Advanced Healthcare Materials

Critical sized craniomaxillofacial bone defects do not heal naturally and often exhibit chronic inflammatory responses that restrict regeneration. It is increasingly apparent that biomaterials must facilitate dynamic crosstalk between immune cells, such as macrophages, and osteoprogenitors to resolve inflammation and accelerate regeneration. Here, we evaluate interactions between macrophages in a neutral (M0) or pro-inflammatory (M1) state with mesenchymal stem cells (MSCs) in a basal or licensed state within a mineralized collagen scaffold. We reveal that MSC-macrophage crosstalk influences significant changes in osteoprogenitor cell differentiation and immune cell polarization. Notably, crosstalk between MSCs and macrophages drives an early-stage inflammatory response, which enhances the immunomodulatory activity of MSCs via secretion of IL-6, an effect that is heightened for already licensed MSCs. The presence of macrophages in the co-cultures upregulated osteogenic (ALPL, BMP2, COL1A2, and RUNX2) and angiogenic genes (ANGPT1) in basal MSC groups. Further, MSC-macrophage interactions subsequently drive increased M2-like macrophage polarization as early as 7 days of culture, as indicated by surface marker expression. These findings show that biomaterial scaffolds can be leveraged as mediators of MSC-mediated immunomodulation with an emphasis on achieving early-stage pro-inflammatory phenotypes that drive subsequent macrophage polarization and markers of increased regenerative potency.

Central nervous system (CNS) disease or injury might be treated by implanted devices, tissue regenerative scaffolds, or drug delivery platforms. However, inflammatory CNS responses limit these interventions and may worsen outcomes following damage to the CNS. Via the foreign body reaction (FBR), macrophages and glial cells trigger a "glial scar" around implants, reducing device performance, scaffold regenerative ability, or drug delivery potential. Previous studies have shown that stiffness of CNS implants significantly affects glial encapsulation, but few studies have investigated materials that truly match brain tissue stiffness. Porous precision-templated scaffolds (PTS) with uniform, interconnected, 40 {micro}m pores have shown favorable healing outcomes and a reduced FBR in numerous soft and hard tissue applications. To quantify the effects of both hydrogel compliance (stiffness) and pore size on glial encapsulation, we implanted poly(2-hydroxyethyl methacrylate-co-glycerol methacrylate) (pHEMA/GMA) PTS of varying stiffness and pore size for 4 weeks in rat brain. We observed reduced astrocyte encapsulation around PTS compared to solid hydrogel rods, reduced pro-inflammatory macrophage polarization for softer hydrogels versus stiffer hydrogels, and the presence of neuronal markers and neurogenesis within the pores. Utilizing soft, precision-porous hydrogels could provide a strategy for mitigating glial scarring and improving implant-based CNS treatments.

Biomaterial implantation can trigger a foreign body response (FBR) that impedes tissue-implant integration. To investigate how implant porosity influences this response, we compared the immune response to subcutaneous implants of microporous annealed particle (MAP) scaffolds and nanoporous hydrogels using mass cytometry, single-cell RNA sequencing, and multiplex cytokine assays. MAP scaffolds promoted vascularization and tissue integration, marked by increased endothelial and regulatory T cells, and reduced proinflammatory immune cells and cytokines. In contrast, nanoporous hydrogels demonstrated enrichment of basophils, natural killer cells, and macrophage populations associated with fibrosis. Transcriptomic and proteomic analyses revealed that MAP scaffolds suppressed activation of the complement-fibroblast-macrophage signaling loop, particularly the C5a signaling crosstalk pathway. This was confirmed using C5-deficient mice, where complement-driven cytokine production was significantly reduced only in nanoporous implants. These findings demonstrate that scaffold porosity modulates immune and complement responses, identifying a key mechanism by which MAP scaffolds reduce FBR and improve biomaterial integration.

