Search Results (102)

Schwann cells (SCs) are critical effectors of peripheral nerve regeneration, and their interaction with biomaterial scaffolds is a key parameter in neural tissue engineering. This pilot study described and evaluated protocols for a morphological, quantitative, and morphometric analysis of SCs seeded on electrospun polyhydroxybutyrate (PHB) scaffolds using variable-pressure scanning electron microscopy (VP-SEM) under a low vacuum, without a metal coating. Six protocols were compared, varying the number of seeded cells (50,000 or 100,000) and the method used to label the seeded face of the scaffold: no marking, graphite pencil, or permanent ink (Sharpie). Confocal microscopy confirmed SC viability and adhesion. The VP-SEM analysis revealed that seeding 100,000 cells significantly increased the number of detectable cells on the scaffold surface. Graphite labeling was associated with higher cell counts and a more stellate morphology, consistent with the biocompatibility of carbon-based materials reported in the literature. Conversely, ink labeling appeared to inhibit SC adhesion. A refined protocol for measuring SC extensions using ImageJ’s ROI Manager and segmented line tools was also established. These findings provide practical methodological insights to improve the reliability and reproducibility of SC morphological analyses on ultra-thin polymeric scaffolds, with implications for peripheral nerve regeneration research. Full article

Tissue engineering represents a central strategy in regenerative medicine to restore damaged or missing tissue through structural and functional replacement. In this study, a two-component bioink platform was developed based on amine–anhydride conjugation as a mild crosslinking reaction between synthetic anhydride-containing oligomers (oSMoMA-x) and natural biopolymers. The compatibility of the oligomers with different amine-containing biopolymers, including chitosan, gelatin, and hydrolyzed collagen peptides, was systematically evaluated. To improve cytocompatibility and enable controlled network formation, oSMoMA oligomers with varying anhydride contents were synthesized and characterized, allowing targeted tuning of material properties through comonomer composition. The resulting hydrogels were comparatively assessed with respect to their rheological and physicochemical properties. While hydrogel formation was achieved with all investigated biopolymers, gelatin-based systems exhibited the most favorable characteristics for bioink development. Two gelatin/oSMoMA bioink formulations with distinct gelation behavior were obtained by employing different base catalysts, enabling control over crosslinking kinetics and material properties. Cytocompatibility was comprehensively evaluated using viability assays, demonstrating enhanced metabolic activity of cells encapsulated in gelatin/oSMoMA-3.5 hydrogels compared to established reference systems, with sustained compatibility for up to seven days. Extrusion-based 3D bioprinting was performed using a modified printhead with integrated temperature control to maintain physiological conditions. The bioinks were successfully printed with embedded murine 3T3 fibroblasts, and post-printing analyses confirmed cell proliferation within the hydrogel constructs. Overall, the results demonstrate the broad compatibility of amin–anhydride-crosslinked oSMoMA systems with different biopolymers and highlight gelatin/oSMoMA bioinks as promising cytocompatible materials for stable 3D bioprinting applications in tissue engineering. Full article

Extrusion-based bioprinting faces significant challenges in achieving the shape fidelity and internal porosity necessary for cell viability, often hindered by subjective assessment methods. This study investigated the relationship between rheological properties and print quality using a natural polymer biomaterial ink composed of 12% gelatin, 5% alginate, and 1% carboxymethylcellulose. We conducted a comparative analysis between traditional pneumatic systems and screw-driven volumetric extrusion, utilizing a suite of quantitative metrics: Spreading Ratio ($SR$), Printability Index ($P_r$), Uniformity Ratio ($UF$), Collapse Angle ($\theta$), and evaluated porosity. Our results demonstrate that the screw-driven system’s positive displacement mechanism provides superior control over filament morphology by enabling precise volumetric modulation. While the pneumatic system exhibited a high $SR$ of 1.82 and the lowest porosity at 59.92%, the screw-driven system allowed for “under-extrusion” to compensate for viscoelastic die swell. Reducing the flow rate to 50% in the screw system lowered the $SR$ to 1.09, nearly matching the nozzle diameter, and increased porosity to 76.46%. Furthermore, the screw-driven system achieved an ideal $P_r$ of 1.0, whereas the pneumatic system produced distorted, rounded pores with a $P_r$ of 1.57. The findings indicate that screw-driven extruders can decouple line complex rheology from the printing process, allowing for finer spatial resolution and better pore interconnectivity. Full article

