Additive manufacturing (3D printing) allows the fabrication of complex 3D geometries, yet the integration of long-range ordered nanostructures within printed materials remains a fundamental challenge. In vat photopolymerization, rapid crosslinking kinetics typically arrest block copolymers in kinetically trapped, disordered morphologies. Here, we introduce Polymerization-Induced Arrangement of Nanostructures with Order-tunability (PIANO), a strategy that overcomes this kinetic mismatch by decoupling nanoscale ordering from network formation. PIANO utilizes a mobility mediator, ethylene glycol, to enhance polymer chain mobility, enabling rapid in situ ordering, while maintaining a hydrogen-bonding network capable of sustaining 3D printing stresses. This approach yields tunable lamellar and hexagonally packed cylindrical morphologies with domain spacings of 20-60 nm. Furthermore, ethylene glycol acts as a latent crosslinker during post-printing annealing, locking the ordered nanostructure while enhancing macroscopic mechanical strength. By reconciling the divergent timescales of molecular self-assembly and additive manufacturing, this strategy provides a robust platform for the hierarchical design of functional systems.

The tumor microenvironment undergoes extensive remodeling during cancer progression, resulting in increased collagen, altered tissue mechanics, and the formation of collagen tracks that permit migration. However, how the extracellular matrix (ECM) regulates cellular plasticity remains less known. Cellular plasticity is essential for successful metastasis, as cells undergo epithelial-to-mesenchymal transition and adherent-to-suspension transition (AST). Studies have begun to use 3D photo-crosslinkable hydrogels, but, unlike in vivo ECM, hydrogel stiffness is inextricably linked to porosity. In this study, we propose a fragmented gelatin methacryloyl (GelMA) scaffold that controls stiffness independently from porosity. When encapsulated as single cells, non-metastatic breast cancer cells do not exhibit growth restriction, whilst pre-engineered metastatic breast cancer cells show altered mechanosensitivity and enhanced migration (p < 0.05). We next study the role of AST in cellular migration and observe similar velocity to invasive metastatic cells. Interestingly, non-metastatic cells showed AST-dependent migration within interstitial spaces, which was enhanced in the stiff scaffold (p < 0.05). AST induction significantly increased Lamin A/C (associated with providing nuclear stability for circulating tumor cells) and reduced nuclear yes-associated protein (YAP) (necessary for cell detachment). Our data highlights the importance of incorporating micro-scale porosity and presents a promising platform for the study of cellular growth and migration.

The spatial organisation of mechanical cues is increasingly recognised as a key regulator of tissue development and disease, yet in vitro systems capable of replicating such environments remain limited. Fibroblast migration and the fibroblast-to-myofibroblast transition, which affect scar formation, are examples of mechanisms affected by mechanical cues. We report a photolithographic platform that enables precise, spatial patterning of the Young's modulus of gelatin methacryloyl (GelMA) hydrogels, using a ruthenium/sodium persulfate (SPS) photoinitiation system. By altering light intensity, we achieved Young's modulus tunability between 4 kPa and 46 kPa in non-patterned gels. By displaying a binary light intensity pattern over the gel, the Young's modulus could be switched from 10 kPa to 45 kPa, representing healthy and fibrotic Young's moduli, over a distance of ∼20 μm (2.3MPa·mm⁻¹). This system could also generate relatively linear Young's modulus gradients that are more physiologically relevant, ∼10 kPa⋅mm-1. Non-patterned and binary-patterned gels confirmed that increasing the Young's modulus drives the fibroblast-to-myofibroblast transition, observed by significantly greater cell volumes and α-smooth muscle actin (α-SMA) stress fibre formation, from encapsulated cardiac fibroblasts after 7 days. In gradient-patterned gels, fibroblasts exhibited a progressive, modulus-dependent increase in both cell volume and α-SMA stress fibre formation, alongside expressing a durotactic response, moving from healthy to fibrotic environments, and also aligning themselves between ±45 degrees to the Young's modulus gradient. Beyond cardiac fibrosis, this versatile platform enables rapid generation of biologically relevant mechanical landscapes for diverse mechanobiological applications, bridging the gap between simplified, mechanically uniform models and the heterogeneous microenvironments of native tissues. STATEMENT OF SIGNIFICANCE: This study developed a platform for spatially controlling the Young's modulus of GelMA using a digital light projection system. Using this system, a change in Young's modulus from 10 kPa to 45 kPa was achieved with a resolution of 20 µm. Natural Young's modulus gradients observed in tissues (∼10 kPa·mm⁻¹) can also be achieved. The fibroblast-to-myofibroblast transition was controlled spatially in the patterned gels, evidenced by changes in cell volume and α-SMA fibrillation. Fibroblasts demonstrated alignment and migration to the Young's modulus gradient. Our photopatterning system demonstrates ease of pattern/gradient definition with adaptability to a diverse range of systems, highlighting the impact of mechanical properties on cardiac scar formation, offering avenues for studying these effects.

