Biomaterials Science

Hydrogels have emerged as attractive vaccine delivery platforms because they enable controlled modulation of antigen availability. However, how different hydrogel environments affect the release and functionality of mRNA-loaded lipid nanoparticles (mRNA-LNPs) remains poorly understood. Here, we investigated the release, stability, cellular uptake, and transfection capability of LNPs released from four hydrogel systems representing distinct crosslinking mechanisms: covalently crosslinked poly(ethylene glycol) (PEG), ionically crosslinked alginate, thermoresponsive Poloxamer 407 (P407), and protein-based Matrigel/collagen hydrogels. All hydrogels enabled release of LNPs over days, with kinetics strongly depending on hydrogel composition and polymer concentration. LNPs were quantitatively recovered from all hydrogel types, except from Matrigel/collagen where incomplete matrix dissolution was the limiting step. Lower polymer concentrations generally accelerated nanoparticle release. PEG offered greatest tunability of release kinetics; at the same time the recovery of the LNP-incorporated fluorescent dye DiI was reduced to about 80 %, indicating partial dye leakage. Alginate hydrogels exhibited recovery of DiI below 50 % and broader particle size distributions after release, while P407 hydrogels largely preserved LNP characteristics. Although quantitative recovery from Matrigel/collagen hydrogels was limited, released LNPs remained readily available for cellular uptake. Notably, LNPs released from low- and intermediate-concentration Matrigel/collagen hydrogels achieved approximately 80-90 % of the eGFP expression compared to mRNA-LNP that were not embedded into a hydrogel. Importantly, cellular uptake and transfection experiments demonstrated that all investigated hydrogels released biologically active mRNA-LNPs capable of mediating protein expression. Moreover, our findings show that hydrogel composition is a critical determinant of mRNA-LNP release, stability, and functional delivery. This work provides design principles for the development of hydrogel-based mRNA delivery systems aimed at sustained antigen availability and prolonged vaccine responses. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/741169v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@138d9eforg.highwire.dtl.DTLVardef@16c0edaorg.highwire.dtl.DTLVardef@1432dd1org.highwire.dtl.DTLVardef@17511b5_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundReactive oxygen species (ROS) overproduction, inflammatory cytokines, and plasma mediators disrupt alveolar tight junction integrity and perpetuate oxidative injury in acute respiratory distress syndrome (ARDS). Biological heterogeneity of ARDS, reflected by hyper- and hypoinflammatory subphenotypes with distinct plasma mediator profiles, may contribute to heterogeneity of treatment effect. Stem cell-derived EVs have been shown to exert regenerative and immunomodulatory effects by horizontal transfer of proteins, lipids and genetic material to injured target cells. This study investigated whether extracellular vesicles derived from bone marrow (MSC-EVs) and adipose tissue (ASC-EVs) mesenchymal stem cells restore barrier integrity and redox homeostasis, and whether efficacy differs across subphenotypes. MethodsBronchial epithelial cells (BEAS-2B), microvascular endothelial cells (HMEC-1), and small airway epithelial cells (SAEC) were challenged with plasma from patients with hyperinflammatory or hypoinflammatory ARDS subphenotypes, including those treated with venovenous ECMO. MSC-EVs or ASC-EVs were added concomitantly with plasma exposure or six hours later, at a dose of 5,000 particles/cell. After 24 h, the expression of actin and tight junction proteins, zonula occludens (ZO)-1, occludin, and vascular endothelial (VE)-cadherin, was evaluated by immunofluorescence. Oxidative stress parameters, antioxidant defenses, and antioxidant enzyme activities, were measured. ResultsPlasma from patients with the hyperinflammatory ARDS subphenotype induced marked disruption of ZO-1, occludin, and VE-cadherin expression, together with actin cytoskeletal rearrangement, across BEAS-2B, HMEC-1, and SAEC cells. Barrier disruption was accompanied by increased oxidative stress markers and altered antioxidant enzyme activity. Both MSC-EVs and ASC-EVs restored tight junction protein expression and cytoskeletal organization when administered either concurrently with ARDS plasma or 6 hours after exposure, and both effectively modulated redox parameters. Delayed administration 6 hours after injury produced greater effects for several oxidative stress endpoints, particularly in cells challenged with hypoinflammatory plasma. ConclusionARDS-associated plasma mediators induce subphenotype-dependent impairment of epithelial-endothelial barrier integrity through oxidative mechanisms. Both MSC-EVs and ASC-EVs mitigate this injury, with efficacy that varies according to ARDS subphenotype and timing of administration.