Open-source bioprinting can broaden access to biofabrication, enabling existing systems to perform high-resolution tissue manufacturing. However, most of these focus on low cost, easy assembly, or specific biomaterial ink rather than making a robust standardized and modularized multifunction platform. In this study, we present CrystalCells, a user-friendly modular open-source bioprinting system centered on the TridentExtruder, a high-performance syringe extruder with extrusion/retraction capability and tool-free automated syringe coupling. The system enables the automated exchange of syringe, temperature-controlling, microscope, and pipette modules. Repeated syringe return-and-pickup cycles showed repositioning errors within ±20 μm, while the extruder generated pressures above 950 kPa and exhibited lower elastic deformation than the Replistruder 4 under the same pressure conditions. CrystalCells supported the extrusion of pre-crosslinked alginate, FRESH printing, and dual-biomaterial inks printing with automated exchange. A microscope module resolved stained HeLa cells and enabled layer-by-layer imaging for defect detection during printing. A thermoelectric module maintained the syringe barrel below 6 °C during the printing of an alginate–collagen biomaterial ink at 23 °C (room temperature), and a pipette module transferred 2–10 μL volumes with errors within ±0.5 μL. These results show that CrystalCells is an open-source modular biofabrication platform integrating printing, imaging, temperature control, and liquid handling within a single workflow. Full article

The development of biomimetic scaffolds requires balancing structural integrity with biological signaling. This study evaluates kefiran, a microbial exopolysaccharide, as a bioactive component in establishing printing feasibility of 3D composite constructs. Kefiran from Romanian artisanal cultures was characterized via ¹H-NMR, HPLC, and SEM/TEM, confirming a high-quality hexasaccharide repeating unit. Three composite inks (K100, K70, and K50) were developed by integrating kefiran, chondroitin sulfate, and Si-substituted hydroxyapatite into an alginate matrix and processed using a Bio X 3D-printer. Results showed that higher kefiran concentrations improved printing feasibility, providing enhanced structural fidelity and stability during the layer-by-layer deposition process. All bioprinted scaffolds demonstrated high cytocompatibility with L929 fibroblasts, maintaining viability above 70%. Notably, kefiran exhibited dual-functional therapeutic potential: concentrations above 500 mg/L showed a concentration-dependent antiproliferative effect against HT-29 cells at 72 h while remaining safe for normal cells. These findings establish kefiran-based biomaterial inks as robust, bioactive platforms for regenerative medicine. By enhancing both the mechanical printability of alginate composites and the biological response of cultured cells, kefiran proves to be a versatile component for advanced tissue engineering and potential biological activity applications. Full article

Squid ink has recently garnered considerable attention as a natural melanin source for the development of biocompatible nanomaterials. Although numerous studies have explored the biological and therapeutic applications of squid ink, the fabrication and modification strategies for squid ink-derived nanoparticles (SINPs) have yet to be comprehensively reviewed. This paper provides an integrated overview of current extraction, purification, and functionalization strategies for SINPs, with a particular focus on how functionalization approaches modulate their physicochemical characteristics and biological behaviors. The review begins by outlining the natural mechanisms of melanin formation and summarizing common extraction methods—including centrifugation, ultrasonication, and dialysis. Subsequently, various surface modification and hybridization techniques—including polymer coating, incorporation of metallic elements (e.g., Se and Fe), and loading of photosensitizers—are compared in terms of their contributions to functional enhancement. Finally, the challenges of reproducibility, batch-to-batch variability, and scalable manufacturing are discussed, outlining future directions for the development of squid ink-derived nanomaterials into standardized biomedical platforms. Full article

