Melt Electrowriting Research Articles

Optimizing melt electrowriting prototypes for printing non‐medical and medical grade polycaprolactone meshes in prolapse repair

Optimizing melt electrowriting prototypes for printing non‐medical and medical grade polycaprolactone meshes in prolapse repair

Engineering hiPSC-derived tissue models for advanced amyloidosis research and therapy development

Abstract Introduction Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is a life-threatening disease currently lacking an optimal therapy, stemming from the absence of representative models. Our work aims to stablish for the first time an all-human 3D model of ATTR-CM where the main cellular and extracellular players of the pathology are present. Methodology A human induced pluripotent stem cell (hiPSC) line was derived from a patient carrying a p.Ser43Asn TTR mutation and differentiated into hepatocytes (hiPSC-HEPs), cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (hiPSC-CFs). Cells were embedded in a hydrogel (tailored for each cell type) and combined with a designed 3D printed scaffold using Melt Electro Writing (MEW) technology. Tissues were maintained in culture for up to 4 weeks and characterized by staining, albumin production, electrophysiological, plus genomic analysis. Exposure to TTR fibrils (TTR-FIB) was evaluated. Results Human hepatic and cardiac functional tissues can be generated using MEW-designs. These constructs exhibited albumin production and an active spontaneous contraction, respectively. Moreover, tissues displayed well distributed cells throughout the 3D-structure, positive for specific hepatic and cardiac mature markers. Interestingly, electrophysiological analysis showed that cardiac tissues displayed shortened calcium transients after TTR-FIB exposure compared to unexposed tissues. However, no differences were found in terms of beat rate, metabolic activity and gene expression for the concentrations used. Conclusion We have developed an advanced model of ATTR-CM capable of recapitulating the multisystem complexity of the disease, which will contribute to advancing our understanding of the mechanisms of the disease, and help discovering and formulating new treatments.

Understanding the Significance of Layer Bonding in Melt Electrowriting.

Melt electrowriting (MEW) is a high-resolution additive manufacturing technology capable of depositing micrometric fibers onto a moving collector to form 3D scaffolds of controlled mechanical properties. While the critical role of layer bonding to achieve mechanical integrity in fused deposition modeling has been widely reported, it remains largely unknown in MEW, in part due to a lack of methods to assess it. Here, a systematic framework is developed to unravel the significance of layer bonding in MEW scaffolds and its ultimate effect on their mechanical properties. Results show that printing parameters, scaffold design, and print path have a strong impact on layer bonding strength of poly(ɛ-caprolactone) MEW scaffolds. This study demonstrates that a small increase of 5µm in fiber diameter can enhance the layer bonding strength by as much as 70%, greatly impacting the overall scaffold properties. A method is also established to control MEW scaffold layer bonding using a heated collector. Importantly, this study reveals that scaffold architecture alone is not responsible for the overall mechanical properties. Finally, a method to obtain tailored layer bond strengths within a given scaffold is established. This has significant implications as provides new possibilities to control mechanical properties of MEW scaffolds through layer bonding.

Medical- and Non-Medical-Grade Polycaprolactone Mesh Printing for Prolapse Repair: Establishment of Melt Electrowriting Prototype Parameters

Pelvic organ prolapse (POP) occurs due to inadequate support of female pelvic organs and is often treated with synthetic implants. However, complications like infections, mesh shrinkage, and tissue erosion can arise due to biomechanical incompatibilities with native tissue. This study aimed to optimize the melt electrowriting process using medical-grade biodegradable Poly(ε-caprolactone) (PCL) with a pellet extruder to print meshes that mimic the mechanical properties of vaginal tissue. Square and diagonal mesh designs with filament diameters of 80 µm, 160 µm, and 240 µm were produced and evaluated through mechanical testing, comparing them to a commercial mesh and sheep vaginal tissue. The results showed that when comparing medical-grade with non-medical-grade square meshes, there was a 54% difference in the Secant modulus, with the non-medical-grade meshes falling short of matching the properties of vaginal tissue. The square-shaped medical-grade PCL mesh closely approximated vaginal tissue, showing only a 13.7% higher Secant modulus and a maximum stress of 0.29 MPa, indicating strong performance. Although the diagonal-shaped mesh exhibited a 14% stress difference, its larger Secant modulus discrepancy of 45% rendered it less suitable. In contrast, the commercial mesh was significantly stiffer, measuring 77.5% higher than vaginal tissue. The diagonal-shaped mesh may better match the stress–strain characteristics of vaginal tissue, but the square-shaped mesh offers stronger support due to its higher stress–strain curve. Overall, meshes printed with medical-grade PCL show superior performance compared to non-medical-grade meshes, suggesting that they are a promising avenue for future advancements in the field of POP repair.