Biomaterials-based tissue engineering aims to recapitulate native tissue architecture and function for both clinical repair and advanced in vitro models. While improvements in biomaterials have been made, including granular hydrogels and ECM-derived scaffolds, current biomaterials lack intentional design choices for effective translation, including regulatory considerations, practical extrusion delivery, and biomimetic characteristics. Here, we develop and characterize a library of granular ECM (gECM) biomaterials for five key tissues (cartilage, bone, skin, liver, and kidney), in which ECM particles are densely packed within a hyaluronic acid hydrogel. We optimize tissue processing methods that preserve proteomic content and structure while also aligning with scale-up manufacturing and regulatory guidelines. We show that gECM hydrogels can be molded, extruded, and 3D-printed while retaining their shape, and they stabilize at physiological temperature and pH. Lastly, we demonstrate that bulk gECM mechanics are driven by tissue type, and gECM hydrogels support viability, proliferation, and tissue-specific cellular activity. Together, these findings establish gECM hydrogels as a translational and biomimetic platform for clinical tissue repair and complex in vitro models.

In vitro reconstruction of human tissue microenvironments that integrate native biochemical and biomechanical cues is essential for disease modelling, regenerative medicine, and personalized therapeutic approaches. However, most currently available engineered matrices fail to recapitulate the complexity and tissue specificity of the human extracellular matrix (ECM). To address this limitation, we developed a novel hydrogel derived from decellularized human adipose tissue (atdECM) designed to support three-dimensional culture of human cells. The decellularization and delipidation processes were first validated, and the biochemical composition and biomechanical properties of atdECM were comprehensively characterized. Human pancreatic organoids were then cultured within atdECM hydrogel, and their structural organization and transcriptional profiles were analyzed and compared with those obtained in Matrigel, the current gold-standard matrix for organoid culture. Proteomic and cytokine analyses demonstrated efficient decellularization while preserving collagen-rich ECM architecture and a diverse repertoire of soluble bioactive factors. AtdECM exhibited physiological stiffness and retained tissue-specific extracellular cues. Pancreatic organoids cultured in atdECM displayed morphological similarities with those grown in Matrigel but exhibited transcriptional profiles more consistent with physiological epithelial homeostasis, with reduced activation of inflammatory and stress-related pathways. Altogether, these findings indicate that atdECM provides a human-derived, tissue-relevant, and permissive microenvironment for human organoid generation. This platform represents a promising alternative to Matrigel for studying human tissue biology and for developing physiologically relevant in vitro models.

Robust development of in vitro mature skeletal muscle with functional neuromuscular junctions is an unmet challenge that must be addressed for advances in skeletal muscle tissue engineering and for the development of skeletal muscle tissue models for disease modelling and drug discovery. Herein, we developed hierarchical, anisotropic biomaterials that induced early maturation of more mature myotubes and the development of neuromuscular junctions (NMJs) during co-culture with motor neurons. We accomplished this by creating micro-nano biomaterial interfaces that presented nanoclusters of integrin-binding ligands to promote mechanotransduction on the surface of aligned electrospun microfibers. Controlling surface topography and nanoscale ligand clustering led to 1.5 to 2.5-fold increases in myoblast proliferation, myotube formation, elongation, and alignment; resulting in spontaneous twitching; and enhanced myotube-neuron connections, including increasing acetylcholine receptor clustering, neurite branching, and myotube contraction compared to control surfaces. These findings highlight the importance of tailoring adhesive peptide distribution and presentation for the in vitro development of NMJs and synaptic organization. This approach offers a valuable platform for fundamental research on muscle development and neuromuscular diseases, paving the way for improved skeletal muscle tissue engineering and drug screening strategies.

Focal injuries to articular cartilage in load-bearing joints fail to heal and often progress to degeneration, underscoring the need for repair strategies that result in restored cartilage structure and function rather than fibrocartilage formation. Granular extracellular matrix (gECM) hydrogels, flowable grafts composed of densely-packed matrix particles, offer a promising approach but lack long-term functional validation in large-animal models. Here, we developed a flowable gECM hydrogel composed of decellularized cartilage microparticles incorporated within a thiol-functionalized hyaluronan matrix. Proteomic analysis confirmed enrichment of cartilage-specific gECM matrisome components. When implanted into critical-sized femoral condyle defects in a goat model and evaluated 12 months post-implantation, both gECM hydrogel and microdrilling (surgical controls) achieved >80% defect filling. However, in contrast to microdrilling, gECM repair tissue exhibited surface tribological (friction, adhesion) and compressive mechanical properties comparable to native cartilage, with a similar proteoglycan-to-collagen ratio, enrichment of type II collagen, minimal type I collagen (typical of a fibrous scar), improved quantitative MRI metrics, and evidence of lateral cartilage integration and subchondral bone remodeling. Together, these findings demonstrate that a flowable gECM hydrogel supports integrative, cartilage-like repair in a load-bearing joint, supporting advancement of this approach toward clinical translation. One Sentence SummaryA granular ECM hydrogel implanted in a goat condyle provided a robust repair, filling the defect tissue with integrated, hyaline-like cartilage at 12 months.