Decellularization removes immunogenic intracellular components of fish tissues while keeping the extracellular matrix (dECM) structure, mechanical integrity, and bioactivity. Fish-derived dECM retains native bioactive components, exhibiting high biocompatibility, low immunogenicity, and biodegradability, while supporting cell adhesion, proliferation, and tissue regeneration. Due to its abundance, minimal ethical concerns, and low zoonotic risks, fish wastes are emerging as sustainable sources of dECM, offering an eco-friendly alternative to mammalian biomaterials. This review highlights advances in decellularizing fish wastes such as skin, scales, bones, viscera, and swim bladders from species including tilapia, tuna, milkfish, carp, goldfish, and sturgeon. Physical, chemical, biological, and hybrid decellularization methods are assessed for cell removal, ECM preservation, and mechanical performance. Recent advances in polymer-dECM composites, crosslinking, and 3D bioprinting have significantly improved scaffold performance, making fish-derived dECM applicable for healing of wounds, regeneration of bone and cartilage, and repair of soft tissues. Despite its potential, challenges remain in optimizing perfusion rates, temperature variations, and tissue-specific protocols, as well as developing eco-friendly decellularization techniques using biodegradable reagents. Future perspectives include expanding decellularized fish tissue sources, innovating bio-inks for 3D bioprinting, and refining tissue-specific processing methods to maximize the potential of fish-derived dECM in regenerative medicine and tissue engineering. Full article

Three-dimensional bioprinting revolutionized tissue and organ replacement by enabling the precise deposition of living cells and biomaterials, making it ideal for biomedical applications. Natural polymers are commonly used as bioink for their biocompatibility and bioactivity. Among them, type I collagen, the most abundant protein of extracellular matrix, is commonly used as bioink. However, mammalian-derived collagens raise concerns related to zoonotic disease transmission, religious restrictions, and immunogenicity. Fish-derived collagen represents a safer and more sustainable alternative, although its rapid degradation and limited mechanical properties remain significant challenges. In this study, the printability of a novel fish collagen ink was assessed for micropatterned scaffolding by extrusion. In order to overcome material-related challenges, the effect of UV-induced crosslinking was investigated. Morphological, rheological, and physicochemical characterizations—including thermal behavior, degradation resistance, exposed chemical groups, and roughness—were performed before and after UV treatment. Results demonstrated that UV crosslinking significantly improved the structural integrity and stability of the printed scaffolds. These findings support the potential of UV-crosslinked fish collagen as biomaterial ink for regenerative medicine and tissue engineering applications. Full article

The development of photocurable hydrogel biomaterial inks with suitable rheology, low cytotoxicity, and tuneable mechanical properties is essential for reliable biofabrication. This study aimed to formulate PEGDA–gelatine–collagen inks using lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as photoinitiator. Rheological characterisation and flow-model fitting were performed, mechanical stiffness modulation under different light intensities was evaluated, complex structures were printed using direct extrusion and FRESH methodologies, and PEGDA/LAP extractables were quantified by NMR after controlled washing procedures. In vitro assays assessed cell viability and proliferation on the resulting scaffolds. The Herschel–Bulkley model best described the flow behaviour across formulations; while viscoelastic measurements showed that increasing light intensity progressively enhanced hydrogel stiffness, enabling fine control over final mechanical properties. NMR analysis showed that washing removed a substantial fraction of residual LAP, in agreement with the biological findings: fibroblasts failed to survive on unwashed scaffolds but exhibited robust proliferation and recovered their characteristic elongated morphology on washed constructs. Among all inks, PeGeCol_10_2 provided the best combination of shear-thinning behaviour, structural integrity, low residual photoinitiator, and tuneable mechanics. Using this formulation, we successfully printed large anatomical models with high fidelity and excellent handling properties, underscoring its potential for soft-tissue prosthetics and broader tissue-engineering applications. Full article