Fabrication of ZnO-loaded, polycaprolactone -based, antibacterial 3D scaffolds for bone tissue engineering using melt-electrowriting

The potential of melt-electrowriting (MEW) to fabricate highly porous 3D composite scaffolds for bone tissue engineering (BTE) with antibacterial attachment properties was explored. Zinc oxide (ZnO) nanoparticles (NPs) were loaded into biodegradable medical grade Polycaprolactone (PCL) loaded with 10 wt% hydroxyapatite (HA) NPs to create antibacterial constructs by the MEW technique. The constructs’ morphology, chemical, thermal and mechanical properties, osteoblasts cell viability and bacterial attachment were investigated. Our results demonstrate a unique homogeneous dispersion of ZnO-HA NPs in the outer shell of the fabricated composite fibres by MEW, leading to significant inhibition of bacterial attachment against microorganisms. This novel composite MEW 3D scaffolds yielded promising multifunctional constructs with high potential in BTE.

Spatial Dimension Cues Derived From Fibrous Scaffolds Trigger Mechanical Activation to Potentiate the Paracrine and Regenerative Functions of MSCs via the FAK-PI3K/AKT Axis

Secretomes from mesenchymal stem cells (MSCs) have significant therapeutic potential and could be the basis for future MSCs treatments. Innovative design of the topology of biomaterials, which mechanically regulate cell behavior and function, can tremendously improve the efficacy of stem cell therapy. However, how spatial dimension cues derived from specific topology command cell mechanotransduction to regulate the paracrine function of MSCs remains unknown. In this study, the three-dimensional (3D) fibrous constructs with box-like pores and precise strand spacing from 150 μm down to only 40 μm were manufactured using melt electrowriting (MEW), which were used to systematically investigate the spatial dimension cues-triggered mechanotransduction of adipose-derived mesenchymal stem cells (Ad-MSCs) and their impact on the paracrine and regeneration function of Ad-MSCs. The results demonstrated that spatial instructions from the 3D fibrous constructs could influence the spatial reorganization of the cytoskeleton, resulting in cell elongation and augmented immunomodulatory and angiogenic paracrine effects of Ad-MSCs, which was most pronounced at a minimum strand spacing of 40 μm. Besides, mechanical activation of the FAK-PI3K/AKT axis significantly enhanced the paracrine function of Ad-MSCs. In vivo experiments demonstrated that the Ad-MSCs trained using well-defined 3D fibrous constructs with a strand spacing of 40 μm significantly promoted skin regeneration via paracrine signals. In conclusion, this study provides a new horizon for deciphering space dimension insights into the interactional mechanisms of mechanotransduction in regulating cell function, which has inspired innovations in biomaterials for improving tissue regeneration. Statement of SignificanceDesigning cell-scale spatial dimension cues in engineered constructs potentiates the paracrine and regenerative functions of Ad-MSCs.FAK-PI3K/AKT signaling serves as the key mechanotransduction checkpoints for spatial dimension cues triggering mechanical activation.Paracrine signals of Ad-MSCs triggered by mechanical activation promotes skin repair and regeneration via the immunomodulation and angiogenesis.

Fabrication of 3D Bioactive Melt Electrowriting Composite Scaffold with High Osteogenic Potential

