponding to C 1s at ~ 284.8 eV and O 1s at ~ 532 eV, consistent with its aliphatic polyester backbone (Si et. al. 2016). In contrast, the PCL/Bioglass spectrum (Fig. 4 g) exhibits additional peaks, notably Si 2p at ~ 102 eV, Ca 2p at ~ 347 eV, and P 2p at ~ 133 eV, confirming successful incorporation of bioglass into the polymer matrix. The relative increase in O 1s intensity in the composite sample indicates enhanced surface oxidation or bonding due to the presence of silicate and phosphate groups from bioglass. Thus, the analysis confirms the effective integration of bioglass particles into PCL matrix, supporting its potential for tissue engineering application, particularly for skeletal tissue regeneration by calcium-phosphate-rich phases (Tolmacheva et al., 2024; Furko et. al., 2025). 3.4. In vitro bioassay The cytocompatibility of the fabricated PCL and PCL/BG composite fibrous scaffolds was further evaluated through hemolysis and protein absorption assays. Both studies are critical for biomedical scaffolds, especially for applications involving direct cell and blood contact. The hemolysis assay results are presented in Fig. 5 a, where the OD of hemoglobin released from lysed red blood cells was measured. The percentage of hemolysis remained below 5%, which is the acceptable threshold for both types of scaffold, as per ASTM standard F756 guidelines, indicating non-hemolytic behaviour. The SEM micrographs further support RBC morphology and adhesion of RBCs on both PCL (Fig. 9b) and PCL/BG (Fig. 5 b) scaffolds without apparent morphological damage or cell lysis. Notably, the PCL/BG scaffold shows more extensive RBC attachment and spreading, possibly due to enhanced surface roughness and ionic bioactivity imparted by the bioglass particles, which can promote better protein adsorption (Baier et. al., 2022), as illustrated in Fig. 5 c. Additionally, the PCL/BG composite scaffold exhibited significantly higher protein adsorption compared to pure PCL. This enhanced adsorption is advantageous for bone regeneration and wound healing applications, where effective interaction with biological fluids is essential for initiating cellular responses and tissue integration. Therefore, biological performance of the 3D-printed PCL/BG fibrous scaffold template was evaluated using the L929 fibroblast cell line. The MTT assay (Fig. 5 e) results depicted in Fig. 5 d demonstrate enhanced cellular viability on PCL/BG electrospun scaffolds compared to PCL scaffold as the control. This improvement can be attributed to the presence of bioactive glass, which enhances surface bioactivity and promotes better cell-scaffold interactions. These findings were further supported by the time-dependent live/dead assay (Fig. 5 f), where predominance of viable cells (stained green) with few dead cells (stained red) were observed, particularly on day 5. Additionally, Rhodamine/DAPI staining (Fig. 5 g) revealed more extensive cytoskeletal spreading and nuclear distribution, indicating improved cellular morphology, attachment, and proliferation on the PCL/BG fibrous scaffold template. Complementary SEM micrographs provided further confirmation, showing direct evidence of cell adhesion and spreading across the fibrous matrix, suggesting a favorable microenvironment for cellular activities critical for tissue regeneration. 3.5. Printing of Anatomical Model Following the evaluation of the biological activity of the MWE fibrous scaffold, the feasibility of onsite scaffold development using this facile technique was further assessed through the printing of a bone anatomical model. This system demonstrates a platform for the fabrication of bioactive cue-based porous patient-specific scaffolds at the damaged site during surgery, as depicted in Fig. 6a. The results highlight the system capability to produce anatomically relevant models on a large scale by tailoring its mechanical properties and design morphology based on site-specific application and loading direction. The detailed G-Code modelling following bone anatomy based on the CT extracted CAD data is shown in Fig. 6b, followed by fabrication of full scale single and multilayed fibrous anatomical model is shown in Fig. 6d-d. However, further advancements are necessary to develop full-scale anatomical models in a short period of time for streamlined clinical translation. This study successfully establishes a facile melt writing (MWE) system for the fabrication of bioactive, ultrathin fibrous 3D scaffolds with high anatomical fidelity. Varying in porosity and design hierarchy evidences the scaffold potential in site-specific tissue healing applications. The integration of bioactive glass into PCL fibers significantly enhanced the biological performance by improving protein adsorption, cell adhesion, and proliferation, as confirmed by in vitro assays. Optimized processing parameters, especially nozzle-to-collector distance, with low voltage enabled uniform fiber formation with potential for live cell printing. Exploring alternative polymer/ceramic combinations, adjusting fiber orientation for anisotropic tissues, and embedding therapeutic agents (e.g., growth factors, antibiotics) within the fiber matrix could be pursued in the near future to further enhancement of biological performance within the scaffold templates. Moreover, long-term scaffold performance in complex tissue regeneration can be validated using small animal models. The ability to fabricate CT-based anatomical models further highlights the system potential for onsite patient-specific scaffold development, supporting the creation of structurally and biologically relevant constructs suitable for clinical use. Further development is required for the rapid fabrication of full-scale constructs suitable for clinical translation. 