Cardiovascular devices such as stents, grafts, and bioprosthetic valves are vital treatments for advanced disease, yet long-term outcomes remain limited by chronic inflammation and poor tissue integration. Conventional drug-eluting devices rely on broad anti-proliferative agents that hinder healing and overlook the underlying mechanisms driving device failure. Growing evidence identifies dysregulated inflammation as a central cause of poor performance. Emerging selective immunomodulatory approaches aim to suppress pathological inflammation whilst promoting vascular regeneration. This review examines novel anti-inflammatory strategies relevant to cardiovascular devices and materials, including dual-drug systems and multimodal agents, and evaluates polymeric coatings, hydrogels, and nanoparticle platforms for targeted delivery. We also highlight translational challenges and propose pathways to integrate immunomodulatory design into clinically scalable cardiovascular devices.

Injectable hydrogels have attracted considerable interest because of their minimally invasive delivery and adaptability to patient-specific defects. This study aimed to investigate the suitability of a photo-crosslinkable tyraminated poly(vinyl alcohol)-gelatin (PVA-GT) polymer network as an injectable platform for tissue engineering and growth factor delivery. Two formulations were developed by varying the concentration of ruthenium/sodium persulfate (Ru/SPS), resulting in formulation-specific physical properties with fast (18 days) and slow (36 days) degradation rates. Their injectability was confirmed across a range of needle sizes (14 to 22G), ensuring versatility for multiple applications. Cytokine release from peripheral blood mononuclear cells and THP-1 demonstrated no inflammatory response due to residual ruthenium. When injected subcutaneously into a mouse model, the fast-degrading formulation exhibited greater cellular and vascular infiltration compared to the slow-degrading one. By leveraging bi-phenol bond formation within the polymer network, the fast-degrading injectable hydrogels were used as a growth factor delivery platform for bone morphogenetic protein-2 (BMP-2). BMP-2-loaded hydrogels were injected into the femoral head of a Legg-Calvé-Perthes disease swine model, preventing the necrosis progression and improving piglets' mobility relative to the sham. These findings highlight the potential of injectable PVA-GT hydrogels as tunable platforms for regenerative therapies, with implications for personalized orthopedic treatments.

Light-based bioprinting has rapidly expanded as versatile platforms to replicate the complex architectures of native tissues, by allowing spatio-temporal localization of biomaterials and cells. These approaches rely on bioresins composed of photo-crosslinkable polymers, photoinitiators, and, where appropriate, photoabsorbers. In this perspective, we summarize recent technological progress in light-based bioprinting, moving beyond mere structural complexity toward the creation of engineered constructs that recapitulate the native tissue function. We discuss the development of bioresins adapted from a long history of tissue engineering and regenerative medicine research, with an emphasis on shifting the field from structural mimicry toward physiologically relevant biological function. We also highlight current limitations, including the constraints of bioprinting workflow, bioresin compositions, and the need to focus more on downstream cellular signaling and function, rather than just basic cytocompatibility. Finally, we suggest several considerations for next-generation bioresin and printing strategies better tailored for clinical translation, including improved control over cellular microenvironments and standardized, regulatory-accepted and reproducible formulations.