Hypertrophic cartilage is a promising bone repair strategy by producing a mineralizable matrix that transitions to bone through endochondral ossification. Current approaches require large cell numbers and costly recombinant factors to induce chondrogenesis. Here, we developed a composite granular scaffold using photocrosslinkable alginate microgels, cell-secreted decellularized extracellular matrix (dECM), and mesenchymal stromal cell (MSC) spheroids under dynamic compressive loading for hypertrophic cartilage formation. Incorporation of dECM into MSC spheroids enhanced expression of chondrogenic markers and supported the hypertrophic phenotype, evidenced by increased VEGFA and SPP1 expression and ALP activity. Dynamic loading further increased spheroid sprouting and scaffold mineralization. Histology confirmed mature hypertrophic cartilage conducive to bone formation. Upregulation of hypertrophic and osteogenic markers was associated with YAP1 activation, linking compressive loading to mechanotransduction to drive hypertrophic cartilage formation. These results demonstrate that dynamic compressive loading, cell aggregates, and scaffold granular macroporosity synergistically yield hypertrophic cartilage.

Critical-sized bone defects and implant-associated complications are often exacerbated by chronic inflammation, which compromises tissue repair and implant integration. Mesenchymal stromal cell (MSC)-derived extracellular vesicles have emerged as promising immunomodulatory nanotherapeutics; however, their clinical translation remains constrained by low yield, heterogeneity, and poor scalability. Here we present a bioengineered MSC-derived nanoghosts platform designed to overcome these translational barriers while enabling tunable osteoimmunomodulatory function. By coupling high-yield nanoghost fabrication with biomimetic MSC conditioning, we demonstrate that oxygen tension (5 or 21% O2) and 3D culture substrates (5 or 15 wt-% GelMA) can reprogram MSC immunophenotype. Nanoghosts generated under hypoxic and 3D conditions displayed enriched anti-inflammatory cargo, preserved MSC viability under inflammatory stress, and partially rescued osteogenic mineralization in the presence of pro-inflammatory cytokines. Together, these findings showcase MSC nanoghosts as scalable and bioactive immunoregulatory nanotherapeutic capable of modulating immune-bone crosstalk, providing a translational strategy to mitigate inflammation-driven impairment of bone regeneration and implant integration. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/724218v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1551655org.highwire.dtl.DTLVardef@12d3371org.highwire.dtl.DTLVardef@8c50bborg.highwire.dtl.DTLVardef@834a8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Significant challenges in spinal cord injury include the loss of neural tissue, disruption of local vasculature, and intrinsic suppression of actin mobilisation in neurons, together preventing axonal regrowth. Here, we develop an implantable biomimetic microRNA (miR) inhibitor-activated scaffold that combines optimised matrix cues with transcriptomically defined RNA-based modulation of intrinsic neuronal pathways as a platform to support neuronal cell delivery and promote neurovascular repair. First, hyaluronic acid macroporous scaffolds functionalized with collagen-IV and fibronectin supported iPSC-derived neuronal spheroid formation and neurite extension. To identify a neurotrophic target, we performed analysis of public miRNA-mRNA interaction datasets, revealing that miR-133a regulates pathways involved in neuronal actin cytoskeletal organisation. MiR-133a inhibitors were complexed with the cell-penetrating peptide RALA to form nanoparticles, demonstrated >95% scaffold loading efficiency, sustained localised release over 28 days and enhanced neurite outgrowth from motor neurons and iPSC neurons. Bulk RNA-sequencing and transcriptomic analysis of iPSC neurons within the scaffolds demonstrated coordinated upregulation of actin-remodelling, cell-matrix adhesion and metabolic pathways, indicative of a cytoskeletally adaptable neuron. When employed in an ex vivo dorsal root ganglia model, scaffold-mediated miR-133a inhibition promoted neurite extension and integration of delivered iPSC neurons with injured neural tissue. Finally, miR-133a-inhibitor-activated scaffolds upregulated neurovascular genes, increased endothelial cell migration and enhanced blood vessel formation in vivo in a chick embryo assay. These findings identify miR-133a as a neurotrophic target, elucidate the underlying mechanisms of action through transcriptomic analysis and demonstrate that biomimetic scaffold-mediated inhibition of miR-133a can enhance neuronal delivery for multifaceted spinal cord repair applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/719922v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@13fcb26org.highwire.dtl.DTLVardef@13239c3org.highwire.dtl.DTLVardef@6e6f5eorg.highwire.dtl.DTLVardef@51ad93_HPS_FORMAT_FIGEXP M_FIG C_FIG