4. Conclusion A robust and versatile MWE-based fabrication platform for the development of bioactive, ultrafine fibrous scaffolds with anatomically relevant geometries has been explored. The incorporation of bioactive cues into the thermoplastic biocompatible polymer matrix significantly enhanced the scaffold biofunctionality. Process optimization, specifically low-voltage operation and controlled nozzle-to-collector distance, enabled consistent fiber deposition with defined porosity and mechanical integrity, critical for replicating the native extracellular matrix with native anisotropy and porosity for cellular migration and tissue integration. Thus, the system highlights its potential in customized scaffold printing towards improving biological performance for tissue engineering applications. Declarations Conflicts of interest The authors declare an Indian Patent application (No. 202431073007) titled “A Facile System and Method for Melt-Electro-Writing of Ultrathin Fibrous 3D Scaffold and Tubular Graft,” based on competing financial interests associated with ICMR and IIT Kharagpur as the funding agencies. The remaining authors declare no competing interests. Competing Interests The authors declare an Indian Patent application (No. 202431073007) titled “A Facile System and Method for Melt-Electro-Writing of Ultrathin Fibrous 3D Scaffold and Tubular Graft,” based on competing financial interests associated with ICMR and IIT Kharagpur as the funding agencies. The remaining authors declare no competing interests. Author Contribution Mr. S. Das: System development, biotemplate fabrication, experimental study, materials characterisation, In Vitro Assay, and manuscript preparation; Mr. N. Kashyap– System customisation, biotemplate fabrication, experimental study; Mr. N. Dogra–In vitro Study and data finalisation; Prof. S. Dhara – Conceptualisation, Study design, Manuscript finalisation. Acknowledgement The authors express their gratitude to DMIHER, Wardha for supporting us in CT data collection. This research was supported by the Indian Council of Medical Research (ICMR) (Proposal ID :2020-4833), Indian Council of Medical Research (ICMR) (ICMR-DHR CoE at IIT Kharagpur). ICMR project Ref. 5/3/8/82/2020-ITR dt. 02-02-2022. The authors also acknowledge IIT Kharagpur for fellowship and Central Research Facility, IIT KGP for support in materials characterization. Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. References Rajasekaran R et al (2023) Polyaniline Doped Silk Fibroin-PCL Electrospun Fiber: An Electroactive Fibrous Sheet for Full-Thickness Wound Healing Study. Chem Eng J 475:146245. https://doi.org/10.1016/j.cej.2023.146245 Cao K et al (2023) Advances in Design and Quality of Melt Electrowritten Scaffolds. Mater Design 226:111618. https://doi.org/10.1016/j.matdes.2023.111618 Greiner A, Wendorff JH (2007) Electrospinning: A Fascinating Method for the Preparation of Ultrathin Fibers. 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Carbohydr Polym 143:270–278. https://doi.org/10.1016/j.carbpol.2016.01.068 Tolmacheva N, Bhattacharyya A, Noh I (2024) Calcium Phosphate Biomaterials for 3D Bioprinting in Bone Tissue Engineering. Biomimetics 9(2):95. https://doi.org/10.3390/biomimetics9020095 Furko M (2025) Bioglasses versus Bioactive Calcium Phosphate Derivatives as Advanced Ceramics in Tissue Engineering: Comparative and Comprehensive Study, Current Trends, and Innovative Solutions. J Funct Biomaterials 16(5):161. https://doi.org/10.3390/jfb16050161 Baier RV et al (2022) Shape Fidelity, Mechanical and Biological Performance of 3D Printed Polycaprolactone-Bioactive Glass Composite Scaffolds. Biomaterials Adv 134:112540. https://doi.org/10.1016/j.bioadv.2022.112540 Additional Declarations Competing interest reported. The authors declare an Indian Patent application (No. 202431073007) titled “A Facile System and Method for Melt-Electro-Writing of Ultrathin Fibrous 3D Scaffold and Tubular Graft,” based on competing financial interests associated with ICMR and IIT Kharagpur as the funding agencies. The remaining authors declare no competing interests. Supplementary Files HandonScaffoldfabrication.mp4 GraphicalAbstract.jpg Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7860311","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":543288312,"identity":"dedca9d3-c578-427a-8c3a-c37c663c8746","order_by":0,"name":"Samir Das","email":"","orcid":"","institution":"Indian Institute of Technology Kharagpur","correspondingAuthor":false,"prefix":"","firstName":"Samir","middleName":"","lastName":"Das","suffix":""},{"id":543288313,"identity":"be686989-510b-4f6d-8e07-05473d9e95cd","order_by":1,"name":"Nitish Das Kashyap","email":"","orcid":"","institution":"Indian Institute of Technology 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1","display":"","copyAsset":false,"role":"figure","size":371500,"visible":true,"origin":"","legend":"\u003cp\u003e(a) CAD Model of engineered nozzle; (b) Visual representation of the fabricated nozzle.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7860311/v1/9d3a8a8c64effbe9c44edf16.png"},{"id":96377460,"identity":"35d7e6d0-0d7b-4e2d-9113-7937a7322ed5","added_by":"auto","created_at":"2025-11-20 11:34:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1175570,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Rheological behaviour of low-viscosity ink under optimized conditions; (b) G-code generation from CAD-based scaffold templates; (c) extrusion process of fibers during 3D-printed melt electrowritten full-scale scaffold fabrication; (d) microscopic image of the 3D-printed fibrous strut, showing the detailed architecture and fiber orientation within the scaffold.