Achieving robust, cytocompatible bonding of hydrogels to solid substrates remains a long-lasting challenge in the development of hybrid solid-hydrogel (HSH) systems for biomedical applications. Current strategies for hydrogel-solid bonding suffer from the complexity of processes, toxicity from residual crosslinkers, and substrate dependency; issues that hinder clinical adoption of HSH structures (HSHs). Overcoming these impediments, a dry, reagent-free strategy is presented to create radical-rich interlayers that enable initiator- and crosslinker-free covalent attachment of hydrogels for the fabrication of robust HSHs. Evidence is provided in which long-lived radicals embedded in ion-assisted plasma polymerized coatings simultaneously drive hydrogel anchoring and in situ crosslinking on diverse non-polymeric substrates, including titanium, stainless steel, and glass. GelMA, chitosan, and PVA-Tyr hydrogels are immobilized with high stability, with coatings remaining intact after two months in aqueous media. Tuning the substrate bias voltage modulates radical concentration, enabling precise control over hydrogel thickness and crosslinking density with no need for extra reagents and/or crosslinkers. Cytocompatibility is confirmed with human mesenchymal stem cells and macrophages, with negligible inflammatory activation detected under the tested conditions. To showcase one application among many, fibroblasts on GelMA-based HSHs exhibited enhanced early attachment, spreading, and proliferation, supporting their application in promoting soft tissue integration. This substrate-independent, additive- and initiator-free strategy embodies high-quality-by-design principles, enabling a universal and scalable platform for the fabrication of HSH systems, particularly suited for applications requiring seamless integration between soft and hard materials, such as biomedical coatings, tissue-interfacing constructs, and next-generation soft robotics.

Image-guided volumetric bioprinting allows for the adaptive fabrication of complex structures for tissue engineering. Seminal work by Florczak et al. introduces Generative, Adaptive, Context-Aware 3D Printing, a workflow that uses computer vision to automatically generate functional, vascular-like networks that conform to living cells within hydrogels, improving their functionality.

ABSTRACT The in vitro engineering of vascularized cardiac tissues holds transformative potential for disease modeling, drug screening, and regenerative therapy. However, despite rapid advances in stem cell biology, biomaterials, and biofabrication technologies, the reconstruction of functional, perfusable vasculature within engineered myocardial tissues remains a central and unresolved challenge. In this review, we move beyond a descriptive catalog of available techniques and instead present a process‐oriented framework for understanding vascularized cardiac tissue engineering. By systematically analyzing how cellular components, biomaterial design, and biofabrication strategies collectively govern vascular formation, perfusion stability, and myocardial function, we examine self‐assembly, mold‐casting, 3D bioprinting, and microfluidic approaches, to critically evaluate their respective advantages and trade‐offs under cardiac‐specific physiological constraints. Finally, application prospects of vascularized cardiac tissues in disease modeling and drug testing are discussed, and current limitations and future directions are proposed to accelerate translational impact. By reframing vascularized cardiac tissue engineering as an integrated manufacturing challenge rather than a collection of isolated technologies, this review aims to provide a coherent conceptual guide for advancing functional human cardiac models.

