Bioactive Materials

Critical-sized bone defects represent a challenge in bone tissue engineering, due to insufficient vascularization that results in implant failure. Scaffold pre-vascularization is a promising strategy to create a functional microvascular network that integrates with host vasculature. In this study, we present a hybrid 3D construct comprising a hyaluronic acid-based hydrogel and a 3D printed polycaprolactone/{beta}-tricalcium phosphate scaffold, to support vascular network formation and osteogenic differentiation. Peptide-functionalized (i.e. RGD, YIGSR, IKVAV, QK) hydrogels were obtained via thiol-ene chemistry, using two crosslinkers (PEG-diSH or MMP-diSH). Preliminary biological experiments assessed human mesenchymal stromal cells (hMSCs), endothelial cells (hUVECs), and their co-culture, on different gel formulations. All cell conditions displayed enhanced spreading and metabolic activity on gel formulations comprising RGD; thus these (i.e. RGD only and a combination of RGD/YIGSR) were selected for further studies. Cells were then mixed with the hydrogel precursor solutions, which were injected to embed the scaffolds and crosslinked using a UV lamp. After 7 days, tubule formation was observed only in co-culture conditions, highlighting the importance of cellular crosstalk for the formation of a vascular network. Significant differences were found across the tested formulations. In the RGD-PEG constructs, hUVECs formed tubule-like structures, surrounded by hMSCs, exhibiting pericyte-like behavior, supported by the upregulation of SMA gene. Conversely, in the RGD/YIGSR-MMP conditions, hMSCs were mostly located on the scaffold fibers, and showed the highest expression of early osteogenic markers (RUNX2 and ALP). Overall, we demonstrated that the hybrid system with tailored hydrogel chemistry can support simultaneous microvascular organization and osteogenic commitment, offering a promising platform for bone tissue engineering applications. However, further studies involving longer culture periods will aim at clarifying the complex interplay between material composition, cell crosstalk and spatial organization and their influence on the maturation and stability of the vascular network.

Biodegradable electrospun nanofiber (NF) scaffolds have emerged as promising materials for tissue engineering applications, including vascular grafts, because their mechanical properties and degradability can be tuned. However, their in vivo degradation behavior remains poorly understood. In this study, we characterized the in vivo degradation profiles of representative biodegradable NF materials widely used in small-caliber vascular graft research, namely polycaprolactone (PCL), poly(D,L-lactide) (PLA), polyglycolic acid (PGA), and a PCL/PLA blend, by monitoring molecular weight changes in subcutaneous and vascular environments. Electrospun NF sheets were implanted subcutaneously in mice, and tubular NF grafts were implanted into the abdominal aorta of rats. Samples were harvested for up to 48 weeks after implantation and analyzed primarily by size-exclusion chromatography (SEC) to assess time-dependent changes in molecular weight. Scanning electron microscopy (SEM) and solid-state 13C nuclear magnetic resonance (NMR) were additionally performed to evaluate ultrastructural and chemical changes associated with degradation. SEC analysis revealed distinct material-specific degradation patterns. PCL showed the slowest degradation and retained a relatively high weight-average molecular weight (Mw) in both environments. PLA exhibited marked environment dependence, with near-complete degradation in the subcutaneous environment by 48 weeks, whereas scaffold structure was maintained in the vascular environment. The PCL/PLA blend showed earlier reduction in the high-molecular-weight fraction than PCL, indicating faster scaffold breakdown. PGA degraded most rapidly and could not be evaluated beyond 2 weeks in the subcutaneous model or in the vascular model because of early graft rupture. SEM analysis further demonstrated that progressive loss of fibrous ultrastructure over time was a common feature across all materials. In addition, NF scaffolds became resistant to organic solvent after implantation in vivo, and solid-state 13C NMR analysis of the solvent-insoluble fractions detected polymer-derived signals together with additional signals consistent with biological constituents. These findings indicate that in vivo degradation of biodegradable NF scaffolds is material dependent, environment dependent, and more complex than simple hydrolytic chain cleavage alone. This study provides a quantitative framework for evaluating NF degradability and offers new insight into the design of biodegradable vascular grafts. HighlightsO_LISEC quantified long-term in vivo degradation of PCL, PLA, PGA, and PCL/PLA. C_LIO_LIDegradation was both material dependent and implantation environment dependent. C_LIO_LIIn vivo nanofiber degradation involved structural and chemical changes beyond hydrolysis. C_LI