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7860311/v1/2df69c7d38640eb542cf2676.png"},{"id":96377462,"identity":"9bba781e-2198-4a0d-be79-0030e5dccdca","added_by":"auto","created_at":"2025-11-20 11:34:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1491528,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Effect of varying tip-to-collector distances on fiber morphology and scaffold architecture during melt electrowriting; (b) Stereo-zoom images of scaffold structures fabricated under different tip-to-collector distances and applied voltages, highlighting variations in fiber alignment and pattern fidelity\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7860311/v1/78a4d272d0999ee881c815f0.png"},{"id":96377464,"identity":"d00161e8-4e33-46be-8ffa-1dcd7c905616","added_by":"auto","created_at":"2025-11-20 11:34:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1628107,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Strut of unit cell of the 3D-printed fibrous scaffold architecture; (b, c) Stereo zoom microscopy images indicating the presence of bioactive glass particles distribution with the PCL matrix with uniform fiber diameter; (d) SEM image of electrospun 3D printed PCL scaffold showing a dense fibrous network, accompanied by EDX analysis revealing dominant peaks for C, N, and O elements; (e) SEM image of the PCL/BG composite scaffold evidence bioglass particles within the fibrous matrix, with EDX analysis confirming the incorporation of BG components such as Si, Ca, Na, and P; XPS spectra for pure (f) PCL (g) and PCL/BG scaffolds demonstrating elemental composition due to bioglass incorporation.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7860311/v1/562d58b855ed0a0a3f8530ee.png"},{"id":96453042,"identity":"93acf64f-094b-4929-bc98-7dd8ac16a1bc","added_by":"auto","created_at":"2025-11-21 09:57:45","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1145175,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Hemolysis assay comparing PCL and PCL/BG fibrous scaffolds, demonstrating non-hemolytic behaviour; (b, c) SEM images of RBCs on PCL and PCL/BG scaffolds, respectively, showing intact RBC morphology; (d) Protein adsorption analysis indicating higher adsorption in the PCL/BG scaffold due to increased surface roughness and ionic bioactivity from bioactive glass particles; (e) Cytocompatibility assessment using MTT assay with L929 cell line on MWE based PCL and PCL/BG scaffolds. Morphological analysis of L929 cells in PCL/BG fibrous scaffold: (f) Live/dead assay demonstrating predominance live cells over dead cells for all time points; (g) Rhodamine/DAPI staining at days 5 displaying well-spread cytoskeletal organization and nuclear distribution, indicating improved cellular attachment and proliferation; (h) SEM micrograph evidence of cell adhesion and spreading across the fibrous scaffold matrix. Data were reported as mean ± s.d. of triplicated (n = 3) experiments, with statistical significance at *p \u0026lt; 0.05 and ***p \u0026lt; 0.001, (B) Evaluation of hemocompatibility and red blood cell (RBC) morphological study.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7860311/v1/6d0555cfa4a08444c6a3b493.jpeg"},{"id":96377487,"identity":"4bcde870-eeee-406d-b4ab-36cee4cee187","added_by":"auto","created_at":"2025-11-20 11:34:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1192878,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Optical Image of 3D electrowriting system, (b) CT-extracted CAD model of reconstracted phalanx bone; (c,d) Fabrication of an anatomically accurate, patient-specific fibrous scaffold based on bone geometry, demonstrating the systems potential for onsite scaffold development during surgery.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7860311/v1/ab51219b1bb26471757b623c.png"},{"id":100362766,"identity":"11f80503-4d83-4726-b5ce-b9b66e6e458d","added_by":"auto","created_at":"2026-01-16 07:48:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8667595,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7860311/v1/0f6690b6-1c01-4f80-8666-be9675556b35.pdf"},{"id":96377485,"identity":"b5b1f4e6-1b9b-48ff-b377-4357a62e4387","added_by":"auto","created_at":"2025-11-20 11:34:02","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19942819,"visible":true,"origin":"","legend":"","description":"","filename":"HandonScaffoldfabrication.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7860311/v1/09868a1252997410a632a591.mp4"},{"id":96377461,"identity":"de7b3c86-c25e-4311-b89c-9b10c910d9a9","added_by":"auto","created_at":"2025-11-20 11:34:01","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1465708,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7860311/v1/89fe1ee7823785ec92063fb2.jpg"}],"financialInterests":"Competing interest reported. The authors declare an Indian Patent application (No. 202431073007) titled “A Facile System and Method for Melt-Electro-Writing of Ultrathin Fibrous 3D Scaffold and Tubular Graft,” based on competing financial interests associated with ICMR and IIT Kharagpur as the funding agencies. The remaining authors declare no competing interests.","formattedTitle":"Near Field Melt-Electrowriting of Bioglass loaded Ultrathin Fibrous 3D-Hierarchy as Tissue Engineering Template – A Practical Approach","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBiological tissues exhibit an anisotropic and hierarchical fibrous architecture of native extracellular matrix (ECM), highlighting the need for fibrous scaffold design in tissue engineering applications. Conventional electrospinning yields continuous fibers with diameters in the nanomicrometer range, as reported by Ragvi et. al., 2023. These processes typically produce randomly oriented fibers that form an interconnected porous network, with pore sizes in few hundred nanometers range (Filippe et. al., 2023). However, these techniques are time-consuming, suffer from design customization, mechanical strength for sufficient cell infiltration, vascularization, and nutrient transport in complex tissue engineering application (Greiner et. al., 2010). Thus, researchers have explored alternative methods such as co-electrospinning (Bazilevsky et. al., 2007), salt-leaching (Hou et. al., 2003), and multi-layering (Kidoaki et. al., 2005) to improve cell infiltration and nutrient diffusion (Huang et. al., 2003), but uneven fiber distribution, lack of 3-dimensional hierarchy with nano-level pore sizes, insufficient mechanical strength, and residual contaminants limit their application in large-scale production (Pham QP et. al., 2006).