Osteoporotic bone degeneration involves progressive deterioration of trabecular microarchitecture, yet most scaffold-based bone tissue engineering studies evaluate osteogenesis in structurally favorable architectures that poorly represent compromised bone environments. Here, we establish a degeneration-inspired Voronoi scaffold platform in which point spacing serves as a single tunable architectural parameter to model transitions from dense mechanically integrated to severely deteriorated trabecular-like microenvironments. Increasing point spacing from 1.25 to 2.5 mm progressively reduced scaffold connectivity and stiffness while shifting deformation behavior from distributed load transfer to localized stress concentration, as confirmed by finite element analysis and mechanical testing. Benchmarking against clinically reported HR-pQCT datasets from postmenopausal women demonstrated that the intermediate 1.75 mm point spacing scaffold represents a clinically relevant compromised trabecular-like state, whereas the 2.5 mm scaffold represents a more severely deteriorated architectural condition. These architecture-dependent mechanical and structural transitions directly regulated hMSC behavior, where high point spacing scaffolds reduced cytoskeletal organization, stress fiber density, and osteogenic mineralization, establishing an architecture-associated osteogenic dysfunction regime. Polydopamine (PDA) coating progressively enhanced cytoskeletal organization and mineralization within architecturally compromised scaffolds without altering scaffold geometry. To quantitatively assess biointerface-mediated functional recovery, a Mineralization Rescue Percentage (MRP) framework was introduced, demonstrating up to 43% restoration of architecture-associated mineralization loss following PDA coating. Collectively, this work establishes a clinically contextualized degeneration-to-rescue biomaterials framework that shifts current scaffold design paradigms beyond structurally favorable architectures toward systematic investigation and functional rescue of architecture-associated osteogenic dysfunction within compromised bone-like microenvironments. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/725650v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@26833forg.highwire.dtl.DTLVardef@72b2b7org.highwire.dtl.DTLVardef@333083org.highwire.dtl.DTLVardef@b5f2d1_HPS_FORMAT_FIGEXP M_FIG C_FIG Statement of SignificanceMost scaffold-based bone tissue engineering studies evaluate osteogenesis in structurally favorable architectures that poorly represent compromised bone microenvironments associated with osteoporosis. Here, a clinically contextualized Voronoi scaffold platform is established in which point spacing serves as a single tunable architectural parameter to model transitions from mechanically integrated to structurally deteriorated trabecular-like states. By decoupling architectural and surface biointerface effects, the study demonstrates that architectural deterioration alone can drive cytoskeletal disruption and osteogenic failure. Importantly, polydopamine-mediated surface engineering partially restored cytoskeletal organization and mineralization within architecturally compromised scaffolds without altering bulk geometry. A Mineralization Rescue Percentage (MRP) framework was further introduced to quantitatively assess biointerface-mediated functional recovery within degeneration-inspired scaffold microenvironments.

Periprosthetic joint infection (PJI) is a severe complication of total knee arthroplasty and is a leading cause of revision surgery, and is associated with significant morbidity. Two-stage exchange using antibiotic-loaded polymethylmethacrylate (PMMA) spacers remains the clinical gold standard, yet the decision to reimplant relies largely on indirect markers and clinical judgment, as no method allows continuous in situ assessment of infection resolution. Here, we report a sensorized knee spacer that transforms PMMA bone cement from a passive structural material into an active, wearable diagnostic device. A miniaturized multimodal sensor unit integrating optoelectronic and physicochemical sensing was embedded within the tibial spacer component and wirelessly coupled, enabling energy-efficient, 24/7 in vivo monitoring during the spacer interval for several months. We developed a reproducible encapsulation and integration strategy compatible with clinically realistic spatial, thermal, and mechanical constraints, without altering the established surgical workflow. The functionality of the embedded camera, spectrometer, and temperature sensors following cement integration was verified. Mechanical integrity and signal stability were confirmed under ISO-compliant dynamic biomechanical loading conditions. In vivo validation of the implantable wearable was preclinically demonstrated in a porcine model using human knee spacer dimensions. These findings establish the technical feasibility of sensor-integrated PMMA spacers and introduce bone cement as an enabling platform for smart orthopedic implants. Continuous, local monitoring of the peri-implant environment may open new pathways for evidence-based decision-making in infection management with temporary implantable wearables.