A key challenge in using melt electrowriting (MEW) technology is incorporating large amounts of bioactive inorganic materials, such as hydroxyapatite (HA). In the present study, following optimization of the fabrication parameters, 40%-HA (HA40) nanoparticles were pre-mixed into medical-grade polycaprolactone (PCL) and processed using the MEW (MEW) technique to mimic the structure and function of the natural extracellular matrix (ECM) for bone regeneration. The HA40 fibrous composite scaffolds showed continuous writing and obtained a well-connected and orderly stacked fibre with a small diameter size (67 ± 8.5µm). A major result of the present study was the successful enrichment and accumulation of the HA particles, which mostly occurred on the MEW fibre external surfaces. This design allows for direct interfacial interaction with human periodontal ligament cells (hPDLCs). We systematically investigated the behaviour and function of hPDLCs on the HA40 composite scaffold, alongside parameters related to mineralization. The HA40 scaffold demonstrated significantly higher metabolic activity and enhanced expression of osteonectin and RUNX2 compared to PCL-only scaffolds, as well as increased levels of ALP and COL1. The study’s findings demonstrate that bioactive composite scaffolds, incorporating 40% HA into m-PCL via MEW, effectively enhance the biological response of the ECM and are promising for potential applications in bone regeneration.

3D Printed Mesh Geometry Modulates Immune Response and Interface Biology in Mouse and Sheep Model: Implications for Pelvic Floor Surgery.

Pelvic organ prolapse (POP) is a highly prevalent yet neglected health burden for women. Strengthening thepelvic floor with bioactive tissue-engineered meshes is an emerging concept. The study investigates tissue regenerative design parameters, including degradability, porosity, and angulation, to develop alternative degradable melt electrowritten (MEW) constructs for surgical applications of POP. MEW constructs are fabricated in hierarchical geometries by two-way stacking of the fibers with three different inter layer angles of 90°, 45°, or 22.5°. Implants printed at 22.5° have higher tensile strength under dry conditions and show better vaginal fibroblast (VF) attachment in vitro. In vivo assessment using preclinical mouse and ovine models demonstrates more effective degradation and improved tissue integration in 22.5° angular meshes compared to 90° and 45° meshes, with evidence of neo-collagen deposition within implants at 6 weeks. The pattern and geometry of the layered MEW implants also influence the foreign body response, where in the anti-inflammatory phenotype shows a greater ratio of anti-inflammatory CD206+ M2 macrophages/pro-inflammatory CCR7+ M1 macrophages. This presents an attractive strategy for improving the design and fabrication of next-generation vaginal implants for pelvic reconstructive surgery.

Advances in melt electrowriting for cardiovascular applications.

Melt electrowriting (MEW) is an electric-field-assisted additive biofabrication technique that has brought significant advancements to bioinspired scaffold design for soft tissue engineering and beyond. Owing to its targeted microfiber placement, MEW has become a powerful platform technology for the fabrication of in vitro disease models up to functional biohybrid constructs that are investigated in vivo to reach clinical translation soon. This work provides a concise overview of this rapidly evolving field by highlighting the key contributions of MEW to cardiovascular tissue engineering. Specifically, we i) pinpoint the methods to introduce microvascular networks in thick 3D constructs benefitting from (sacrificial) MEW microfibers, ii) report MEW-based concepts for small-diameter vascular grafts and stents, iii) showcase how contracting cardiac tissues can profit from the tunable structure-property relationship of MEW scaffolds, and iv) address how complete regenerative heart valves can be built on complex fiber scaffold architectures that recapitulate J-shaped tensile properties and tissue heterogeneity. Lastly, we touch on novel biomaterial advancements and discuss the technological challenges of MEW to unlock the full potential of this transformative technology.

Electrospinning and melt electrowriting of a tunable triblock-copolymer composed of poly(ε-caprolactone) and poly(L-lactic acid) for biomedical applications

Both electrospinning (ES) and melt electrowriting (MEW) garner emerging interest within the field of biomedical applications. Poly(ε-caprolactone) (PCL) is still the gold standard for both techniques, demonstrating excellent processability due to its solubility, melt flow properties and thermal stability. However, when evaluating biomaterials for soft tissue applications, PCL exhibits limitations such as low elasticity and extended biodegradation times. Additionally, the limited availability of materials that are processable by MEW constrains their broader clinical translation. To address this limitation, a tunable triblock-copolymer of PCL and poly(L-lactic acid) (PLLA) is synthesized and processed using ES and MEW. This synthesis employs a PCL-diol backbone with a molar mass of 30 000 g mol-1, to which various lengths of PLLA end-blocks are added on both termini (PLLA-b-PCL-b-PLLA). The successful incorporation of these tunable end-blocks is verified through spectroscopic analysis. The ES of PLLA-b-PCL-b-PLLA reveals excellent fiber formation, with fiber diameters ranging between 2–4 µm as the PLLA molar mass increases. The tunable nature of the polymer allows for MEW at 90 °C, producing fibers with diameters ranging between 40–50 µm. Additionally, the materials exhibit adjustable mechanical properties and biodegradation rates by varying the PLLA segment lengths. Finally, in vitro cell testing using MC3T3-E1 cells demonstrates the polymer's excellent biocompatibility.