\u003c/p\u003e\u003cp\u003eOn the other hand, near-field electrospinning is an advanced technique that enables the precise fabrication of 3D fibrous scaffolds with microscale architectures (Brown et. al., 2011). Unlike conventional electrospinning, MEW utilizes a molten polymer jet that solidifies upon deposition, allowing controlled fiber alignments with spatial arrangements (Rnjak-Kovacina J et. al., 2011). This process is particularly advantageous for fabricating tailorable scaffold shape and hierarchy for patient-specific applications with tunable mechanical properties, making it ideal for tissue engineering applications (Stankus JJ et. al., 2008). Nonetheless, the requirement for heat treatment to maintain ink viscosity, combined with the application of high voltage, increases the overall system cost. Additionally, the potential interaction between electrical heating systems and high-voltage components could lead to short-circuit hazards (Wang et. al., 2022).\u003c/p\u003e\u003cp\u003eIn this context, advanced research is focused on enhancing the scalability of MEW for commercial use by optimizing processing parameters and integrating bioactive elements into 3D fibrous architectures (Lifson et. al., 2019). The present study introduces a facile system for fabricating bioactive cue-incorporated, mechanically robust melt-spun fibers through 3D patterning. The newly developed nozzle design enables the controlled melting of low-temperature biocompatible thermoplastic polymers, such as polycaprolactone (PCL). By circulating non-electroconductive, high\u0026ndash;heat-capacity fluids (e.g., oil or silicone gel) around the nozzle, localized heating is maintained while minimizing heat loss and reducing the risk of electrical hazards. This approach facilitates the fabrication of anatomically precise scaffolds capable of supporting tissue regeneration. Optimization of the process parameters further demonstrates the potential of these scaffolds for on-site wound healing applications and highlights their promise for future clinical translation in healthcare.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Experimental setup\u003c/h2\u003e\u003cp\u003eThe elevated temperature electrowriting system was developed by integrating a high-voltage power supply with the 3D printer to fabricate bioactive ultrathin fibrous scaffolds. A customized nozzle with a fluid-based 3D printer provided precise control for layer-by-layer fiber deposition, ensuring accurate scaffold architecture, as mentioned in the patent entitled \u0026ldquo;A facile system and method for melt-electro-writing of ultrathin fibrous 3D scaffold and tubular graft\u0026rdquo; by our group. The engineered metal nozzle is designed to accommodate the thermoplastic polymer and is equipped with a heating jacket to ensure efficient melting within an optimal processing time. An emulsion pump-based hot fluid circulation system was utilized to maintain a consistent temperature around the nozzle periphery, effectively preventing polymer solidification during extrusion. This approach also eliminated short-circuit risks typically encountered with electrically heated platens. This system maintains the polymer in a molten state, enabling precise fiber deposition under high voltage. The CAD model of the nozzle is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, while a visual representation of the assembled nozzle is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Viscosity measurement\u003c/h2\u003e\u003cp\u003eAn engineered syringe with an internal diameter of 14.5 mm was employed under controlled conditions as a capillary viscometer, hereafter referred to as a syringe capillary rheometer, following the method reported by Teng et. al. (1997). This approach was chosen to closely replicate direct ink-based printing, in which extrusion is driven by a screw mechanism through a blunt capillary nozzle (22G \u0026times; 15 mm; outer diameter 0.7 mm, inner diameter 0.4 mm). The shear rate and shear stress were calculated using Equations (1) and (2), respectively, across extrusion velocities (V) ranging from 10 to 120 mm/s at temperatures between 80\u0026deg;C and 240\u0026deg;C. The apparent viscosity was subsequently determined using Eq.\u0026nbsp;(3), mentioned in Fig.\u0026nbsp;2c.\u003c/p\u003e\u003cp\u003eγ=\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{4AV}{\\pi\\:{r}^{3}}\\)\u003c/span\u003e\u003c/span\u003e \u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;..(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eτ= \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{Fr}{{\\pi\\:}{R}^{2}\\text{L}}\\)\u003c/span\u003e\u003c/span\u003e \u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eη=\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{{\\tau\\:}}{{\\gamma\\:}}\\)\u003c/span\u003e\u003c/span\u003e \u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;.\u0026hellip;\u0026hellip;(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eWhere A is the cross-sectional area of the nozzle, V is the velocity of extrusion, r is the radius of the capillary, F is the plunging force, L is the length of the nozzle, and R is the radius nozzle.