Low-pH and hypoxic conditions commonly develop in oral surgical sites and mucosal wounds, impairing cell viability and delaying healing. This study presents a simple, cell-free, and clinically translatable hydrogel patch incorporating microencapsulated calcium peroxide granules to locally deliver oxygen and buffer acidity. Calcium peroxide particles in the range of 50 to 150 micrometers, were coated with a thin PLGA shell to moderate reactivity and embedded into a GelMA-AlgMA composite membrane. In acidic artificial saliva, pH 5.2, patches containing 0.25% calcium peroxide released oxygen steadily for up to 8 hours and restored pH to physiological levels within 90 minutes. When applied to a DPSC-seeded collagen wound model exposed to lactic-acid challenge, the patches significantly improved metabolic activity and cell viability compared to acidified controls, without signs of cytotoxicity. These findings indicate that calcium peroxide-integrated hydrogels offer a low-cost, practical approach to counteract hypoxia and acidosis in oral wound environments, supporting early regenerative processes and providing a translationally viable platform for future preclinical development.

Engineering skeletal muscle tissue regeneration, particularly in dystrophin-deficient muscles is dependent on facilitating myogenesis and recovery of myotube structure and function, which can be challenging due to compromised cell-extracellular matrix (ECM) interactions. The current study explored the potential impact of enhancing dystrophin-associated protein complex and focal adhesion formation and the interaction with associated target receptors to improve cellular response in both normal and Duchenne muscular dystrophy (Dmd) mutant myoblasts. This was achieved by multivalent dual ligands functionalization of RAFT-synthesized copolymer with fibronectin- and laminin-derived adhesion peptides (RGD, AG73, and A2G80) and their clustering at the biointerface. Our findings demonstrated the synergistic effect of integrin-syndecan/dystroglycan engagement and their clustering on enhancing myoblast adhesion, proliferation, and differentiation, partially overcoming the deficits caused by loss of dystrophin. Furthermore, enhanced focal adhesion formation and elevated receptor localization, particularly dystroglycan, at the sarcolemma were associated with improved structural organization, mechanical stability, and neuromuscular connectivity of myotubes. These results suggest a novel insight into harnessing next-generation molecularly engineered biomaterials with robust interaction with cells mechanosensors for advancing skeletal muscle tissue engineering, offering potential applications in the regeneration of dystrophic muscle and the development of neuromuscular disease models for drug testing. O_FIG O_LINKSMALLFIG WIDTH=174 HEIGHT=200 SRC="FIGDIR/small/717576v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@16c6b87org.highwire.dtl.DTLVardef@107a84borg.highwire.dtl.DTLVardef@1b9e4ddorg.highwire.dtl.DTLVardef@160a9a7_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract/ToCC_FLOATNO Current work developed molecularly engineered biomaterial surfaces with nanoscale clustering of integrin-, syndecan-, and/or dystroglycan-binding peptides for skeletal muscle tissue regeneration. By controlling peptide distribution and type at the biointerface, cell adhesion, proliferation, and differentiation were modulated in dystrophin-deficient myoblasts. Accordingly, the results demonstrated significant improvement in myotube structural organization, mechanical stiffness, and their innervation in response to heteronanoclusters. C_FIG