Lipid nanoparticles (LNPs) have demonstrated strong potential in COVID-19 mRNA vaccines nevertheless they still face the challenges in low mRNA delivery efficacy. Virus-like porous silica (VLPSi) nanoparticles (NPs) represent a promising biomimetic delivery platform because their spiked morphology may enhance cellular internalization and promote endosomal membrane disruption. However, the application of VLPSi for mRNA has been rarely explored. In this study, hybrid lipid-VLPSi NPs were developed by combining VLPSi with either lipoplexes (LPs) or LNPs. The effects of lipid types, mass ratio of different compositions, and amine modifications of VLPSi on mRNA delivery were studied. The results demonstrated that both LP and LNP could be successfully integrated with VLPSi to form hybrid delivery systems for mRNA transfection. VLPSi could significantly enhance mRNA delivery of both LPs and LNPs due to improved cellular uptake, structural stabilization of the mRNA complex, and enhanced endosomal escape mediated by the rigid virus-like surface architecture. Among the tested lipid formulations, the ionizable lipid ALC-0315 and helper lipid DOPE with mass ratio of 5:3 was the most effective lipid composition to be integrated with VLPSi, showing the highest mRNA delivery performance. In addition, amino modification of VLPSi was found to be a critical factor for efficient mRNA delivery. Hybrid LNPs containing amino-modified VLPSi showed significantly higher transfection efficiency than those containing unmodified VLPSi. Notably, amino-modified LNP-VLPSi achieved up to fivefold higher gene expression than conventional LNPs. Overall, this study establishes VLPSi as an efficient platform for amplifying lipid-mediated mRNA delivery. Owing to its straightforward integration into widely used LNP systems, VLPSi offers an adaptable and effective strategy for advancing next-generation mRNA therapeutics.

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

Cultivated meat is an emerging biotechnology that aims to produce edible tissues in an ethical and sustainable manner. However, the recreation of skeletal muscle tissue that replicates the protein composition and sensory characteristics of traditional meat is a major challenge. Skeletal muscle tissue engineering requires non-animal-based scaffolds which are inexpensive and food-safe, while meeting specific mechanical requirements with respect to viscosity, stress-relaxation and stiffness. While many of these characteristics can be fulfilled by alginate-based biomaterials, a key limitation of alginate is its lack of intrinsic attachment sites for animal cells, preventing efficient adhesion, differentiation and tissue formation. Here, we established a screening platform to evaluate extracellular matrix (ECM)-mimicking peptides as functionalisations of alginate scaffolds in 2D. Our platform enables high-throughput assessment of cell/peptide interactions, serving as a predictive tool for 3D tissue constructs. Our screen identified two RGD-containing sequences (vitronectin- and fibronectin-mimicking peptides) as most effective in promoting attachment and myogenic fusion of bovine satellite cells. Notably, these peptides outperformed more complex mixtures containing up to seven different ECM-mimicking peptides. Our findings provide a streamlined approach for optimising biomaterial functionalisations for cultivated meat applications, and lay the groundwork for future advancements in scalable, sustainable skeletal muscle tissue engineering.

Therapeutic cancer vaccines represent a promising approach to boost patients own immune system to fight cancer. However, many vaccine candidates have shown limited success in clinical trials in large part due to the insufficient antigen delivery to overcome tolerance and hypoxia mediated immunosuppressive mechanisms. Cryogel-based delivery scaffolds have emerged as a promising platform for cancer vaccines due to their biocompatibility and macroporous structure that allows for effective delivery to infiltrating antigen-presenting cells. However, these systems are limited by rapid, diffusion-mediated burst release of encapsulated recombinant proteins and local hypoxia-driven immunosuppression within the scaffold. Herein, we demonstrate that click conjugation of a tumor-associated protein within cryogel-based vaccines, combined with our new O2-generating platform (Click O2-CryogelVAX), helps overcome immune suppression and weak antigenicity and primes effective anti-cancer immune responses. Sustained antigen delivery promotes cellular memory and Th1-mediated anti-cancer responses. By reversing hypoxia-driven immunosuppression, O2 acts as a powerful co-adjuvant to enhance humoral immunity. Together, Click O2-CryogelVAX supports a robust antitumor response that inhibits tumor growth and prolongs survival in a therapeutic prostate cancer model. These findings support the further research and development of Click O2-CryogelVAX as an effective delivery platform for therapeutic cancer vaccines.