Calcium phosphate cements (CPCs) and biomaterials, such as mesoporous bioactive glass (MBG), are critical for bone tissue engineering. This study aimed to 3D-print CPC scaffolds modified with MBG to enhance their osteogenic potential and regenerative ability. MBG powder was synthesized and characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), and nitrogen adsorption–desorption techniques. A commercial CPC ink (hydroxyapatite/α-tricalcium phosphate) was mixed with 5% MBG (w/w; CPC/MBG), and, after rheological assessment, the mixture was used to obtain scaffolds via 3D printing. These scaffolds were then tested for chemical, morphological, and mechanical properties, as well as ion release analysis. Unmodified CPC 3D-printed scaffolds served as controls. Biological experiments, including cell viability, DNA content, cell adhesion/spreading, and osteogenic gene expression, were performed by seeding alveolar bone-derived mesenchymal stem cells onto the scaffolds. Statistics were performed using Student’s t-test and ANOVA with post hoc tests (α = 5%). MBG characterization showed a typical mesoporous structure with aligned microchannels and an amorphous structure. Both formulations released calcium and phosphate ions; however, CPC/MBG also released silicon. Cell viability, adhesion/spreading, and DNA content were significantly greater in CPC/MBG scaffolds compared to CPC (p < 0.05) after 3 and 7 days of culture. Furthermore, CPC/MBG supported increased expression of key osteogenic genes, including collagen (COL1A1), osteocalcin (OCN), and Runt-related transcription factor 2 (RUNX2), after 14 days (p < 0.05). The combination of CPC ink with MBG particles effectively enhances the biocompatibility and osteogenic potential of the scaffold, making it an innovative bioceramic ink formulation for 3D printing personalized scaffolds for bone regeneration. Full article

Biomaterials are either cross-linked ionically, chemically, or physically, or they can be functionalized with amino acids to overcome inherent biocompatibility and stability limitations. Hydrogels for scaffold fabrication have been effectively utilized to promote tissue integration and cellular processes for soft tissue regeneration. Despite significant progress, poor remodeling limitations persist, hence the need for cross-linkers with dynamic adaptability, native tissue mimicry, and controllable degradation. The aim of this review is to highlight cysteine’s capability and potential to cross-link biomaterials using thiol chemistry while discussing the different cross-linking strategies to aid in the fabrication of robust hydrogel inks and bioinks. Furthermore, cysteine’s limitations and research scarcity in soft tissue scaffolds are highlighted for its chemical significance and potential role. The review examines cysteine’s thiol reactions, including disulfide bonds, thiol–ene, thiol–yne, and Michael additions, and cross-linking ability, with a specialized focus on adipose tissue regeneration. The fabrication methods reviewed include 3D bioprinting, electrospinning, films, and nanostructured scaffolds, with a primary focus on 3D bioprinting of hydrogel scaffolds. Cysteine cross-linking enhances the scaffolds’ stability, printability, biocompatibility, degradability, and biological performance of scaffolds with an 85% increase in Young’s modulus. Cysteine adequately enhances the mechanical properties and degradation rates of adipose tissue scaffolds. This review addresses the underexplored use of cysteine cross-linking in soft tissue scaffolds, beyond its common bone tissue applications. Full article

Three-dimensional (3D) bioprinting has become one of the most advanced and useful innovations that allows the creation of personalized macroscopic and microscopic constructs at different scales that match a patient’s anatomy. Intensive research efforts are currently underway to develop highly printable and biocompatible materials. Among the variety of bioprinting materials (i.e., biomaterial inks), naturally derived hydrogels have attracted great interest due to their beneficial properties in terms of biocompatibility, cost-effectiveness, and biodegradability. In this proceeding paper, we provide an overview of the formulation and use of three functional polysaccharides as ink-based hydrogels. First, 3D bioprinting is summarized as revolutionary technology that is able to create cell-laden structures layer by layer in a specific pattern that mimics native tissue and organs. Cellulose, chitosan, and lignin are presented below, followed by an overview of their applicability in 3D bioprinting, focusing on printability and the resulting printed 3D structures as illustrated in various published figures. In the same way, a comparative overview of 3D bioprinting applications is summarized. Finally, a section dedicated to comparisons, limitations, and crosslinking strategies is provided. It is worth noting that this proceedings paper provides a brief overview rather than a comprehensive review, as it is limited by page constraints and is based on the content of our poster presented at the 1st International Online Conference on Bioengineering. Full article