Osteoarthritis, characterized by cartilage degradation and synovial inflammation, has spurred interest in mechano-piezoelectric bio-hydrogel therapies that can both relieve symptoms and reverse progression. However, current approaches lack sufficient piezoelectric output and dual cartilage/inflammation targeting. To address this, we demonstrated a mechano-piezoelectric peptide hydrogel composed of nanofibers integrating piezoelectric cues with mesenchymal stromal cells (MSCs) recruitment and PIEZO2 mechanosignaling. Molecularly, the hydrogel's seed peptide incorporated four functions: COL2A1 targeting, MMP-13 responsiveness, MSCs homing, and self-assembly. Overexpressed MMP-13 in the osteoarthritis niche triggers gelation, promoting MSCs recruitment and drug retention. Fluorination modulates hierarchical nanofiber assembly, enhancing mechanical and piezoelectric properties, as confirmed by morphological, biophysical, and computational analysis. The trifluoromethyl-modified, 4-octyl itaconate (4-OI) loaded formulation reverses osteoarthritis via PI3K/AKT activation and Wnt/{beta}-catenin suppression, as shown by improved Osteoarthritis Research Society International (OARSI) scores, bone microarchitecture, and cartilage matrix. This synergy of mechano-piezoelectric cues and 4-OI offers a clinically promising strategy for osteoarthritis.

Articular cartilage degeneration is a hallmark of degenerative joint diseases, yet its limited regenerative capacity poses significant challenges for tissue engineering. While mesenchymal stem cells (MSCs) are the most commonly employed cell type in cartilage tissue engineering, they exhibit donor variability, restricted expansion capacity, and tendencies toward fibrocartilage formation and hypertrophic differentiation. Human induced pluripotent stem cell-derived chondroprogenitors (iCPs) represent a promising alternative, offering scalable production of developmentally relevant cells with intrinsic chondrogenic commitment. However, their performance within three-dimensional biomaterial scaffolds remains largely unexplored. Here, we compared chondrogenic differentiation of iCPs and MSCs within alginate hydrogels of varying stiffness (0.37 - 4.55 kPa) exhibiting physiologically relevant stress relaxation properties. Intermediate stiffness (2% alginate, ~2.17 kPa) optimally supported chondrogenesis for both cell types. While MSCs differentiated as single cells, iCPs spontaneously self-organized into cartilaginous aggregates without requiring a separate pellet pre-culture step, showing significantly higher hyaline indices and reduced COL10 expression, despite initial low viability in hydrogels. To further enhance chondrogenesis, we functionalized 2% alginate gels with RGD and HAVDI peptides mimicking integrin- and cadherin-mediated signaling. HAVDI/RGD functionalization significantly enhanced hyaline cartilage marker expression in both cell types, with iCPs exhibiting superior matrix composition characterized by elevated aggrecan and SOX9 expression and reduced COL10 and MMP13 compared to MSCs. These findings establish iCPs as a promising cell source for cartilage tissue engineering and disease modeling, particularly within biomaterials integrating mechanical and bioactive cues that recapitulate the native cartilage microenvironment.

Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality worldwide. This clinical burden is predominantly attributable to atherothrombotic events resulting from platelet hyperactivation, a process integral to the pathophysiology of atherosclerosis. Cilostazol (CLZ) is a clinically established phosphodiesterase-IIIA inhibitor with antiplatelet and vasodilatory activity. However, its extremely low aqueous solubility results in dissolution-limited and variable oral absorption, while dose-dependent adverse effects and cardiovascular safety restrictions may constrain its clinical use. Lipid-polymer hybrid nanoparticles (LPHNPs) represent a highly promising drug delivery system to address these biopharmaceutical limitations. However, their interactions with blood components remain poorly understood, with surface charge among the most controversial physicochemical parameters implicated in platelet toxicity. Here, we applied an integrated ex vivo and in silico screening strategy to design LPHNPs based on poly(lactic acid) and poly(ethylene glycol)-modified lipids displaying positive (amine), nominally neutral (methoxy), and negative (carboxyl) surface charges to systematically identify safe boundaries at the nano-human platelet interface and evaluate their suitability as CLZ carriers. Positively charged nanoparticles precipitated immediately during self-assembly due to uncontrolled ionic pairing and were therefore structurally unviable. Neutral nanoparticles maintained a stable size (~113 nm hydrodynamic diameter) but unexpectedly induced spontaneous platelet aggregation, limiting their applicability. In contrast, negatively charged nanoparticles exhibited exceptional colloidal stability (hydrodynamic size of ~120 nm and zeta potential of -48 mV), achieved significant inhibition of ADP-induced platelet aggregation, and adhered to the platelet surface without inducing activation or membrane disruption. Steered molecular dynamics simulations, performed under identical non-equilibrium pulling conditions, showed the highest interfacial resistance to CLZ extraction for the negatively charged model, qualitatively consistent with the sustained CLZ release profile observed in vitro. These findings highlight the importance of precise surface charge tuning in the development of safe cardiovascular nanomedicines based on LPHNPs, emphasizing the need for comprehensive platelet compatibility testing before further biological evaluation.

By decoupling biological function from cellular viability, fixed abiotic cellular materials have emerged as a promising platform for next-generation immunotherapies, preserving essential immunomodulatory activities while enabling unprecedented control over manufacturing, storage, and standardization. Nevertheless, the coupled effects of fabrication history and post-fabrication environmental cues on their biological functionality remain elusive. Here, we show that both preparation route and storage time critically affect the bioactivity of mesenchymal stromal cell (MSC)-derived microtissues. By exploring different devitalization agents, we demonstrate that an ethanol-based strategy provides a green fixation option with comparable preservation of volumetric and fixed cell architecture, as well as immunomodulatory function. Processing protocols did not merely substitute conventional fixation but tailored specific immune-related bioactivity. An unexpected processing protocol and time-dependent enhancement of immunomodulatory performance was observed, with ethanol-fixed microtissues maintaining a stable baseline bioactive profile while increasing functional potency over 8 weeks of storage. We establish a framework for the design of immunomodulatory abiotic cell-derived materials with potential for the treatment of immune-mediated diseases and regenerative therapies, and we hope that the principles uncovered here may contribute to a broader understanding of how devitalized tissues can be engineered, preserved, and functionally tuned, whether lab-made or derived from natural biological systems.

Macrophages are highly dynamic cells that maintain tissue homeostasis by regulating both initiation and resolution of inflammation. During efferocytosis, macrophages recognize the eat me signal, phosphatidylserine (PS), exposed at the surface of apoptotic cells, leading to the resolution of inflammation and acquisition of a pro-resolving phenotype. Inspired by this endogenous mechanism, PS-based biomaterials have demonstrated immunomodulatory potential. However, the molecular mechanisms underlying PS-mediated macrophage reprogramming remain poorly understood. Here, submicron PS-exposing polymeric particles (PSPs; [~]300 nm) were developed to improve the suitability of PSP formulations for future systemic administration while preserving their immunomodulatory activity. PSPs were efficiently internalized by macrophages through both actin- and dynamin-dependent pathways. PSP treatment significantly reduced IL-6 and IL-12p70 production in LPS-stimulated macrophages, whereas induction of the classical anti-inflammatory M2 marker CD206 was limited. Transcriptomic analysis revealed coordinated attenuation of inflammatory signaling pathways, including downregulation of Myd88, Nfkb1, Rel, and Irf8, together with activation of NRF2-associated antioxidant pathways characterized by increased expression of Nfe2l2, Hmox1, Prdx1, Gclm, and Gclc. Activation of antioxidant-associated genes together with reduced Irf8 expression suggests that PSP promotes inflammatory resolution through coordinated redox adaptation and selective attenuation of inflammatory signaling. Collectively, these findings provide mechanistic insight into PS-mediated macrophage reprogramming and support the future development of systemically administered therapies for chronic inflammatory diseases, including vascular inflammatory disorders. HighlightsO_LISubmicron PSPs retain immunomodulatory activity of apoptotic cell-mimicking biomaterials. C_LIO_LIPSPs are rapidly internalized through actin- and dynamin-dependent pathways. C_LIO_LIPSPs attenuate inflammatory signaling and selectively suppress IL-6 and IL-12p70 production. C_LIO_LIPSPs induce NRF2-associated antioxidant and glutathione responses. C_LIO_LITranscriptomics reveals an early redox-adaptive macrophage program. C_LI