Pulmonary delivery of lipid nanoparticles (LNPs) remains an area of significant interest, given the broad range of genetic disorders that could be addressed through localized administration of therapeutic nucleic acids to the lung. In this study, we investigated how incorporation of the clinically used lung surfactant cocktail Poractant alfa affects the in vitro and in vivo transfection performance of mRNA-loaded LNPs. The resulting lung surfactant-enhanced LNPs (Surf-LNPs) exhibited substantial improvements in particle assembly, yielding an order of magnitude higher particle concentration at equivalent input conditions compared to conventional (Onpattro-like) LNP formulations. In vitro, Surf-LNPs demonstrated several-fold increases in mRNA transfection efficiency and protein expression while maintaining excellent cytocompatibility. These enhancements are attributed to an elevated apparent pKa and the surface-active properties of surfactant protein B (SP-B), which promote more rapid and efficient endosomal escape relative to conventional LNPs. In vivo evaluation following intranasal administration further revealed enhanced mCherry expression in the lungs of mice treated with Surf-LNPs compared to conventional LNPs. Ultimately, these findings establish lung surfactant incorporation as a simple yet powerful formulation strategy to improve pulmonary gene delivery using LNPs, with the potential to significantly advance the translation of inhaled nucleic acid therapeutics.

BackgroundBone graft biomaterials play a critical role in bone regeneration by influencing osteoblast differentiation and mineralization. However, comparative data regarding the osteogenic potential of commonly used graft materials under standardized conditions remain limited. Method and materialIn this in vitro experimental study, osteoblast-like cells (MG-63) were cultured with four bone graft materials, including Bio-Oss, Cerasorb, Bio-Tiss Cerabone, and Pro Osteon. The relative mRNA expression of osteogenic markers (COL1 and OPN) was evaluated at 1, 7, 14, and 21 days using real-time PCR. Alkaline phosphatase (ALP) activity and mineralization capacity were also assessed using colorimetric assay and Alizarin Red staining. Data were analyzed using one-way ANOVA and Tukey post hoc test (P < 0.05). ResultsSignificant differences were observed among the tested materials across all evaluated parameters. Bio-Oss and Cerasorb demonstrated higher gene expression levels and ALP activity compared to Bio-Tiss Cerabone and Pro Osteon (P < 0.05). Mineralization analysis showed significantly greater calcium deposition in the Bio-Oss and Cerasorb groups, whereas Pro Osteon consistently exhibited the lowest osteogenic performance. ConclusionBone graft biomaterials significantly influence osteogenic activity in osteoblast-like cells. Bio-Oss and Cerasorb showed superior osteogenic potential, while Pro Osteon demonstrated weaker performance. These findings highlight the importance of material properties in optimizing bone regeneration.

Matrix-bound nanovesicles (MBVs) are a type of small extracellular vesicle (EV) embedded in the extracellular matrix (ECM) throughout the body. MBVs have been previously isolated from various tissues and in vitro-cultured cell sheets, demonstrating remarkable attributes in regenerative medicine. However, differences between MBVs and conditioned culture medium-derived EVs (liquid-EVs) have yet to be characterized, and the field currently lacks specific protein markers that can identify MBVs from other EV subtypes. Here, we isolate MBVs and liquid-EVs from bone marrow mesenchymal stem cell (MSC) sheets and define differences in size, protein, and zeta potential between these EVs. We show that there is a correlation between cell-driven ECM deposition and MBV and liquid-EV production. We also find that MBVs are smaller, contain less protein per particle, and possess lower zeta potential than liquid-EVs. Interestingly, MBVs also comprise a distinct tetraspanin profile compared to liquid-EVs, with MBVs containing more CD63 and little to no CD81. Finally, we define that CD63, LAMP1, Alix, ITG{beta}1, and GRP94 and their abundance, may be markers specifically used to identify MBVs from liquid-EVs. Our study paves the way for the characteristic differentiation between MBVs from liquid-EVs, elucidates their differences in biogenesis, and reveals a potential connection between EV and ECM production.