This study focuses on advancing the understanding of breast cancer through 3D in vitro models, which provide biomimetic environments superior to many 2D cultures and animal models. Ex vivo analyses show that malignant breast tissues exhibit increased stiffness with higher tumour grade. Tumour stiffening is associated with altered cell phenotype, promoting progression, invasion, and metastasis. This research aims to design 3D models that mimic the evolving tumour microenvironment to study how matrix stiffness affects breast cancer cell behaviour. Using gelatin-methacryloyl (GelMA) hydrogels, we investigated the phenotypic responses of MCF7 and MDA-MB-231 cells in 3D models of clinically relevant stiffness. A visible-light photoinitiation system enabled precise control of hydrogel mechanics while supporting biocompatibility and long-term cell viability. Over a 21-day culture period, MCF7 cells exhibited partial epithelialmesenchymal transition in stiff hydrogels, showing altered morphology, downregulating E-cadherin and upregulating N-cadherin and Vimentin. Comparatively, MDA-MB-231 cells showed no such changes. Phenotype remained stable in soft hydrogels for both cell lines. This study demonstrates the impact of microenvironmental stiffness on breast cancer cell phenotype and highlights 3D GelMA hydrogels as a platform to investigate tumour microenvironment dynamics. The findings provide insights into how matrix stiffness influences EMT and breast cancer behaviour in biomimetic settings.

Cartilage tissue engineering requires biomaterials that can effectively maintain the tissue-specific functions of chondrocytes to enable the restoration of cartilage structure and function. Decellularised extracellular matrix (dECM)-derived hydrogels serve as tissue-specific biomaterials capable of preserving native biochemical cues and maintaining physiological chondrocyte phenotype in three-dimensional culture. However, their sol-gel transition relies heavily on collagen fibrillogenesis, a slow and poorly controllable process that limits mechanical tunability and suffers from inter-batch variability. Therefore, further efforts are required to functionalise cartilage dECM to achieve reproducible and controllable physicochemical properties. Here, we present a light-activated cartilage dECM hydrogel system based on ruthenium/sodium persulfate (Ru/SPS)-mediated dityrosine crosslinking, enabling rapid hydrogel formation under visible light irradiation while providing tunable mechanical properties and improved biological functionality. Comparison of the decellularisation protocols indicated that Triton X-100 combined with ammonium hydroxide efficiently eliminated residual DNA while preserving a substantial proportion of the native cartilage proteome. Pepsin-solubilised cartilage dECM hydrogels formed via dityrosine-based photo-crosslinking exhibited rapid gelation behaviour and superior mechanical characteristics compared to conventional thermally gelled dECM. The photo-crosslinked dECM hydrogels were cytocompatible, supported human bone marrow-derived mesenchymal stem cells (hBMSCs), and favoured cartilage-specific phenotypes, as demonstrated by the upregulation of chondrogenic genes, includingCOL2A1andACAN, compared with gelatin methacrylate (GelMA) hydrogels. Importantly, this photo-crosslinking strategy overcomes the incompatibility between oxygen-sensitive redox-based photochemistry and hypoxic culture conditions, enabling the incorporation of oxygen-scavenging microcapsules to establish low-oxygen microenvironments. Under hypoxia, the cartilage dECM hydrogels promoted a more articular-like phenotype in hBMSC-derived chondrocytes, with transcriptomic features associated with TGF-β/SMAD2/3 and IGF-1/2-IGF-1R signalling. Collectively, these findings establish photo-crosslinked cartilage dECM hydrogels as a biomaterial platform with tunable mechanical properties and favourable biological functionality for cartilage tissue bioengineering and biomimeticin vitrocartilage models.

Early fracture repairs are characterized by dynamic immune-skeletal interactions. While immune cells are known to be critical, how macrophage polarization (M1 to M2) and metabolism jointly shape the microenvironment repairs remains unclear. Here, we integrated three mouse long bone fracture sc/snRNA-seq datasets with multi-algorithm consensus annotation. Fracture expanded and rewired intercellular communication, with redistribution of incoming signaling toward immune populations, especially macrophage subsets, and increased relative flow through TGF-β, BMP, and FN1 pathways. From days 1 to 7 post-injury, macrophages followed a graded M1-to-M2 continuum, while M1-like cells remained prevalent across this interval. Distinct transcriptional programs were associated with M1-like and M2-like macrophages, with Creb3l2/Fos enriched in M1-like cells and Maf/Mafb enriched in M2-like cells alongside differential metabolic features. Data-driven prioritization across integrated public mouse omics datasets nominated Pbx3, Creb3l2, Nfix, Maf, and Mafb as candidate regulators associated with macrophage polarization, with spatial enrichment in macrophage-associated niches. A fracture-associated repair module comprised skeletal stem/progenitor cells (SSPCs), fibroblasts, macrophages, and osteoclasts, and was accompanied by predicted metabolite-mediated communication, with communication involving glutamine, sterol/cholesterol, and GABA prioritized as relatively increased and communication involving heme and 27-hydroxycholesterol as relatively reduced. SSPC lineage tracing revealed Taco1 as an early dynamic marker and branch-specific drivers, Runx2/Egfr (osteogenesis), Ebf1 (chondrogenesis), and Stat5a (adipogenesis). Collectively, these findings provide a computational atlas of early fracture healing, suggest that macrophages may play an important coordinating role during this stage, and prioritize transcriptional and metabolic candidates for future experimental validation.