The increasing demand for novel tissue engineering (TE) applications in bone tissue regeneration underscores the importance of exploring advanced manufacturing techniques and biomaterials for personalised treatment approaches. Three-dimensional printing (3DP) technology facilitates the development of implantable devices with intricate geometries, enabling patient-specific therapeutic solutions. Although Fused Filament Fabrication (FFF) and Direct Ink Writing (DIW) are widely utilised for fabricating bone-like implants, the need for multiple processing steps often prolongs the overall production time. In this study, a single-step melt-extrusion 3DP technique was performed to develop multi-material scaffolds including bioceramics, hydroxyapatite (HA), and β-tricalcium phosphate (TCP) in both their bioactive and calcined forms at 10% and 20% w/w, within polycaprolactone (PCL) matrices. Printing parameters were optimised, and physicochemical properties of all biomaterials and final forms were evaluated. Thermal degradation and surface morphology analyses assessed the consistency and distribution of the ceramics across the different formulations. The tensile testing of the scaffolds defined the impact of each ceramic type and wt% on scaffold flexibility performance, while in vitro cell studies determined the cytocompatibility efficiency. Hence, all 3D-printed PCL–ceramic composite scaffolds achieved structural integrity and physicochemical and thermal stability. The mechanical profile of extruded samples was relevant to the ceramic consistency, providing valuable insights for further mechanotransduction investigations. Notably, all materials showed high cell viability and proliferation, indicating strong biocompatibility. Therefore, this additive manufacturing (AM) process is a precise and fast approach for developing biomaterial-based scaffolds, with potential applications in surgical restoration and support of segmental bone defects. Full article

Organ failure constitutes a significant global concern requiring urgent attention. While organ transplantation offers prospective treatment, it remains suboptimal. The scarcity of donor organs and the need for lifelong immunosuppressive treatments highlight the necessity for innovative approaches in regenerative medicine. In response, tissue engineering has emerged as a promising alternative, particularly through advancements in three-dimensional (3D) and four-dimensional (4D) printing technologies. These approaches enable the fabrication of complex, patient-specific constructs for regenerating tissues such as skin, bone, cartilage, and vascularized organs. This review systematically examines 3D printing techniques, commonly used biomaterials (e.g., hydrogels, bio-inks, and polymers), and their applications in dermal, cardiovascular, bone, and neural regeneration. In addition to discussing 3D technology, an introduction to 4D bioprinting is provided, enabling advanced biomedical applications and establishing itself as an innovative tool that enhances the classic approach to 3D bioprinting in the context of regenerative medicine. Finally, key challenges and ethical considerations are discussed to provide a comprehensive perspective on the current state and future of printed scaffolds in regenerative medicine. Full article

Approximately 20–30% of cultivated oyster mushrooms (Pleurotus ostreatus) are classified as low grade due to morphological and visual imperfections or mechanical damage, representing significant waste in mushroom production systems. This review examines the structural and biochemical properties of P. ostreatus, particularly focusing on cell wall components including chitin, β-glucans, and mannogalactans, which provide crucial rheological characteristics for 3D printing. The literature results demonstrate that these natural polysaccharides contribute essential viscosity, water-binding capacity, and mechanical stability required for printable edible inks. Notably, the mushroom stipe contains significantly higher concentrations of glucans compared to the cap, with 57% more α-glucans and 33% more β-glucans. The unique combination of rigidity from chitin, elasticity from β-glucans, and water retention capabilities creates printable structures that maintain shape fidelity while delivering nutritional benefits. This approach addresses dual challenges in sustainable food systems by reducing agricultural waste streams while advancing eco-friendly food innovation. The integration of mushroom-derived biomaterials into 3D printing technologies offers a promising pathway toward developing nutrient-rich, functional foods within a regenerative production model. Full article