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

Glioblastoma (GBM) presents significant therapeutic challenges due to its aggressive nature, complex microenvironment and the limitations of conventional drug delivery systems. In this study, hybrid nanoparticles were developed by combining synthetic liposomes with macrophage-derived extracellular vesicles (EVs) to harness the strengths of both platforms. Two distinct liposomal formulations, DPPC:Chol:DSPE-mPEG2000 (F1) and DPPC:DPPS:Chol:DSPE-mPEG2000 (F2), were used as the basis for the synthesis. EVs derived from J774 macrophages were integrated with F1 and F2 to create hybrid nanoparticles (H-F1 and H-F2). Doxorubicin (DOX) was encapsulated using a pH gradient and a remote loading procedure. The mean particle size of H-F1-DOX and H-F2-DOX was 158.2 {+/-} 1 nm and 162.8 {+/-} 9 nm, respectively. The polydispersity index (PDI) was 0.130 {+/-} 0.012 and 0.084 {+/-} 0.033, while the zeta potential values were -14.9 {+/-} 0.7 mV and -26.7 {+/-} 3.1 mV, respectively. H-F2-DOX exhibited the highest encapsulation efficiency (EE%), reaching 76.5{+/-}3.4%. The encapsulated hybrids remained stable up to one week, at +5{degrees}C. The release of DOX from H-F2-DOX in DMEM supplemented with 10% serum showed pH sensitivity, with total DOX release of 64.9 {+/-} 5.3% at pH 7.4 and 90.7 {+/-} 6.5% at pH 5.5. The cell viability assay demonstrated that all formulations exhibited strong cytotoxic effects against GBM cells under normoxic conditions, with H-F2-DOX showing the most potent effect under hypoxia-mimetic conditions.

Postoperative gastric leak after bariatric surgery is a serious complication associated with prolonged treatment, repeated interventions, and substantial morbidity. Endoscopic internal drainage using double pigtail stents is widely adopted. However, current stents, originally designed for biliary use and often based on simple cylindrical geometries, are not optimized for post-bariatric gastric leak anatomy, mechanical support, or fluid drainage. Here, we present BRIDGE (Biodegradable aRchitected Internal DrainaGE), a stent concept integrating triply periodic minimal surface (TPMS) architectures to control mechanical compliance, kink resistance, and drainage performance. Using computational modeling, mechanical testing, and benchtop flow studies, we evaluate TPMS designs and identify volume fraction as a key parameter balancing flexibility, structural integrity, and hydraulic performance. TPMS-integrated designs tolerated a 7.1-fold smaller bend radius than a commercial stent without kinking and achieved up to a 2-fold increase in drainage. We also developed a stereolithography-printable biodegradable resin and fabricated a prototype lattice-integrated stent. TeaserA biodegradable, 3D-printed stent with an architected lattice design improves flexibility, kink resistance, and abscess drainage while eliminating the need for device removal.

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.