Gelatin methacrylate (GelMA) hydrogels offer many advantageous properties such as excellent biocompatibility, tunable stiffness, and rapid fabrication, making them attractive materials for neural systems-on-a-chip devices used to study axonal outgrowth in three-dimensional (3D) environments. In this study, we evaluate properties of GelMA hydrogels crosslinked with visible blue light and application of these hydrogels for encapsulation of primary dorsal root ganglion (DRG) explants inside a custom microscale device. We selected low GelMA polymer concentrations of 3 % and 6 % w/v for hydrogel preparation to create soft materials and demonstrate the effect of degree of functionalization (DoF) on stiffness of GelMA in a blue light-induced crosslinking system. Additionally, a decrease in crosslinking efficiency and stiffness of GelMA was observed upon reconstitution of the polymer in DMEM cell culture media compared to PBS. To evaluate the axonal outgrowth, DRG explants were encapsulated in various GelMA formulations and cultured for 7 days. Following incubation, the explants were fixed and immunochemically labelled with anti-beta-III-tubulin antibody. We further describe an application of custom-developed semi-automated image analysis tool together with linear mixed effects modeling to evaluate axonal outgrowth across different GelMA formulations. Among the formulations tested in our study, 3 % GelMA 95 % DoF hydrogels supported the longest neurite outgrowth suggesting that matrix properties influenced neurite extension.

Intranasal administration provides a noninvasive route for local and systemic drug delivery, yet its clinical effectiveness remains limited by rapid mucociliary clearance and short drug residence times within the nasal cavity. Existing stimulus-responsive nasal hydrogels seek to address this challenge but rely on physiological triggers such as temperature, pH, or ionic strength to induce gelation, resulting in variable performance due to natural differences in the nasal cavity. Here, we present a polyethylene glycol (PEG)-based hydrogel platform that overcomes these limitations through rapid in situ gel formation that is independent of physiological stimuli. The two liquid precursor solutions remain sprayable during administration and rapidly crosslink upon deposition to form a cohesive hydrogel depot. Although this dual-component design presents challenges for compatibility with conventional nasal spray devices, we demonstrate successful delivery using commercially available dual-barrel spray systems. The hydrogel maintained spray performance and regional nasal deposition comparable to conventional aqueous formulations while significantly reducing mucociliary transport to prolong mucosal residence. Importantly, reduced mucociliary transport was achieved without impairing ciliary function or compromising epithelial barrier integrity, addressing key safety considerations for repeated intranasal administration. In addition, the hydrogel altered epithelial uptake and transport of biologics, suggesting its potential to improve the nasal delivery of macromolecular therapeutics that typically exhibit poor bioavailability. The results of these study establish a versatile and clinically translatable approach for enhancing intranasal drug delivery across a broad range of therapeutic applications.

Photocrosslinkable gelatin derivatives are promising bioinks for soft tissue engineering, but their use in cell-laden digital light processing (DLP) requires a balance between tunable mechanics, printability and cytocompatibility. Here, we report a small library of photocrosslinkable gelatin-based precursor formulations for cell-laden DLP, with the broader aim of generating a versatile platform for soft tissue engineering rather than a formulation restricted to one tissue model. A bifunctional methacryloyl-norbornene gelatin (GelMANB) was developed to combine methacryloyl chain-growth polymerisation with norbornene-mediated thiol-ene crosslinking and was benchmarked against gelatin methacryloyl (GelMA). GelMANB formulations containing different thiolated crosslinkers were evaluated to determine the influence of crosslinking chemistry as well as polymer concentration on network formation and physico-chemical properties. Thiol-ene formulations enabled broad tuning of hydrogel stiffness and exhibited rapid photocrosslinking, while crosslinker selection further influenced swelling and tensile behaviour. Selected GelMANB/dithiothreitol (GelMANB/DTT) and GelMANB/thiolated gelatin (GelMANB/GelSH) formulations were subsequently processed via DLP alongside GelMA. Optimised exposure conditions enabled reproducible fabrication of porous scaffolds that retained dimensional stability under osmotic pressures relevant to several soft tissues. Following cell-laden printing, human foreskin fibroblasts remained viable after seven days, reaching 95.0{+/-}5.9% viability in GelMANB/DTT and 94.9{+/-}2.7% in GelMANB/GelSH, compared with 87.2{+/-}6.0% in GelMA. In 2-mm-thick hydrogel discs, GelMANB/GelSH supported the most sustained fibroblast elongation, reaching 30.9{+/-}6.3% elongated cells at day 7, although elongated cells were mainly confined to the outer surface layers. Porous scaffolds with 400 m struts and 1.3 mm pores reduced the characteristic hydrogel thickness and enhanced elongated morphology across all formulations, highlighting GelMANB as a modular platform for cell-laden soft tissue biofabrication.