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Physico-Chemical Characterisation of the Scaffold\u003c/h2\u003e\u003cp\u003eThe surface morphology and elemental analysis of the samples were conducted using scanning electron microscopy (SEM) (Sigma 300 VP-FESEM, Zeiss, Germany) coupled with energy dispersive X-ray spectroscopy (EDS) with an acceleration voltage of 10 kV in both backscattering and secondary electron imaging modes. Furthermore, the microstructure of the samples was observed through stereo-zoom microscopy (Leica Microsystems, Germany). The confirmation of functional groups was further investigated by X-ray photoelectron spectroscopy (XPS) (PHI 5000 VERSA PROBE III, ULVAC PHI, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Hemocompatibility and In-vitro protein adsorption Assay\u003c/h2\u003e\u003cp\u003eSamples of identical size and weight were placed in a 24-well plate for evaluation. The hemocompatibility study was carried out to assess the blood compatibility of MWE samples as per ASTM standard F756 guidelines. Blood was collected in ethylenediaminetetraacetic acid (EDTA)-coated vials to prevent coagulation. Red blood cells (RBCs) were isolated by centrifugation at 4000 rpm for 10 minutes at 37\u0026deg;C, followed by washing with phosphate-buffered saline (PBS, pH 7.4). The assay evaluated hemolytic activity of the scaffold samples in comparison to positive (Triton X-100) and negative (PBS) controls, and the optical density of the supernatant was measured at 540 nm. The percentage of hemolysis was calculated using the following formula (Sperling et. al., 2023):\u003c/p\u003e\u003cp\u003eHemolysis ratio (%) =\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{(\\text{s}\\text{u}\\text{s}\\text{p}\\text{e}\\text{n}\\text{s}\\text{i}\\text{o}\\text{n}-\\text{n}\\text{e}\\text{g}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}{(\\text{p}\\text{o}\\text{s}\\text{i}\\text{t}\\text{i}\\text{v}\\text{e}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}-\\text{n}\\text{e}\\text{g}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l})}\\)\u003c/span\u003e\u003c/span\u003ex100%\u003c/p\u003e\u003cp\u003eFor the protein adsorption study, each well was filled with 300 \u0026micro;L of a 0.1% bovine serum albumin (BSA, fraction V, Merck) solution prepared in PBS (pH 7.4). Following incubation at 37\u0026deg;C, samples were rinsed with PBS to remove unbound proteins. Adsorbed proteins were then desorbed using 300 \u0026micro;L of a 2% sodium dodecyl sulfate (SDS) solution. Protein concentration was quantified using the bicinchoninic acid (BCA) protein assay kit (Sigma-Aldrich), which consists of BCA reagent and a 4% copper sulfate pentahydrate solution mixed in a 50:1 ratio. The absorbance of the resulting purple-blue complex was measured at 562 nm using a microplate reader (BIO-RAD, iMark) (Smith et. al., 1985).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. \u003cem\u003eIn vitro\u003c/em\u003e cytocompatibility Assessment using L929 cell lines\u003c/h2\u003e\u003cp\u003eThe scaffold was sterilized using ethanol, followed by sterile PBS wash. After sterilization, the samples were put into a 24-well plate filled with complete DMEM low-glucose media (Gibco, Life Technologies), and placed in an incubator at an operating temperature of 37\u0026deg;C within 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Each well was seeded with 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells to incubate for 1, 3, and 5 days, with the media refreshed every 24 h.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.5.1. Cell viability and morphological Analysis\u003c/h2\u003e\u003cp\u003eCytotoxicity and cellular viability of the materials were evaluated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The samples were incubated with L929 cells obtained from NCCS, Pune, in complete DMEM high glucose cell culture medium (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic. Following incubation, MTT reagent (0.5 mg/mL) was added, and optical density was measured at 570 nm using a Thermo Scientific Multiskan GO plate reader. Cell proliferation over different time intervals was assessed using a live/dead assay Kit (Life Technologies). In order to assess the cellular morphology over scaffold surface, the cells cultured samples were fixed with 4% paraformaldehyde, and permeabilized using 0.1% Triton X-100, and rinsed with PBS (pH 7.4). Cytoskeletal and nuclear staining was performed using Rhodamine/DAPI, staining as per Invitrogen protocol. Fluorescence microscopy (Axio Observer Z1, Carl Zeiss) was used to visualize cell attachment and distribution. Subsequently, the cell-laden scaffolds were dehydrated and imaged using SEM to further evaluate cell adherence and morphological characteristics.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Design and fabrication of scaffold temptress following anatomy\u003c/h2\u003e\u003cp\u003eThe Digital Imaging and Communications in Medicine (DICOM) files of anonymized patient-specific defective phalanx data were collected from Datta Meghe Institute of Medical Sciences, Wardha, after the informed consent of the patient and with permission of the Ethics Committee of the institute. Reconstruct of the customized phalanx into 3D CAD model was performed using Materialise Mimics Research version 19. The whole phalanx bone is divided into two phalanx bones, i.e., distal and intermediate, in a sagittal section, which is more prone to damage. After the removal of defects form MP phalanx model, refining of the curvature of the 3D reconstruction was done by mashing. Next, the reconstructed phalanx model was saved in. stl file format and imported into the open-source slicing software CURA (v15.04.5, Ultimaker, the Netherlands), to generate the GCODE using optimized printing parameters.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Statistical Analysis\u003c/h2\u003e\u003cp\u003eThe statistical analysis was performed to assess significant disparities between the experimental and control groups using a one-way analysis of variance (ANOVA) followed by t-test in GraphPad Prism software (version 5.02) with statistical significance at *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003eThe facile system for developing ultrathin, fibrous scaffolds was established using optimized printing parameters, as mentioned in the following section:\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Fabrication of Scaffold template\u003c/h2\u003e\u003cp\u003eThe rheological characterisation of the polymer ink revealed shear-thinning behaviour, as shown in Fig.