Non-compressible hemorrhage (NCH) remains a leading cause of preventable trauma mortality because conventional gauze and oxidized cellulose act as passive barriers, whereas fibrin sealants depend on patient-derived fibrinogen; neither is reliable in coagulopathic patients. Recent convergence of bioink engineering, 3D/4D bioprinting, and artificial intelligence (AI)-assisted material design enables the fabrication of biomimetic hemostatic constructs with programmable composition, multiscale architecture, and dynamic responsiveness. This review critically examines how intelligent bioprinting—integrating stimuli-responsive bioinks, multi-axis extrusion/digital light processing printing, and AI-guided bioink optimization—fundamentally differs from conventional biomaterial fabrication in recapitulating the structural mechanics of natural thrombi. We first analyze the structure–function relationships of native fibrin/platelet/red blood cell networks that should be mimicked by bioprinted constructs. We then systematically compare bioink formulations (natural biopolymers, synthetic peptides, cell-membrane-coated nano-vectors, and inorganic–organic composites) across four bioprinting-relevant dimensions: printability, post-printing bioactivity, wet-tissue adhesion strength, and burst pressure. We further evaluate cross-tier preclinical validation, including microfluidic thrombosis-on-chip, small-animal models, and lethal large-animal NCH models (porcine liver/spleen/junctional injury), with explicit differentiation from rodent wound-healing studies. Finally, we identify translational bottlenecks (rheological paradox, Good Manufacturing Practice scale-up, regulatory pathways) and articulate future directions: AI-guided bioink discovery, organ-on-chip patient-specific screening, and deployable 4D-printed hemostats. By bridging biomimetic design, programmable fabrication, and quantitative translational evaluation, intelligent bioprinting offers a manufacturable and clinically translatable route to next-generation NCH therapeutics.
Ewing sarcoma (EwS) is a rare pediatric malignancy that develops in a physiologically stiff bone environment. Conventional in vitro models fail to faithfully recapitulate the complexity of this microenvironment and therefore have limited translational relevance for preclinical drug screening. In this study, we present a novel three-dimensional (3D) in vitro EwS model using extrusion-based 3D bioprinting with a collagen-based bioink. Rheological and morphometric analyses were performed to establish a printability framework, optimizing bioink formulations and cell densities for effective biofabrication. Alamar Blue assay was used to identify optimal cell densities for the evaluation of cell viability and growth in short- and long-term studies. The optimized formulation (medium:bioink = 1:10) containing TC-71 cells at a final density of 1.4 × 106 cells/mL enabled reproducible biofabrication of structurally stable constructs with improved viscoelastic properties and shape fidelity. The 3D tumor microenvironment was modulated by adjusting hydrogel viscoelasticity, which supported sustained cell growth and remodeling activities as indicated by increased expression of MMP-9 and MMP-13. Under these conditions, viable multicellular spheroids formed within the hydrogel by day 3 and exhibited sustained growth through day 14, enabling both short- and long-term studies. The immunohistochemical analysis confirmed homogeneous cell distribution, and together with the gene expression analysis, recapitulated key tumor features. As proof of concept, treatment with talazoparib, a potent poly (ADP-ribose) polymerase inhibitor, led to reduced proliferation (Ki-67 labeling) and activation of the DNA damage response (γH2AX expression) in the 3D EwS model, demonstrating the feasibility of this platform for drug screening. Comparative analysis with a conventional two-dimensional (2D) model further illustrated the enhanced physiological relevance of the 3D environment. Notably, the analysis of Ki-67 expression revealed that the 3D model exhibited slower proliferation kinetics compared to the 2D culture, with significantly lower Ki-67 levels at day 3 and comparable levels to the 2D control by day 10. Furthermore, treatment with 20 nM talazoparib in the 3D model significantly reduced Ki-67 expression and increased γH2AX positivity by day 10, indicating a delayed DNA damage response relative to the rapid accumulation of DNA damage observed in the 2D cultures. Overall, this study provides a reproducible and biologically relevant 3D-bioprinted EwS model, supporting its application as a predictive preclinical tool for therapeutic evaluation.