Interleukin-4 (IL-4) is a key immunoregulatory cytokine that promotes type 2 inflammation, drives macrophage polarization toward an anti-inflammatory M2 phenotype, and supports tissue repair. However, clinical translation of IL-4 therapies to modulate the immune response is limited by the need for precise control over its delivery to avoid immune dysregulation. Here, we report an affinity-based strategy to modulate IL-4 delivery and bioactivity using engineered affibody proteins. A yeast surface display library was screened via magnetic- and fluorescence-activated cell sorting to identify two IL-4-specific affibodies with moderate binding affinities (dissociation constants, KD = 459 and 141 nM). Circular dichroism confirmed expected alpha-helical folding, and biolayer interferometry characterized the kinetics of IL-4 binding. Structural modeling using AlphaFold3 and RosettaDock and molecular dynamics simulations using GROMACS predicted distinct binding sites for each IL-4-specific affibody on the IL-4 protein and suggested potential interference with receptor complex formation. Bioactivity studies using murine bone marrow-derived macrophages demonstrated that IL-4 complexed with affibodies maintained Ym1 gene expression but significantly reduced Ym1 protein levels, indicating partial inhibition of IL-4 signaling. To enable controlled cytokine delivery via affinity interactions, affibodies were conjugated to polyethylene glycol maleimide (PEG-mal) hydrogels, which were loaded with IL-4. Affibody-conjugated hydrogels achieved high IL-4 loading efficiency (>90%) and exhibited sustained release over 7 days. Increasing affibody-to-IL-4 ratios significantly reduced both the rate and total amount of cytokine release. Overall, this work establishes IL-4-specific affibodies as versatile tools for tuning cytokine presentation and modulating bioactivity and provides a promising approach for regulating inflammatory responses and advancing cytokine-based therapies with improved temporal control. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/723637v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@12bdb14org.highwire.dtl.DTLVardef@3c09eeorg.highwire.dtl.DTLVardef@1b00934org.highwire.dtl.DTLVardef@2c4840_HPS_FORMAT_FIGEXP M_FIG C_FIG

Hydrogels are widely used as three-dimensional cell culture systems to understand the impact of cellular mechanotransduction for tissue engineering applications. Photoinitiated thiol-ene click chemistry is a commonly utilized hydrogel crosslinking mechanism that provides spatial and temporal control over hydrogel network formation and resulting mesh size and compressive properties. Despite historically documented efficiency as step-growth reactions, these reactions do not always proceed as predicted. To understand the impact of cell confinement and microenvironmental mechanics on cellular function, thiol-ene network formation must be thoroughly characterized. To this end, the objective of this work was to investigate the crosslinking dynamics to determine hydrogel network formation as assessed via mesh size and mechanical properties using a pentenoate-functionalized hyaluronic acid thiol-ene reaction. Hydrogel parameters including polymer concentration and thiol:-ene crosslinker molar ratio were modulated (4, 6, or 8 polymer weight percent and 0.15:1, 0.5:1, or 1:1 molar ratio of thiol groups to reactive -ene groups) to tune network properties including shear storage modulus and relative mesh size. Molecular Dynamics (MD) simulations were used to simulate the thiol-ene crosslinking reaction and establish a method for predicting thiol-ene reaction efficiency. Lastly, the feasibility of this hydrogel system for in vitro modeling was confirmed via assessment of metabolic activity of encapsulated primary human meniscal cells.

Tetrahedral DNA nanostructures (TDNs) are promising nanocarriers due to their structural precision, biocompatibility, and efficient cellular uptake. However, their stability under physiological conditions remains a key challenge. In this study, TDNs were synthesized via a one-pot thermal annealing method and characterized using native PAGE, dynamic light scattering (DLS), and zeta potential analysis, confirming uniform size ([~]13 nm) and negative surface charge. Their stability was systematically evaluated across different biological media (DMEM complete, serum-free DMEM, and E3), temperatures (4 {degrees}C, 25 {degrees}C, and 37 {degrees}C), and pH conditions (4.0, 7.0, and 8.5) over 24 h. Results revealed rapid degradation in serum-containing medium, increased instability at higher temperatures, and reduced stability under acidic conditions, while serum-free, lower-temperature, and neutral to mildly basic environments enhanced structural integrity. These findings highlight the strong environmental dependence of TDN stability and provide insights for optimizing their design for biomedical applications.

Pathologic arterial stiffening is a hallmark of vascular disease that contributes to maladaptive vascular remodeling and neointimal hyperplasia through vascular smooth muscle cell (VSMC) phenotypic switching. Yet, because vascular disease progression is governed by both biomechanical and extracellular matrix (ECM) alterations, existing in vitro models often fail to recapitulate the full complexity of the diseased vascular microenvironment. Here, we developed a bioactive decellularized extracellular matrix (dECM) and methacrylated hyaluronic acid (MeHA) composite scaffold platform with tunable stiffness that preserves native vascular ECM components while enabling controlled investigation of stiffness-dependent cell behavior. Proteomic analyses confirmed retention of key vascular matrisome components, including collagens and glycoproteins, following decellularization. Electrospun vascular dECM scaffolds maintained an aligned fibrous architecture and spanned stiffness ranges representative of healthy and pathologically stiffened arterial microenvironments. Within this matrix-preserving platform, human VSMCs cultured on stiff dECM scaffolds exhibited increased spreading, altered morphology, enhanced nuclear localization of YAP and survivin, and broad transcriptional changes consistent with a shift toward a proliferative, matrix-remodeling VSMC phenotype. Together, this bioactive, matrix-preserving platform enables mechanobiologically relevant modeling of stiffness-driven vascular remodeling and indicates YAP and survivin as candidate regulators of maladaptive VSMC mechanotransduction.