\u0026nbsp;2a. The apparent viscosity decreased consistently with increasing extrusion velocity and temperature, indicating suitability of the formulation for direct ink-based 3D printing. This trend suggests that higher shear rates reduce flow resistance, thereby facilitating controlled fiber extrusion. Based on optimized viscosity of 112 Pa-s, as reported by Ding et. al., 2018, the temperature and velocity of printing were determined to 134 \u003csup\u003eo\u003c/sup\u003eC and 90 mm/sec, respectively. The CAD-based design and corresponding G-code generation (Fig.\u0026nbsp;2b) enabled accurate translation of the scaffold template into a full-scale 3D printed construct. Further, the modified system is connected to a high-voltage power supply (Glassman, US), generating an electric field that draws out polymer fibers onto the grounded collector as shown in Fig.\u0026nbsp;2c. The nozzle-to-collector distance is carefully controlled to achieve the desired fiber diameter and morphology. Under the influence of the electric field, the polymer solution forms ultrathin fibers, typically under 10 microns in diameter, making them suitable for biomedical scaffold pplications. Figure\u0026nbsp;2d shows the uniform grid pattern with consistent spacing and structural integrity for a full-scale 3D fibrous scaffold template. The magnified optical images evidence the fiber morphology of unit cell within the gridded scaffold templates. A highly ordered porous architecture with well-defined, continuous fibers and strong interconnections at the cross-over points is clearly observed. This structural organization is essential for maintaining mechanical integrity while simultaneously supporting nutrient diffusion and facilitating cell migration under biological conditions, as reported by previous studies on fiber-based tissue scaffolds (Tamayol et. al., 2013). Further, the viscosity was measured using capillary rheometer techniques as mention in Eq.\u0026nbsp;3 to determine the optimized flow condition during melt-electrowriting.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Effect of Nozzle to Collector Distance on fibrous extrusion\u003c/h2\u003e\u003cp\u003eThe distance between the nozzle tip and collector, commonly known as the tip-to-collector distance, is a crucial parameter in the MWE process, which significantly influences fiber formation and overall scaffold architecture, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Variations in this distance affect the electrostatic forces acting on the polymer jet, thereby impacting fiber diameter, morphology, and uniformity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). At longer distances (e.g., 4 cm and 3.5 cm), the electrostatic field weakens, leading to a reduced concentration of charged polymer molecules at the collector. This results in loosely connected, tangled fibers with poor definition and lower structural uniformity. In contrast, reducing the tip-to-collector distance strengthens the electric field even at lower voltages, enhancing fiber alignment and uniformity as shown under Stereo-zoom microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Specifically, a reduced tip-to-collector distance can decrease the voltage required for fiber extrusion and shorten the processing time. Such optimized conditions are also favorable for achieving precise, fibrous, and pattern-based live cell printing with high cell viability, as reported by He et. al., 2017. Overall, the results confirm that reducing the tip-to-collector distance enhances fiber control, leading to structurally defined scaffolds with desirable mechanical properties for biological performance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Physico-chemical Characterization of fibrous Scaffold templates\u003c/h2\u003e\u003cp\u003eThe unit cell of the scaffold was observed using a stereo zoom microscope (Leica, Germany), as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ea. Melt-spun fiber diameters were measured in the range of 25 \u0026micro;m to 40 \u0026micro;m, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eb. The distribution of bioglass particles within the core of the melt-spun fibers is evident in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ec. The incorporation of bioactive glass into the fiber core is particularly significant, as it enhances cell migration and supports mineralization, thereby improving the scaffold potential for site-specific tissue regeneration (Gentile et. al., 2012).The surface morphology and elemental composition of the 3D-printed fibrous scaffold made of PCL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) and PCL/Bioglass (PCL/BG) composite were analysed using SEM. SEM images confirmed uniform fiber formation across the scaffold in both types of samples, with well-defined porosity. The presence of bioactive glass particles embedded within the fiber matrix was visually confirmed and validated through elemental composition via EDX, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ee. Furthermore, the XPS analysis reveals significant differences in surface composition between PCL and PCL/Bioglass printed scaffold templates. The PCL spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ef) shows prominent peaks corresponding to C 1s at ~\u0026thinsp;284.8 eV and O 1s at ~\u0026thinsp;532 eV, consistent with its aliphatic polyester backbone (Si et. al. 2016). In contrast, the PCL/Bioglass spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eg) exhibits additional peaks, notably Si 2p at ~\u0026thinsp;102 eV, Ca 2p at ~\u0026thinsp;347 eV, and P 2p at ~\u0026thinsp;133 eV, confirming successful incorporation of bioglass into the polymer matrix. The relative increase in O 1s intensity in the composite sample indicates enhanced surface oxidation or bonding due to the presence of silicate and phosphate groups from bioglass. Thus, the analysis confirms the effective integration of bioglass particles into PCL matrix, supporting its potential for tissue engineering application, particularly for skeletal tissue regeneration by calcium-phosphate-rich phases (Tolmacheva et al., 2024; Furko et. al., 2025).