Quantum Sensing Unravels Antioxidant Efficacy Within PCL/Matrigel Skin Equivalents.

Skin equivalents (SE) that recapitulate biological and mechanical characteristics of the native tissue are promising platforms for assessing cosmetics and studying fundamental biological processes. Methods to achieve SEs with well-organized structure, and ideal biological and mechanical properties are limited. Here, the combination of melt electrowritten PCL scaffolds and cell-laden Matrigel to fabricate SE is described. The PCL scaffold provides ideal structural and mechanical properties, preventing deformation of the model. The model consists of a top layer for seeding keratinocytes to mimic the epidermis, and a bottom layer of Matrigel-based dermal compartment with fibroblasts. The compressive modulus and the biological properties after 3-day coculture indicate a close resemblance with the native skin. Using the SE, a testing system to study the damage caused by UVA irradiation and evaluate antioxidant efficacy is established. The effectiveness of Tea polyphenols (TPs) and L-ascorbic acid (Laa) is compared based on free radical generation. TPs are demonstrated to be more effective in downregulating free radical generation. Further, T1 relaxometry is used to detect the generation of free radicals at a single-cell level, which allows tracking of the same cell before and after UVA treatment.

A versatile 5-axis melt electrowriting platform for unprecedented design freedom of 3D fibrous scaffolds

Melt electrowriting (MEW) is an electrohydrodynamic additive manufacturing technology for the fabrication of precise microfiber scaffold architectures but is so far restricted to printing onto simple collector geometries and, therefore, constrained in design freedom. This is due to current limitations in print head designs, kinematic systems, and software to generate motion commands. We address these aspects by upgrading a low-cost 5-axis kinematic system and complete it with a custom-designed print head and a collector fabrication workflow to enable collision-free MEW onto arbitrarily shaped multicurvature 3D geometries. A universal graphical approach enables generation of Gcodes customized to the 5-axis MEW requirements. The print head is validated by producing polycaprolactone fibers with diameters ranging from 5.6 ± 1.7 µm to 50.7 ± 5.5 µm and by writing both linear and non-linear fiber patterns onto collectors featuring concave and convex surfaces. Stable printing conditions are achieved by continuously reorienting the print head and collector surface perpendicular to each other at a constant working distance. Ultimately, we demonstrate the system’s potential in shaping anatomically relevant scaffold geometries by stacking microfibers onto collectors resembling a trileaflet valve and a bifurcating vessel. This multi-axis MEW platform unlocks exciting avenues towards unprecedented design freedom for MEW scaffolds.

Muticomponent Melt‐Electrowritten Vascular Graft to Mimic and Guide Regeneration of Small Diameter Blood Vessels

AbstractCardiovascular disease is a leading cause of morbidity. Current treatments include vessel substitution using autologous/synthetic vascular grafts, but these commonly fail in small diameter applications, largely due to compliance mismatch and clot formation. In this study, a multicomponent vascular graft, that takes inspiration from native vessel architecture, is developed to overcome these limitations. Melt electrowriting (MEW) is used to produce tubular scaffolds with vascular‐mimetic fiber architecture and mechanics, which is combined with a lyophilized fibrinogen matrix with tailored degradation kinetics to generate a hybrid graft. The MEW framework not only contributes to graft mechanics, but also provides contact guidance to direct cell/neotissue orientation and mimic the native tunica media. This construct is further functionalized with heparin, which in combination with the smooth extracellular matrix (ECM) surface, reduced platelet adhesion and clot formation providing a substrate for endothelization, thereby mimicking the function of the intima. Lastly, an outer electrospun layer representing the adventitia is added to improve elasticity and reduce permeability. This graft satisfies ISO implantability requirements, matches the compliance of native vessels, and reestablishes physiological flow with minimal clot formation in a preclinical model. Therefore, this graft represents an innovative off‐the‐shelf alternative to address the unmet clinical need for small‐diameter vascular grafts.