Background and ObjectivesCardiovascular cells differentiated from human induced pluripotent stem cells (iPSCs), including cardiomyocytes, are valuable for evaluating human cardiac pharmacology and toxicity. Early assessment of cardiotoxicity, especially for novel drugs like anticancer agents, is essential for improving drug development efficiency and reducing costs. This study aimed to develop a highly sensitive bioassay system capable of evaluating the physiological function of human cardiac tissue in vitro. MethodsHuman iPSCs were differentiated into cardiovascular cell types (cardiomyocytes, vascular endothelial cells, and vascular mural cells) and assembled into a cardiac tissue model on aligned fiber device. This tissue was cultured dynamically to induce the formation of vascular network-like structure. By combining the fiber device with our previously developed heart-on-a-chip microdevice (HMD), we created a new model of HMD (Aligned Fiber-based HMD; AF-HMD) with improved throughput and stability. Pulsatile force changes induced by drug exposure were quantified by tracking the displacement of fluorescent microbeads within the microchannels. ResultsAF-HMD demonstrated functional responses to known cardiac agonists and toxicants, such as doxorubicin. The device also replicated clinically relevant cardiotoxic events, including the synergistic effects of trastuzumab and doxorubicin, showing marked reductions in contractile force and beat rate, mirroring clinical observations. ConclusionsThe AF-HMD system provides a sensitive and reproducible platform for evaluating cardiotoxicity in drug development. It offers a promising tool for preclinical screening, with potential applications in personalized medicine and predicting cardiotoxic risk in cancer therapy.

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.

Conventional fluorescent imaging probes, including organic dyes and semiconductor quantum dots, suffer from inherent limitations such as photobleaching, cytotoxicity, poor aqueous dispersibility, and complex synthetic routes, necessitating the development of next-generation nanoscale fluorophores suitable for biological imaging. Carbon dots (CDs) have emerged as a compelling alternative owing to their nanoscale dimensions, tunable photoluminescence, excellent biocompatibility, and amenability to green synthesis from biomass-derived precursors. Herein, we report a comparative synthesis and systematic physicochemical evaluation of nitrogen-doped and undoped carbon dots derived from chamomile (Matricaria chamomilla L.) extract, prepared via solvothermal and microwave-assisted routes. Among the four synthesized variants--CM ST-U, CM ST-N, CM MW-U, and CM MW-N--the solvothermally synthesized nitrogen-doped carbon dots (CM ST-N) exhibited markedly superior optical performance, characterized by a high fluorescence quantum yield of 57.2%, which is among the highest reported for biomass-derived nitrogen-doped carbon dots. Comprehensive characterization using UV-visible spectroscopy, photoluminescence (PL) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), zeta potential analysis, and atomic force microscopy (AFM) confirmed the nanoscale dimensions (~8.3 nm), surface-rich functional groups, successful nitrogen incorporation (10.86 %), and moderate colloidal stability (zeta potential: -17.3 mV). Photoluminescence stability studies across seven solvent systems including biologically relevant media--phosphate-buffered saline (PBS), Dulbeccos modified Eagles medium (DMEM), and serum-free medium (SFM) demonstrated sustained fluorescence emission over 72 hours. In vitro cytotoxicity assessment using the MTT assay on RPE-1 retinal pigment epithelial cells confirmed high cell viability (>70%) across a broad concentration range (10-500 {micro}g mL-1) over multiple exposure durations. Collectively, these results establish CM ST-N as a highly fluorescent, biocompatible, and colloidally stable nanoprobe with strong potential for fluorescence-based bioimaging applications.