Pediatric cancer survivors treated with gonadotoxic chemotherapy or radiation face lifelong premature ovarian insufficiency (POI), leading to elevated risk of cardiovascular disease, osteoporosis, and metabolic dysfunction. Pharmacological hormone replacement therapy (HRT) cannot replicate the pulsatile, bidirectional signaling of the hypothalamic-pituitary-gonadal (HPG) axis, leaving a critical therapeutic gap. Immune-isolating hydrogel capsules offer a promising strategy for the implantation of donor ovarian tissue without immunosuppression yet they require optimization for human applications. Here, we engineer a microporous immune-isolating capsule by incorporating thermosensitive gelatin microgels as sacrificial porogens. Microfluidic fabrication yielded monodisperse microgels that dissolved at 37{degrees}C generating disconnected micropores within a non-degradable poly(ethylene glycol) (PEG) matrix. Critically, the diffusion of FSH-scale analogs (40 kDa) increased by almost two-fold through the microporous capsules relative to nanoporous controls, while antibody-scale molecules (150 kDa) were blocked, demonstrating size-discriminating permeability. In ovariectomized mice implanted with encapsulated ovarian xenografts for 20 weeks, microporous capsules restored dynamic HPG-axis signaling evidenced by elevated levels of estradiol and progesterone, FSH suppression, and fluctuating hormone levels that resembled physiological patterns. Microporosity also improved graft viability, increasing stromal cellularity and reducing follicular apoptosis. These findings support microporous immune-isolating capsules as a platform for physiologically authentic therapy for POI.

Critical-sized craniomaxillofacial (CMF) defects affect the skull, face, and jaw, arising from conditions such as cleft palate, oncologic resections, and high energy impacts, and due to their large size and irregular geometry, cannot heal naturally by the body, thus requiring surgery. The field of biomedical research has long recognized the need to develop higher order biomaterial model systems for improved disease characterization and translational therapeutic/material progress. There is, however, difficulty in developing these workflows at the scale of conventional two-dimensional cell culture screening systems while simultaneously approaching a level of complexity necessary to consider translation to in vivo animal models. Here, we describe a three-dimensional (3D), in vitro model system to investigate the impact of stromal cell migration from one microenvironment to another at a medium-throughput scale. Importantly, we demonstrate the ability of this workflow to be utilized as a screening tool for collagen-based biomaterial motifs of interest in promoting craniomaxillofacial bone defect repair. Taken together we provide a strategy for interpreting cell-to-cell, cell-to-material, and material-to-material interactions across a multidimensional spatiotemporal scale.

Macrophages play a central role in early immune response after injury that can shape the success or failure of craniomaxillofacial (CMF) bone repair. While mineralized collagen glycosaminoglycan (GAG) scaffolds have been developed to support osteogenesis, here we define how scaffold pore size, pore alignment, and glycosaminoglycan (GAG) composition influence human monocyte-derived macrophage polarization. We establish flow cytometry, secretome, and gene expression benchmarks to assess primary macrophage polarization toward M1 versus M2 phenotypes in response to cytokine cocktails in 2D culture and 3D scaffolds. We then define the kinetics macrophage polarization in response to scaffold pore architecture and composition in the absence of exogenous cytokines. All scaffold variants support an early pro-inflammatory response followed by a shift toward M2-like phenotypes over seven days reflected by increased CD206 expression, secretion of pro-healing factors such as CCL18, and upregulation of M2a- and M2c-associated genes. Anisotropic scaffolds with smaller pores more robustly drove angiogenic and extracellular matrix related gene expression as well as earlier emergence of M2-like phenotypes. Scaffold GAG chemistry provided an additional tuning mechanism, with chondroitin-6-sulfate variants promoting the greatest late-stage M2 surface marker expression, heparin variants accelerating early M2 and pro-angiogenic phenotypes, and chondroitin-4-sulfate variants dampening both M1 and M2 phenotypes at early timepoints. These findings demonstrate that mineralized collagen scaffolds intrinsically guide macrophage polarization toward pro-regenerative states but that scaffold structure and composition can be used to shape the kinetics and intensity of these responses. These insights provide a critical foundation for immuno-instructive biomaterial designs that enhance CMF bone repair.