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.4. \u003cem\u003eIn vitro\u003c/em\u003e bioassay\u003c/h2\u003e\u003cp\u003eThe cytocompatibility of the fabricated PCL and PCL/BG composite fibrous scaffolds was further evaluated through hemolysis and protein absorption assays. Both studies are critical for biomedical scaffolds, especially for applications involving direct cell and blood contact. The hemolysis assay results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, where the OD of hemoglobin released from lysed red blood cells was measured. The percentage of hemolysis remained below 5%, which is the acceptable threshold for both types of scaffold, as per ASTM standard F756 guidelines, indicating non-hemolytic behaviour. The SEM micrographs further support RBC morphology and adhesion of RBCs on both PCL (Fig.\u0026nbsp;9b) and PCL/BG (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) scaffolds without apparent morphological damage or cell lysis. Notably, the PCL/BG scaffold shows more extensive RBC attachment and spreading, possibly due to enhanced surface roughness and ionic bioactivity imparted by the bioglass particles, which can promote better protein adsorption (Baier et. al., 2022), as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ec. Additionally, the PCL/BG composite scaffold exhibited significantly higher protein adsorption compared to pure PCL. This enhanced adsorption is advantageous for bone regeneration and wound healing applications, where effective interaction with biological fluids is essential for initiating cellular responses and tissue integration. Therefore, biological performance of the 3D-printed PCL/BG fibrous scaffold template was evaluated using the L929 fibroblast cell line. The MTT assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ee) results depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ed demonstrate enhanced cellular viability on PCL/BG electrospun scaffolds compared to PCL scaffold as the control. This improvement can be attributed to the presence of bioactive glass, which enhances surface bioactivity and promotes better cell-scaffold interactions. These findings were further supported by the time-dependent live/dead assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ef), where predominance of viable cells (stained green) with few dead cells (stained red) were observed, particularly on day 5. Additionally, Rhodamine/DAPI staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eg) revealed more extensive cytoskeletal spreading and nuclear distribution, indicating improved cellular morphology, attachment, and proliferation on the PCL/BG fibrous scaffold template. Complementary SEM micrographs provided further confirmation, showing direct evidence of cell adhesion and spreading across the fibrous matrix, suggesting a favorable microenvironment for cellular activities critical for tissue regeneration.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Printing of Anatomical Model\u003c/h2\u003e\u003cp\u003eFollowing the evaluation of the biological activity of the MWE fibrous scaffold, the feasibility of onsite scaffold development using this facile technique was further assessed through the printing of a bone anatomical model. This system demonstrates a platform for the fabrication of bioactive cue-based porous patient-specific scaffolds at the damaged site during surgery, as depicted in Fig.\u0026nbsp;6a. The results highlight the system capability to produce anatomically relevant models on a large scale by tailoring its mechanical properties and design morphology based on site-specific application and loading direction. The detailed G-Code modelling following bone anatomy based on the CT extracted CAD data is shown in Fig.\u0026nbsp;6b, followed by fabrication of full scale single and multilayed fibrous anatomical model is shown in Fig.\u0026nbsp;6d-d. However, further advancements are necessary to develop full-scale anatomical models in a short period of time for streamlined clinical translation.\u003c/p\u003e\u003cp\u003eThis study successfully establishes a facile melt writing (MWE) system for the fabrication of bioactive, ultrathin fibrous 3D scaffolds with high anatomical fidelity. Varying in porosity and design hierarchy evidences the scaffold potential in site-specific tissue healing applications. The integration of bioactive glass into PCL fibers significantly enhanced the biological performance by improving protein adsorption, cell adhesion, and proliferation, as confirmed by \u003cem\u003ein vitro\u003c/em\u003e assays. Optimized processing parameters, especially nozzle-to-collector distance, with low voltage enabled uniform fiber formation with potential for live cell printing. Exploring alternative polymer/ceramic combinations, adjusting fiber orientation for anisotropic tissues, and embedding therapeutic agents (e.g., growth factors, antibiotics) within the fiber matrix could be pursued in the near future to further enhancement of biological performance within the scaffold templates. Moreover, long-term scaffold performance in complex tissue regeneration can be validated using small animal models. The ability to fabricate CT-based anatomical models further highlights the system potential for onsite patient-specific scaffold development, supporting the creation of structurally and biologically relevant constructs suitable for clinical use. Further development is required for the rapid fabrication of full-scale constructs suitable for clinical translation.