Periodontitis is a chronic inflammatory disease characterized by plaque biofilm dysbiosis, immune dysregulation, oxidative stress, alveolar bone resorption, and impaired wound healing. Although conventional periodontal therapies can reduce microbial burden and slow disease progression, predictable regeneration of the cementum-periodontal ligament (PDL)-alveolar bone complex remains difficult in irregular, infected, and inflammation-compromised defects. Three-dimensional (3D) printing offers a programmable strategy for fabricating periodontal scaffolds with patient-specific geometry, tunable pore architecture, spatially distributed materials, and region-specific biological functions. This review summarizes recent advances in 3D-printed scaffolds for periodontitis therapy and periodontal tissue regeneration, with emphasis on spatial programming and microenvironmental regulation. We first analyze the pathological barriers dictating scaffold design requirements, including persistent biofilms, unresolved inflammation, oxidative stress, disrupted bone homeostasis, and defective healing. We then discuss major printing modalities, material platforms, and biomimetic design strategies, including patient-specific customization, multiphasic compartmentalization, PDL fiber guidance, vascularization-oriented pore networks, and integrated barrier control. Functional strategies involving antibacterial activity, immuno-redox regulation, tissue-specific regenerative cues, neurovascular support, and spatiotemporal delivery are highlighted. Finally, we outline challenges in material optimization, disease-relevant modelling, functional evaluation, manufacturing standardization, and clinical translation. Future 3D-printed periodontal scaffolds should evolve from passive defect fillers into microenvironment-driven precision regenerative systems for functional periodontal reconstruction.
Bone tissue engineering (BTE) has emerged as a promising approach for repairing critical bone defects that remain challenging to treat using conventional grafting strategies. Recent advances in three-dimensional (3D) printing have enabled the fabrication of patient-specific and biomimetic bone constructs with precise control over structural architecture, material composition, and biological functionality. However, native bone exhibits a highly hierarchical organization spanning multiple length scales, and current 3D printing approaches often remain limited by insufficient integration between macroscopic mechanical reconstruction, microscale cellular regulation, and nanoscale bioactive signaling. In this review, we provide a comprehensive overview of multiscale 3D printing strategies for bone tissue reconstruction, highlighting how additive manufacturing technologies facilitate the recapitulation of hierarchical bone structures. We first summarize the historical evolution of 3D printing in BTE and discuss advances in bioinks, biomaterials, and printing technologies. Subsequently, we analyze multiscale reconstruction strategies from three perspectives: macroscale fabrication for personalized anatomical restoration and mechanical support, microscale construction for cellular organization and vascular network formation, and nanoscale engineering for biomimetic extracellular matrix interfaces and molecular regulation. Furthermore, we discuss emerging strategies integrating smart responsive materials, bioactive cues, and computational design to enhance the regenerative capacity of 3D-printed bone constructs. Finally, current challenges and future perspectives are presented, focusing on vascularization, immune regulation, artificial intelligence-assisted scaffold optimization, four-dimensional bioprinting, and clinical translation. This review highlights the potential of multiscale 3D printing as a transformative platform for developing functional bone tissues and advancing personalized regenerative medicine.
Digital light processing (DLP) three-dimensional (3D) printing is an additive manufacturing technique that fabricates objects layer by layer using digitally projected light patterns. It effectively addresses the demand for complex geometries and is widely adopted in the biological field. However, traditional macromolecular bioinks still suffer from printing inaccuracies due to light scattering and runaway free-radical reactions. In this study, we introduced silk fibroin (SF) into the conventional poly(ethylene glycol) diacrylate (PEGDA)/chitosan methacrylate (CSMA) bioink to enhance the precision of DLP 3D printing. The incorporation of SF achieved a significant improvement in mechanical property (alcohol-induced β-sheet formation increased the compressive modulus from 9.5 kPa to 208.1 kPa) and anti-swelling performance (volume swelling ratio from about 366.5% to 144.6%) of cell-free PEGDA/CSMA/SF hydrogel via alcohol treatment, meeting the enhanced mechanical requirements and addressing the commonly neglected issue of geometric deformation of 3D-printed hydrogels caused by swelling in moist environments. Ethanol treatment induced SF β-sheet structure formation and reduced PEGDA/CSMA/SF hydrogel pore size (from about 78.3 μm to 23.6 μm), thereby enhancing the hydrogel network crosslinking. The PEGDA/CSMA/SF bioink exhibited antibacterial potential, inhibiting Escherichia coli by about 41.3% and Staphylococcus aureus by about 45.8% within 12 h in vitro. The porous scaffolds were printed with a designed PEGDA/CSMA/SF bioink and effectively accelerated defect repair in rat full-thickness skin wound models (about 19.1% of the untreated control group). Histological analysis revealed that the PEGDA/CSMA/SF hydrogel scaffold promoted angiogenesis and collagen deposition, upregulated vascular endothelial growth factor A expression, and downregulated tumor necrosis factor-α expression. The straightforward approach of introducing SF into traditional bioinks represents a promising strategy for DLP 3D printing in tissue regeneration.