The plasticity of dendritic cell (DC) functional state is a major hurdle in DC therapy, yet how DCs acquire distinct states independent of ontogeny remains poorly understood. Here, we demonstrate that changes in matrix stress relaxation mechanically educate DCs to adopt distinct, persistent functional states even after the removal of mechanical cues. Stem cell-derived DCs cultured in a fast-relaxing environment exhibited enhanced antigen presentation, faster migration, and higher expression of T cell-recruiting chemokines. Slow-relaxing DCs, biased towards pro-inflammatory cytokine secretion, were enriched for gene signatures associated with lipid accumulation and stress response. These mechanical responses were conserved across human and murine DCs. Using ovalbumin (OVA) as the model antigen, fast-relaxing DCs elicited a CD8+-biased response in vitro, with higher antigen-specific CD8+ T cell activation and proliferation. In vivo adoptive cell transfer of mechanically educated DCs demonstrated that the fast-relaxing matrix licensed DCs to induce a potent draining lymph node T cell response with more antigen-specific T cells and higher restimulation potential. We further showed that DCs sensed matrix stress relaxation through PI3K signaling and actin branching, mediated by the concerted signaling of IL-4 and GM-CSF. Together, these findings demonstrate the role of matrix stress relaxation on the functional state of DCs and suggest a novel approach to enhance ex vivo cellular engineering by targeting mechanical signaling. Graphical AbstractStem cell-derived dendritic cells (DCs) generated ex vivo are engineered using biomaterial platform with tunable matrix stress relaxation. Mechanical education of DCs is licensed by cytokine signaling, actin branching, and PI3K signaling. Fast-relaxing DCs exhibit higher antigen presentation and faster migration, which enhances their capacity to prime and activate antigen-specific CD8+ T cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/725170v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@bb6709org.highwire.dtl.DTLVardef@1698c8eorg.highwire.dtl.DTLVardef@8adb0dorg.highwire.dtl.DTLVardef@336d3a_HPS_FORMAT_FIGEXP M_FIG C_FIG

This study focuses on the development of 3D culture model dedicated to liquid cancers drug screening. The challenge addressed was to effectively retain non adherent small cells within a 3D-scaffold with tailorable mechanical properties, while proposing a fast and effective tool for drug screening. To that aim, we developed a macroporous alginate-chitosan polyelectrolyte complex (PEC) scaffold combined with a low-viscosity alginate (LVA) cell seeding solution. We hypothesized that LVA could undergo in situ pore gelation via calcium ions retained from the PEC fabrication process, enabling effective retention and homogeneous cell distribution, leading to an improved platform for drug screening and personalized medicine. First, we evaluated scaffold suitability for LVA infiltration and gelation. Microtomography revealed a highly porous architecture (98%) enabling LVA homogeneous penetration and complete gelation within 30 min, as confirmed by SEM, microscopy, rheology, and micro-rheology. Next, we assessed cell retention and biocompatibility using primary human chronic lymphocytic leukemia (CLL) cells. LVA-assisted seeding increased cell density 2.6-fold compared to medium alone, with homogeneous distribution, >80% viability over 7 days, and preserved differentiation into nurse-like cells. Finally, we demonstrated a proof of concept for drug screening. The Alginate-PEC scaffold (A-PEC scaffold) supported both qualitative live/dead imaging and rapid quantitative viability measurement with the Alamar Blue assay. Drug responses reproduced microenvironment-dependent protection effects observed in vivo. This integrated scaffold and seeding method provides a promising 3D platform for in vitro liquid cancer studies and drug screening on patient-derived hematological cancer cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/722037v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@9b71d4org.highwire.dtl.DTLVardef@14e1dd0org.highwire.dtl.DTLVardef@1876a56org.highwire.dtl.DTLVardef@15656bc_HPS_FORMAT_FIGEXP M_FIG C_FIG

Articular cartilage has limited self-repair capacity, and current treatments fail to fully restore its structure and function. 3D hydrogels that support chondrocyte viability and extracellular matrix (ECM) deposition offer a promising strategy for cartilage regeneration. Here, we developed a photo-crosslinkable silk fibroin-hyaluronic acid hydrogel for 3D encapsulation of primary human chondrocytes. Hydrogels were formulated with varying silk fibroin methacrylate (SilMA, 10-20% w/v) and hyaluronic acid methacrylate (HAMA, 1-2% w/v) concentrations and characterized for rheological, mechanical, and morphological properties. All SilMA-HAMA hydrogel formulations exhibited shear-thinning behavior and rapidly gelled (<20 s) under UV irradiation while maintaining high porosity, thereby ensuring injectability and efficient nutrient diffusion. Notably, the Youngs modulus of the cell-laden scaffolds increased from [~]18 kPa to [~]1200 kPa over culture, indicating mechanical maturation driven by chondrocyte-mediated matrix deposition. This maturation was further confirmed by histological analysis and qPCR, which demonstrated enhanced ECM production and chondrogenic gene expression. Taken together, these results highlight SilMA-HAMA hydrogels as a promising biomimetic platform that couples mechanical reinforcement with biological functionality for cartilage tissue engineering.