Edible plant derived extracellular vesicles (PDEVs) are emerging as biocompatible, orally deliverable nanocarriers with therapeutic potential; however, their phytometabolite cargo, gastrointestinal stability, and source specific biological functions remain poorly characterized. Here, PDEVs were isolated from four phytochemically distinct plant based foods (black carrot, ginger, garlic, and turmeric), selected for their diverse bioactivity, and characterized by transmission electron microscopy, nanoparticle tracking analysis, and zeta potential. Gastrointestinal stability was evaluated in simulated digestion model. Source specific phytometabolites were profiled by untargeted LC MS MS metabolomics. Functionally validated in ammonia stressed epithelial cells and steatotic hepatocytes. PDEVs exhibited characteristic cup shaped morphology with particle sizes ranging from 60 to 214 nm and zeta potentials of -6.0 to -49.0 mV. PDEVs retained colloidal stability, supporting their suitability for oral delivery. We identified 572 phytometabolites with distinct source specific signatures, including lignin and quercetin in carrot EVs, [6] gingerol and silymarin in ginger EVs, diosgenin in garlic EVs, and curcumin in turmeric EVs. These metabolites found associated to antioxidant, anti inflammatory, epithelial barrier, lipid metabolic, and apoptotic pathways. Functional validation demonstrated carrot EVs significantly enhanced epithelial barrier integrity by increasing claudin (>8-fold, p<0.05), occludin (>2-fold, p<0.05). Ginger EVs restored ZO 1 while suppressing cyclin D1 and MMP9(p<0.05). Garlic and turmeric EVs attenuated inflammatory signaling by reducing STAT3, AKT1, and TNF , whereas turmeric EVs additionally decreased caspase 3 and PTGS2(p<0.01). In steatotic hepatocytes, garlic EVs significantly reduced PNPLA3 (p<0.001) and SREBP 1c while increasing PPAR- (p=0.002). Hence, our results indicate that edible PDEVs are gastrointestinally stable, phytometabolite enriched nanocarriers with distinct source specific functional properties, supporting their potential as orally deliverable nutraceuticals for improving gut liver functions.

Articular cartilage damage often progresses to osteoarthritis (OA), a degenerative joint disease characterized by chronic pain, limited mobility, and reduced quality of life. Tissue engineering approaches using poly (ethylene glycol) (PEG)-based hydrogels offer tunable mechanical properties and bioactive functionalization, yet the influence of surface charge on cartilage regeneration remains underexplored. Moreover, recombinant fibroblast growth factor 18 (FGF18) has successfully improved cartilage tissue thickness in clinical trials, but required high dosages and recurring injections may limit compliance. Here, we developed a granular microgel-based platform with bioorthogonally tethered FGF18 to evaluate the interplay of microgel surface charge and growth factor presentation on chondrogenesis. Azide groups were incorporated onto the microgel surfaces to enable site specific FGF18 conjugation across microgel scaffolds with distinct surface charges. When seeded with mesenchymal stromal cells, microgel scaffolds functionalized with FGF18 outperformed their unmodified counterparts, evidenced by higher GAG content, collagen content, and compressive modulus. In a murine microfracture model, anionic and zwitterionic microgel scaffolds tethered with FGF18 increased cartilage regeneration compared to nonionic microgels. We detected increased collagen II content within defects treated with tethered FGF18 microgel scaffolds. This work demonstrates the role of surface charge and growth factor presentation in directing cell behavior and tissue repair, advancing the design of biomaterials for cartilage regeneration. HIGHLIGHTSO_LICovalent tethering of FGF18 to microgel surface enables localized bioactivity C_LIO_LIFGF18 tethered microgel scaffolds enhance extracellular matrix deposition and chondrogenic differentiation of murine mesenchymal stromal cells in vitro C_LIO_LILocalized FGF18 presentation improves cartilage tissue formation and mechanical properties in vivo, with anionic and zwitterionic microgel scaffolds outperforming nonionic scaffolds C_LIO_LIFGF18 presentation is a more potent stimulus than microgel surface charge for cartilage regeneration C_LI