Accessible melt electrowriting three-dimensional printer for fabricating high-precision scaffolds

Melt electrowriting (MEW) is an established 3D printing technology for producing high-resolution scaffolds for tissue engineering. Commercial MEW devices are expensive while having limited processing capabilities, which limits their accessibility and widespread use. Herein, we report an easy-to-assemble and inexpensive ($2000) MEW printer based on an off-the-shelf automated dispensing machine with user-friendly controls and a custom-designed MEW printhead capable of printing polymers with a melting point of up to 260 °C. Using the gold standard printable material, poly(ε-caprolactone) (PCL), this printer can deposit 3 μm diameter fibers at 50 μm spacing, demonstrating that the highest quality of scaffolds can be produced on the low-cost MEW system. Stability in printing is improved by maintaining a relative humidity of 30–35 %, ensuring nozzle conditions are optimal, and minimizing bubbles in the polymer melt. Key printing parameters such as a 3 kV applied voltage, 75 °C heating temperature, 100 kPa air pressure, and 30 G nozzle size are critical for high accuracy, achieving 100 μm fiber spacing and layer stacking up to 30 layers without fiber breakage. The printer effectively creates complex scaffold geometries, showcasing its precision and suitability for advanced tissue engineering applications. Overall, this study promotes low-cost MEW technology by establishing a guideline on building an accessible but capable MEW printer and providing a crucial experimental foundation toward achieving high-accuracy scaffolds.

Artificial Trabecular Meshwork Structure Combining Melt Electrowriting and Solution Electrospinning.

The human trabecular meshwork (HTM) is responsible for regulating intraocular pressure (IOP) by means of gradient porosity. Changes in its physical properties, like increases in stiffness or alterations in the extracellular matrix (ECM), are associated with increases in the IOP, which is the primary cause of glaucoma. The complexity of its structure limits the engineered models to one-layered and simple approaches, which do not accurately replicate the biological and physiological cues related to glaucoma. Here, a combination of melt electrowriting (MEW) and solution electrospinning (SE) is explored as a biofabrication technique used to produce a gradient porous scaffold that mimics the multi-layered structure of the native HTM. Polycaprolactone (PCL) constructs with a height of 20-710 µm and fiber diameters of 0.7-37.5 µm were fabricated. After mechanical characterization, primary human trabecular meshwork cells (HTMCs) were seeded over the scaffolds within the subsequent 14-21 days. In order to validate the system's responsiveness, cells were treated with dexamethasone (Dex) and the rho inhibitor Netarsudil (Net). Scanning electron microscopy and immunochemistry staining were performed to evaluate the expected morphological changes caused by the drugs. Cells in the engineered membranes exhibited an HTMC-like morphology and a correct drug response. Although this work demonstrates the utility of combining MEW and SE in reconstructing complex morphological features like the HTM, new geometries and dimensions should be tested, and future works need to be directed towards perfusion studies.

In-situ electret scaffolds with controllable electric fields printed by MEW for bone tissue regeneration

Physiologically relevant electrical microenvironments play an integral role in manipulating bone metabolism. Although implanted biomaterials using conductive or piezoelectric materials to mimic natural tissue electrical properties have been introduced into the field of bone regeneration, the use of electret materials to provide stable and durable electrical stimulation has rarely been studied in biomaterial design. In this study, the ZnO/PCL composite scaffolds with in-situ electret were fabricated using melt electro-writing to explore its efficacy in bone regeneration. By adjusting the electret concentration, the surface potential of the composite scaffolds could be tuned to the biopotential for promoting bone regeneration, and the prepared scaffolds exhibited good electrical stability over an observation period of up to 42 days. In vitro biological experiments showed that the in-situ electret scaffolds promoted the cell viability and osteogenic differentiation of mesenchymal stem cells. At the same time, the in-situ electret composite scaffolds can induce the phenotype transformation of RAW 264.7 cells from M1 to M2, creating a favorable microenvironment for bone tissue regeneration. The composite scaffolds significantly promoted bone regeneration in vivo through sustained endogenous electrical stimulation. These findings suggest that the in-situ electret scaffolds that maintain a stable and physiological electrical microenvironment are promising candidates for enhanced bone regeneration.