Hydrogels are an attractive biomaterial for use in soft tissue engineering applications but fall short when used in large volume applications, exhibiting limited cellular infiltration and exaggerated fibrotic capsule responses. Embedding microgels within bulk hydrogels to generate microporosity enables greater cellular infiltration and provides more control over cell fate. Microgels can be further encapsulated with therapeutics to provide bioactive cues to the surrounding local microenvironment, synergistically complementing their intrinsic physical properties. MCC950, a NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome inhibitor, has been previously shown to selectively halt foreign body driven inflammation while preserving inflammation beneficial to minimizing the fibrotic capsule response and promoting tissue repair within implanted biomaterials. Here, we fabricated a silk fibroin-based hydrogel construct composed of MCC950-encapsulated microgels encased within a bulk hydrogel filler. While the encapsulation of MCC950 into silk constructs resulted in few changes in mechanical properties, the eluted drug retained bioactivity in vitro against human THP-1 cells. In vivo implantation of MCC950-encapsulated microgel-hydrogel constructs in a subcutaneous mouse model over 2 weeks showed synergistic effects between the physical and biological cues, resulting in a decrease in fibrous capsule formation and increase in cellular infiltration, attributed to the decrease in NLRP3 expression around the scaffold. Together, these results demonstrate the potential of MCC950-encapsulated silk microgel-hydrogel constructs for soft tissue engineering applications. Furthermore, these findings highlight a synergistic interplay between targeted NLRP3 inhibition and the microporous scaffold architecture that collectively drives reduced fibrosis and a more pro-regenerative immune microenvironment conducive to positive tissue remodeling. STATEMENT OF SIGNIFICANCE: Hydrogels are promising for soft tissue engineering but often lack cell infiltration at larger volumes. Embedding microgels within bulk hydrogels creates a microporous structure that enhances cellular infiltration. Therapeutics can also be encapsulated to provide bioactive cues to synergistically complement the physical architecture. In this study, silk-based microgel-hydrogel constructs were encapsulated with MCC950, a selective immunomodulatory drug, to minimize the fibrotic response and promoting tissue repair. Scaffolds were optimized for mechanical strength, drug release with eluted MCC950 retaining bioactivity against human THP-1 cells. 2-week mouse subcutaneous implantations demonstrated effects between architectural and bioactive cues, decreasing capsule thickness and increasing cell infiltration. These findings highlight the synergistic between physical and bioactive cues towards creating a pro-regenerative microenvironment conducive to tissue remodeling.