Tympanic membrane perforations remain a common clinical problem, and although surgical intervention through tympanoplasty achieves high success rates, it is associated with donor-site morbidity, surgical complexity and limited restoration of the native radial and circumferential collagen architecture. In this study, 3D extrusion printing was utilized to create an active scaffold and attempt to promote collagen organization through shear-mediated structural alignment. An alginate-carboxymethyl cellulose (CMC) hydrogel with bovine SIS-dECM was prepared and investigated for its suitability as a bioink alternative to tympanoplasty grafts. The physiochemical, rheological and printability characteristics of the hydrogel were assessed. Successful decellularization was confirmed by histological analysis. The incorporation of the SIS-dECM into the hydrogel led to increased swelling, lower apparent viscosity, yield stress and flow stress while maintaining favourable printability and filament stability. Polarized optical microscopy was also used to study the influence of printing speed on the alignment of collagen to mimic the native tympanic membrane radial collagen architecture. Compared with the cast controls, the printed samples presented stronger birefringence signals. Biological evaluation demonstrated that the 15% dECM hydrogel exhibited the highest live cell area percentage and live/dead ratio after 48 h. In addition, the chick chorioallantoic membrane assay demonstrated that the dECM-containing hydrogels improved vascular density. The findings establish a printable, biologically active dECM bioink capable of generating bulk collagen organization through extrusion printing as a platform for tympanic membrane regeneration.

Angiogenesis following injury has been shown to be driven by fibrillar proteins of the extracellular matrix (ECM), such as collagen. However, the use of protein-based biomaterials presents some challenges, such as uncontrolled degradation and limited tuneability. We demonstrate how to create patterned interpenetrating networks (IPNs) based on covalently crosslinked alginate and physically crosslinked collagen that provide suitable mechanical properties to support migration of endothelial cells (ECs) in a spatially controlled manner. Low molecular weight alginate is functionalized with norbornene (N) or tetrazine (T), which enables two independent covalent crosslinking methods: UV-mediated and degradable crosslinks with matrix metalloproteinase (MMP) sensitive peptides (Deg) and slower spontaneous N:T non-degradable crosslinks (noDeg). Using photolithography, patterns in degradation, collagen fibrillogenesis, microarchitecture and matrix viscoelasticity are created. The potential of such 3D patterned alginate-collagen (Alg-Col) IPNs to spatially guide EC invasion and proliferation was tested in a microfluidics platform resembling an early healing setting. Only regions combining collagen fibrillogenesis, alginate degradability and viscoelasticity demonstrated EC cell invasion similar to the ones found in vivo following injury. The 3D patterned Alg-Col IPNs are compatible with microfluidics, offer an strategy to widen the applications of protein-based hydrogels and present a versatile platform for tissue engineering and disease modeling.

Sensory and motor neurons differ significantly in their morphology, structural organization, functional properties, and mode of action. However, despite this heterogeneity, many in vitro studies focus only on a single neuronal subtype, mainly being sensory neurons, limiting the translational potential and relevance of these studies for the evaluation of therapeutic options for spinal cord injury. In this study, we investigate the differentiation, maturation, and neuronal outgrowth of human induced pluripotent stem cell (iPSC)-derived motor and sensory neurospheres using polyethylene glycol (PEG)-microgels with various bioactive coatings. Our results show subtype-specific responses to the PEG-microgel scaffolds, with respect to motor and sensory neurosphere morphology and size. Furthermore, we compare the formation of the PEG-microgel/scaffolds when starting from iPSCs-derived precursor neuron spheres versus undifferentiated iPSCs. We observe notable differences in structural organization, maturity, and neuronal outgrowth between the two approaches, as well as between motor and sensory neurospheres. Together, these results underline the importance of studying motor and sensory neurons separately and highlight the need for a controlled, tunable culture platform to assess the impact of the microenvironment and to improve the physiological relevance of in vitro platforms for neuron-based research.