The Design and Fabrication of Engineered Tubular Tissue Constructs Enabled by Electrohydrodynamic Fabrication Techniques: A Review

AbstractElectrohydrodynamic processes have emerged as promising methods for fabricating polymetric fiber‐based artificial tubular tissues. Existing review articles focus on the biological applications and processing materials associated with electrohydrodynamic processes in artificial tubular constructs, while overlooking the design and fabrication of these constructs. To address this gap, this review article emphasizes the design and fabrication of tubular tissue constructs enabled by employing electrohydrodynamic processes. This article begins by presenting an overview of two electrohydrodynamic processes: solution electrospinning (SE) and melt electrowriting (MEW). It then delves into the control of the fiber diameter enabled by SE and MEW, offering insights into the manipulation of processing parameters to achieve desired fiber diameters. Additionally, the review highlights cutting‐edge strategies for electrohydrodynamic processes to create tubular structures with customized microarchitectures. This includes fiber alignment control for SE and pore morphology design for MEW. Moreover, the review covers the creation of customized macroscale tubular geometries through collector geometry design. Lastly, a comprehensive survey is presented for designing multiphasic tubular structures specifically for electrohydrodynamic techniques or in tandem with other techniques. The objective of this review is to offer a thorough understanding of the design considerations and potential applications of tubular structures fabricated by electrohydrodynamic processes.

A Melt Electrowriting Toolbox for Automated G‐Code Generation and Toolpath Correction of Flat and Tubular Constructs

A Melt Electrowriting Toolbox for Automated G‐Code Generation and Toolpath Correction of Flat and Tubular Constructs

Integration of Melt Electrowritten Polymeric Scaffolds and Bioprinting for Epithelial Healing via Localized Periostin Delivery.

Management of skin injuries imposes a substantial financial burden on patients and hospitals, leading to diminished quality of life. Periostin (rhOSF), an extracellular matrix component, regulates cell function, including a proliferative healing phase, representing a key protein to promote wound healing. Despite its proven efficacy in vitro, there is a lack of scaffolds that facilitate the in situ delivery of rhOSF. In addition, there is a need for a scaffold to not only support cell growth, but also to resist the mechanical forces involved in wound healing. In this work, we synthesized rhOSF-loaded mesoporous nanoparticles (MSNs) and incorporated them into a cell-laden gelatin methacryloyl (GelMA) ink that was bioprinted into melt electrowritten poly(ε-caprolactone) (PCL) microfibrous (MF-PCL) meshes to develop mechanically competent constructs. Diffraction light scattering (DLS) analysis showed a narrow nanoparticle size distribution with an average size of 82.7 ± 13.2 nm. The rhOSF-loaded hydrogels showed a steady and controlled release of rhOSF over 16 days at a daily dose of ∼40 ng/mL. Compared with blank MSNs, the incorporation of rhOSF markedly augmented cell proliferation, underscoring its contribution to cellular performance. Our findings suggest a promising approach to address challenges such as prolonged healing, offering a potential solution for developing robust, biocompatible, and cell-laden grafts for burn wound healing applications.

Antibacterial and Osteogenic Dual-Functional Micronano Composite Scaffold Fabricated via Melt Electrowriting and Solution Electrospinning for Bone Tissue Engineering.

The utilization of micronano composite scaffolds has been extensively demonstrated to confer the superior advantages in bone repair compared to single nano- or micron-sized scaffolds. Nevertheless, the enhancement of bioactivities within these composite scaffolds remains challenging. In this study, we propose a novel approach to combine melt electrowriting (MEW) and solution electrospinning (SES) techniques for the fabrication of a composite scaffold incorporating hydroxyapatite (HAP), an osteogenic component, and roxithromycin (ROX), an antibacterial active component. Scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR) confirmed the hierarchical architecture of the nanofiber-microgrid within the scaffold, as well as the successful loading of HAP and ROX. The incorporation of HAP enhanced the water absorption capacity of the composite scaffold, thus promoting cell adhesion and proliferation, as well as osteogenic differentiation. Furthermore, ROX resulted in effective antibacterial capability without any observable cytotoxicity. Finally, the scaffolds were applied to a rat calvarial defect model, and the results demonstrated that the 20% HAP group exhibited superior new bone formation without causing adverse reactions. Therefore, our findings present a promising strategy for designing and fabricating bioactive scaffolds for bone regeneration.