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eA robust and versatile MWE-based fabrication platform for the development of bioactive, ultrafine fibrous scaffolds with anatomically relevant geometries has been explored. The incorporation of bioactive cues into the thermoplastic biocompatible polymer matrix significantly enhanced the scaffold biofunctionality. Process optimization, specifically low-voltage operation and controlled nozzle-to-collector distance, enabled consistent fiber deposition with defined porosity and mechanical integrity, critical for replicating the native extracellular matrix with native anisotropy and porosity for cellular migration and tissue integration. Thus, the system highlights its potential in customized scaffold printing towards improving biological performance for tissue engineering applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflicts of interest\u003c/h2\u003e\u003cp\u003eThe authors declare an Indian Patent application (No. 202431073007) titled \u0026ldquo;A Facile System and Method for Melt-Electro-Writing of Ultrathin Fibrous 3D Scaffold and Tubular Graft,\u0026rdquo; based on competing financial interests associated with ICMR and IIT Kharagpur as the funding agencies. The remaining authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003eThe authors declare an Indian Patent application (No. 202431073007) titled \u0026ldquo;A Facile System and Method for Melt-Electro-Writing of Ultrathin Fibrous 3D Scaffold and Tubular Graft,\u0026rdquo; based on competing financial interests associated with ICMR and IIT Kharagpur as the funding agencies. The remaining authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMr. S. Das: System development, biotemplate fabrication, experimental study, materials characterisation, In Vitro Assay, and manuscript preparation; Mr. N. Kashyap\u0026ndash; System customisation, biotemplate fabrication, experimental study; Mr. N. Dogra\u0026ndash;In vitro Study and data finalisation; Prof. S. Dhara \u0026ndash; Conceptualisation, Study design, Manuscript finalisation.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors express their gratitude to DMIHER, Wardha for supporting us in CT data collection. This research was supported by the Indian Council of Medical Research (ICMR) (Proposal ID :2020-4833), Indian Council of Medical Research (ICMR) (ICMR-DHR CoE at IIT Kharagpur). ICMR project Ref. 5/3/8/82/2020-ITR dt. 02-02-2022. The authors also acknowledge IIT Kharagpur for fellowship and Central Research Facility, IIT KGP for support in materials characterization.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRajasekaran R et al (2023) Polyaniline Doped Silk Fibroin-PCL Electrospun Fiber: An Electroactive Fibrous Sheet for Full-Thickness Wound Healing Study. 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Biomaterials Adv 134:112540. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bioadv.2022.112540\u003c/span\u003e\u003cspan address=\"10.1016/j.bioadv.2022.112540\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":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":"Melt electrowriting, Computer-aided design, Biofunctionalization, In-vitro study, Anatomical model","lastPublishedDoi":"10.21203/rs.3.rs-7860311/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7860311/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Personalized Three-dimensional (3D) printed scaffolds represent an advancement in tissue engineering and regenerative medicine, offering customizable implants that closely match the unique anatomical and pathological needs of individual patients. 3D printing of polymeric scaffolds enables the precise fabrication of complex structures with customizable porosity and mechanical properties. Despite the potential of 3D printing, achieving interconnected fibrous hierarchy that closely mimic the natural extracellular matrix remains a challenge. In this context, melt electrospinning and its utilization in near field electrowriting could be an emerging technique in the field of tissue engineering for tailorable architecture and mechanical properties that meet the specific requirements of various tissue types. But the challenges remain in optimizing processing parameters to ensure reproducibility and functionality of the scaffolds in cost effective way. Hence, we aim to refine this knowledge by developing a specialized 3D printing system that reduces polymer viscosity through controlled heating while enhancing electrical conductivity. Bioactive cues were integrated into fibrous scaffolds to improve biological activity and maintain mechanical strength for native tissue models. Process parameters were systematically optimized, and scaffold properties were characterized through physicochemical analyses and cellular assays, demonstrating effective cell–matrix interactions and anisotropy comparable to native tissue, outperforming conventional electrospun scaffolds.","manuscriptTitle":"Near Field Melt-Electrowriting of Bioglass loaded Ultrathin Fibrous 3D-Hierarchy as Tissue Engineering Template – A Practical Approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-20 11:33:56","doi":"10.21203/rs.3.rs-7860311/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":"4c57e25e-9048-44f8-be33-c982c1c791f4","owner":[],"postedDate":"November 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T07:39:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-20 11:33:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7860311","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7860311","identity":"rs-7860311","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}