Extrusion bioprinting requires simultaneous control of material processability, rapid post-deposition stabilization, geometric fidelity, and cell compatibility. Here, we defined an experimental biofabrication window for three visible-light photocrosslinkable protein formulations based on silk fibroin (SF), gelatin methacryloyl (GelMA), and collagen methacryloyl (ColMA) using a extrusion-bioprinting workflow and ruthenium/sodium persulfate (Ru/SPS) photochemistry. Material-specific photocrosslinking conditions were first established under 430-nm irradiation and subsequently evaluated by ATR-FTIR spectroscopy, unconfined compression, short-term ARPE-19 cytocompatibility, and printing assays. Stable bulk hydrogels were obtained using 1-Ru/SPS, 45 mW, and 5 min for SF; 2.5- Ru/SPS, 45 mW, and 7 min for GelMA; and 2.5- Ru/SPS, 45 mW, and 6 min for ColMA. The selected formulations formed compliant hydrogels with initial compressive moduli of 0.60, 0.52, and 2.55 kPa for SF, GelMA, and ColMA, respectively, without significant differences among groups. Short-term XTT assays identified the unreacted Ru/SPS-containing precursor as a major biological constraint, whereas photoinitiator-free SF and GelMA maintained metabolic activity close to the culture-medium reference. GelMA and SF generated interconnected lattices with printability indices of 0.893 and 0.963, respectively. Detailed SF analysis showed high positional accuracy (96.9%) despite substantial post-deposition spreading, demonstrating that positioning accuracy and dimensional fidelity are distinct properties. Cell-laden printing was achieved with both SF and GelMA, and independent extrusion testing showed no statistically significant reduction in short-term ARPE-19 viability after SF bioprinting. Following the complete printing and photocrosslinking workflow, both formulations contained predominantly viable cells, but only SF retained the printed grid-associated cellular architecture under the tested conditions. These findings support the concept that biofabrication performance is an operational property arising from the intersection of photocrosslinking, cytocompatibility, extrusion compatibility, geometric fidelity, and post-fabrication structural stability.

Synthetic blood contacting devices frequently fail due to the lack of requisite biochemical and biomechanical cues needed to support transanastamotic endothelialization. During transanastomotic endothelialization, endothelial cells experience dynamic changes in extracellular mechanical cues as they migrate from compliant native vessels onto stiffer blood contacting device surfaces. However, how substrate stiffness and mechanical memory from prior mechanical environments influence temporal integrin remodeling and downstream endothelialization processes necessary to establish a stable endothelial layer remains poorly understood. In this study, human coronary artery endothelial cells (HCECs) were cultured on substrates spanning physiologically relevant stiffnesses to determine how substrate mechanics regulate collagen binding integrins and endothelialization. Increasing substrate stiffness promoted time dependent upregulation of 2{beta}1 integrin expression, whereas 1{beta}1 expression remained unchanged. Enhanced 2{beta}1 expression on stiff substrates was accompanied by increased vinculin associated focal adhesion maturation and accelerated endothelialization, characterized by increased proliferation, migration, and progression to confluence prior to reaching quiescence after 1 week. To better model transanastomotic migration and investigate mechanical history effects, cells initially expanded on compliant hydrogels were transferred to stiff substrates. Although these cells exhibited transient reductions in 2{beta}1 expression at early timepoints compared with tissue culture polystyrene expanded controls, no persistent differences in focal adhesion maturation, proliferation, migration, confluence, or quiescence were observed. Collectively, these findings demonstrate that substrate stiffness is a primary regulator of the early endothelialization processes required to establish a stable endothelial monolayer, whereas the influence of mechanical history is transient and ultimately superseded by the current mechanical environment. These findings also identify 2{beta}1 mediated mechanotransduction as a potential design target for blood contacting biomaterials that promote rapid endothelialization while supporting long-term endothelial cell quiescence.

Bone formation and remodeling depend on dynamic biochemical and biomechanical signaling from the collagen-rich osteoid that precedes mineralization, yet the role of osteoid nonlinear mechanics in regulating osteocyte-like network formation remains poorly understood. Here, we engineered a synthetic bottlebrush polymer hydrogel (BB) that mimics key mechanical features of osteoid and compared it to collagen type-I (Col1) matrices with matched shear modulus (~70 Pa) and strain-stiffening behavior. Human bone marrow-derived mesenchymal stem/stromal cells (hMSCs) were cultured for 28 days in growth (GM) or osteogenic media (OM) to examine network formation, and functional connectivity using live cell fluorescence recovery after photobleaching. hMSCs cultured in BB networks and OM showed upregulation of early osteocyte markers compared to Col1. We find that strain-stiffening materials with minimal stress relaxation promote osteocyte-like cell differentiation with functional connectivity, establishing osteoid-mimetic BB hydrogels as a promising matrix to study human osteocytogenesis and osteocyte mechanotransduction in vitro.