Collagen bioinks are widely used in biofabrication, but their relatively soft mechanical properties can lead to structural instabilities under cell-generated contraction forces. While synthetic functional groups can be conjugated for covalent crosslinking, these methods often disrupt natural protein fibrillogenesis, thereby compromising collagen fibre architecture. This work presents a strategy for the direct covalent stabilisation of native collagen bioinks with dityrosine bonds via visible-light photocrosslinking with ruthenium (Ru) and sodium persulfate (SPS), avoiding the need for polymer pre-functionalisation. Multimodal characterisation, including high-resolution microscopy, spectroscopy, mass spectrometry, and nanoindentation, identified photocrosslinking conditions that enhance collagen fibrillogenesis and reduce off-target polymer oxidation. Interestingly, the biofabrication process itself affected ultimate collagen fibre architecture, with shear-induced alignment during extrusion enhancing fibril proximity and self-assembly, overcoming inhibitory effects the crosslinkers had on fibrillogenesis via ionic and electrostatic interactions. Leveraging these insights, embedded bioprinting was used to fabricate cardiac constructs with high cell viability (>80%), where dityrosine crosslinking could be tuned to modulate geometric shape changes under cell-generated forces (1-15% shrinkage). Finally, the platform was used to bioprint anatomically accurate double-ventricle human heart models with robust shape fidelity. This research establishes a versatile photocrosslinking framework for bioprinting cardiac constructs with tunable shape stability using native collagen bioinks. ### Competing Interest Statement The authors have declared no competing interest. European Research Council, https://ror.org/0472cxd90, 101077900 Research Ireland, Future Digital Challenge Grant 22/NCF/FD/10991G, 13/RC/2073_P2 Galway University Foundation

Volumetric bioprinting (VBP) enables the rapid photopolymerization of 3D constructs by modifying the illumination patterns within a build volume. However, only a few unmodified, pristine protein-based bioinks can be used for VBP, making the resulting (bio)printed volumes sometimes incompatible with further modification steps required for extended applications and thus limiting the wider adoption of VBP. We have recently developed new methods for VBP, in which unmodified protein-based (bio)inks with tyrosine groups, including those based on silk, decellularized extracellular matrix (dECM) and gelatin, can be (bio)printed, in their pristine state, by using the tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate/sodium persulfate photoinitiator system to form sophisticated shapes and architectures. Here, we provide step-by-step instructions to complete the VBP process and include the characterization of these bioinks. After treatment, the volumetrically printed silk sericin constructs show properties including reversible shrinkage and expansion, or shape-memory, whereas the volumetrically printed silk fibroin constructs exhibit broadly tunable mechanical performances ranging from a few hundred pascals to hundreds of megapascals. Both types of silk-based (bio)inks as well as dECM (bio)inks are cytocompatible. We further cover several demonstrations that show the potential uses of volumetrically (bio)printed silk and dECM constructs in clinical and biomedical applications.

Modulating how macrophages sense mechanical cues offers a novel strategy to control fibrosis around implanted biomaterials. We term this approach ‘mechano-immunotherapy’, which involves the desensitization of immune mechanosensory pathways to control the host response. Here, we use RN-1734 (RN), a model small molecule to demonstrate the proof-of-concept that pharmacologically disrupting macrophage mechanosensation can mitigate fibrosis. In vitro, RN reduced calcium influx and pro-inflammatory cytokine secretion in J774. a2 macrophages. These effects were strictly context-dependent with efficacy observed only in macrophages on high-stiffness (10% w/v) GelMA hydrogels, with no significant impact on those in softer (5% w/v) hydrogels. In vivo, RN selectively attenuated fibrotic capsule formation around implanted electrospun scaffolds but not smooth hydrogels. Notably, despite hydrogels releasing ∼6-fold more drug than scaffolds, fibrosis was reduced only in the scaffold group, suggesting that therapeutic efficacy is driven by the inhibition of high mechanosensory input rather than the loaded drug concentration alone. Spatial transcriptomics revealed that macrophages acted as the primary mechanosensors at the tissue-implant interface. Unsupervised global principal component analysis revealed that RN acted predominantly on early day 3 macrophages. The strongest effect was observed in surface-adhered mechanosensitive macrophages, where RN treatment enhanced their M2-like phenotype and promoted their dispersal from clustered aggregates into broader distribution within the scaffold. This redistribution was accompanied by a marked reduction in the recruitment of interstitial macrophages from the surrounding tissue, which were enriched for matrix-forming gene signatures. Together, these findings suggest that pharmacological desensitization of immune mechanosensors may represent a promising, context-specific approach to improve biomaterial integration.