Background: Post-operative uterine adhesions remain a major clinical challenge, often leading to infertility and severe pelvic pain. Current anti-adhesion barriers lack the mechanical strength, tissue specificity, or bioactivity required for optimal regeneration of uterine tissue. To address these limitations, we developed a biomimetic, multilayer scaffold that replicates the structural and functional complexity of the myometrial and serosal layers of the uterus by combining a tunable polycaprolactone (PCL) mesh with a bioactive hydrogel derived from decellularized uterine extracellular matrix (dUECM) reinforced with sodium alginate (Alg). Materials and Methods: Composite scaffolds were fabricated using near-field melt electrowriting (MEW) to create precision-engineered PCL meshes with various infill angles (90{degrees}, 60{degrees}, 45{degrees}, and 30{degrees}), followed by impregnation with an alginate-dUECM hydrogel. A comprehensive physicochemical characterization was conducted using Fourier Transform Infrared Spectroscopy (FTIR), Thermogravimetric Analysis (TGA), and contact angle measurements. Mechanical properties were evaluated in both dry and wet states via uniaxial tensile testing. Swelling and degradation were assessed over 10 days. hTERT-HM cells were used for in vitro evaluation through Live/Dead staining, MTT, SEM, and immunocytochemistry for -SMA and DAPI. Results: The successful incorporation of the hydrogel into the PCL mesh was confirmed by the results obtained from FTIR and TGA analyses; these findings were further supported by contact angle studies, which showed a marked increase in hydrophilicity, resulting in a reduction of the static contact angle from 113 {+/-} 4{degrees} in the pure PCL to 60 {+/-} 12{degrees} in the hydrogel-infiltrated samples in the 30{degrees} group. Mechanical testing showed that the wet composite scaffolds maintained higher stiffness than hydrated native uterine tissue. Cell culture assays confirmed excellent cytocompatibility and proliferation, especially in 30 infill architecture. Immunofluorescence revealed strong -SMA expression and cytoskeletal organization, indicating phenotypic maturation of uterine smooth muscle cells for all groups. Conclusion: Among all groups, the 30 MEW mesh impregnated with Alg-dUECM hydrogel demonstrated the most balanced combination of mechanical resilience, degradation profile, wettability, and cellular compatibility. This structure most closely recapitulates the biological characteristics of uterine outer myometrium tissue, positioning it as a highly promising scaffold for regeneration and biofunctional anti-adhesion barrier applications.

One crucial step during early osteogenesis is the embedding of osteoblasts within a collagen-rich extracellular matrix (osteoid), where they subsequently differentiate into a functional network of osteocytes. However, reconstructing 3D osteocyte networks in vitro remains a major challenge. We recently developed a synthetic microporous hydrogel to support the in vitro culture of 3D bone cell networks. Although matrix biodegradability facilitates cell-material interactions, the influence of micropores on bone tissue morphogenesis and differentiation remains poorly understood. Here, we investigate the effect of cell-scale micropores on bone cell morphogenesis and osteogenic differentiation in vitro. By exploiting polymerization-induced phase separation (PIPS) between 4-arm polyethylene glycol vinyl sulfones and dextran in the presence of hyaluronan, we generated matrix metalloproteinase-sensitive hydrogels with cell-scale micropores. Increasing the dextran concentration enlarged the average pore size from 4 m to 8 m, accompanied by a slight decrease in mechanical stiffness. Following encapsulation within these hydrogels, primary human osteoblasts remained highly viable. Hydrogels with larger pores supported extensive 3D cell network formation, whereas hydrogels with smaller pores exhibited enhanced osteogenic differentiation following 21 days of osteogenic culture. Together, these findings highlight that bone cells are sensitive to microporous physical cues and even minor changes over pore sizes can make an impact on osteocyte-like morphogenesis and differentiation in vitro.