Biomaterials Science

BackgroundProtein fibrillation represents a critical challenge in therapeutic insulin delivery, yet the structural determinants and immunological consequences of insulin-derived fibrils (IDFs) formed in the presence of phenolic preservatives remain poorly characterized. ObjectiveThis study investigated the structural characteristics of IDFs formed with (IDF (+)) and without (IDF (-)) phenolic preservatives and elucidated their differential immunomodulatory mechanisms in bone marrow-derived macrophages (BMDMs). MethodsIDF structural properties were characterized using Thioflavin T fluorescence and nanoparticle tracking analysis (Spectradyne nCS1). BMDMs were treated with serial dilutions of IDF (+), IDF (-), or m-cresol. Cytotoxicity, reactive oxygen species (ROS) production, MIP-1 levels, and expression of signaling pathways were quantified. ResultsStructural analysis revealed similar aggregation states between IDF (+) and IDF (-). However, IDF (+) induced greater cytotoxicity and ROS production than IDF (-), which produced minimal ROS. Both fibrils increased MIP-1 chemokine levels. Additionally, IDF (-) upregulated NRF2 whereas m-cresol downregulated STAT6 compared to control. Together, these results support the existence of distinct mechanisms of macrophage activation and suggest that protein aggregates can directly induce macrophage responses independent of ROS production. ConclusionsInsulin fibrils activate macrophage inflammatory pathways through ROS-independent mechanisms. Phenolic preservatives enhance fibril cytotoxicity and likely ROS production while differentially modulating inflammatory signaling. These findings suggest that strategies to remove or reduce the effects of IDFs in insulin infusion therapy may increase longevity and biocompatibility of these devices.

Collagen type II (Col-II) and collagen type I (Col-I) are major components of articular cartilage present at different ratios at its surface. Understanding how each of these components mediates the assembly of molecular films derived from synovial fluid (SF), the native lubricant of synovial joints, is critical to explain the loss of mechanical performance in pathological conditions, guide the design of biomaterial implants meant to be in contact with SF, and develop molecular therapies to restore SF properties. This work demonstrates that Col-II articular surface model assists in scaffolding of full SF-derived films, while Col-I model lacks SF film scaffolding capabilities. However, when Col-II and Col-I are exposed to recombinant lubricin (rLub) alone, the major boundary lubricant in SF, both adsorbed and retained similar amounts. These insights, deduced from quartz crystal microbalance with dissipation, diffuse reflectance circular dichroism, and atomic force microscopy, reveal possible mechanisms underlying the loss of mechanical performance of synovial joints in pathology, where Col-I becomes the major collagenous component of the articular cartilage surface, as well as considerations for designing functional biomaterial implants. Furthermore, this work reinforces the idea of rLub as an intra-articular osteoarthritis therapy with the ability to bind to Col-II and Col-I, irrespectively. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC="FIGDIR/small/726594v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@138a77borg.highwire.dtl.DTLVardef@7b8512org.highwire.dtl.DTLVardef@15d7060org.highwire.dtl.DTLVardef@17ccf20_HPS_FORMAT_FIGEXP M_FIG C_FIG

Matrix-bound nanovesicles (MBVs) are a recently discovered subclass of small extracellular vesicles (EVs) that reside within the extracellular matrix of non-mineralized tissues throughout the body. Functionally, MBVs exhibit unique immunomodulatory properties that have been leveraged therapeutically to treat various tissue pathologies, including periprosthetic osteolysis, rheumatoid arthritis, and skeletal muscle injury. However, like other EVs, the therapeutic efficacy of MBV applications is limited by delivery methods, namely bolus injections, that offer poor control of EV persistence and bioavailability at the site of administration. We hypothesized that a superior MBV delivery platform could be developed by entrapping MBVs in a tunable, engineered alginate matrix to control retention and release of MBVs on therapeutically relevant timescales. To this end, we encapsulated dermal fibroblast MBVs in bioinert alginate hydrogels of varying stiffness and stress relaxation rates to determine the impact of matrix mechanical properties on MBV release and retention over a 14-day period. We found that stiffer matrices increased MBV release compared to their softer counterparts. Additionally, fast-relaxing matrices exhibited release of MBVs in the first four days of release experiments, in contrast with slow-relaxing matrices, which promoted long-term sequestration of nearly all encapsulated MBVs regardless of differences in matrix stiffness. Our results offer promise that alginate hydrogels can be utilized for more precise control of MBV delivery in the body and may overcome limitations associated with current EV administration methods.

Sustainable, biodegradable elastomers are needed to replace fossil-based alternatives and reduce the environmental impact of traditional vibration damping materials. We investigate agarose-based hydrogels as eco-friendly vibration absorbers, examining the combined effects of polymer concentration (1-7 wt%), relative humidity (55-98%), and mechanical pre-stress on their dynamic mechanical properties. Frequency-dependent viscoelastic and vibration transmissibility tests, supported by Gaussian Process Regression (GPR), reveal that increasing agarose concentration enhances the storage modulus (E') by over an order of magnitude, reaching[~] 5 MPa depending on humidity and applied prestress. Remarkably, the damping efficiency--characterised by the loss factor (tan(d))--exhibits a highly non-monotonic trend. Maximum energy dissipation is observed at intermediate network densities, with tan(d) up to 0.21 and a loss modulus of[~] 515 kPa at 5 w% and 75% relative humidity, comparable to synthetic elastomers. GPR analysis shows that prestress controls nonlinear stiffening and transmissibility resonance behavior, while shifting peak damping from 5 wt% to 1 wt% agarose as prestress increases. These findings underscore the mechanical tunability and sustainability of agarose hydrogels, providing potential design guidance for biodegradable vibration mitigation materials.

Extracellular vesicles (EVs) are nanoscale, cell-secreted mediators of intercellular communication with growing promise as therapeutic agents. Manufacturing practices, including EV isolation and storage approaches, are critical determinants of product consistency, purity, and potency. In this study, neural stem cell (NSC)-derived EVs were isolated from conditioned NSC culture media via oscillator-based isolation (OSC), ultracentrifugation (1 or 2 hours), and ultrafiltration, and were stored lyophilized or cryopreserved. Nanoparticle yield, size distribution, and subpopulation composition were evaluated by nano-flow cytometry, quantifying total nanoparticles, membrane-bound EVs and CD63+ EVs. Purification was calculated via particle-to-protein ratios, morphology was evaluated by transmission electron microscopy, and potency was assessed using a microglia morphology assay. Particle yield was comparable across isolation methods, though protein clearance varied, with OSC demonstrating purification relative to conditioned media. Lyophilized samples retained structural integrity, size, and population profiles comparable to cryopreserved samples. Lyophilized and cryopreserved EVs exhibited dose-dependent immunomodulatory activity in our microglia morphology assay, with significant effects observed at 200,000 EVs per cell. These findings highlight the importance of isolation method in EV product quality and support lyophilization as a viable storage strategy which overcomes the logistical limitations of cryopreservation, thereby advancing the development of a robust pipeline for therapeutic EV manufacture.

Fibrosis is a progressive and often fatal pathological process characterized by excessive extracellular matrix deposition, tissue stiffening, and irreversible organ dysfunction. Effective antifibrotic therapies remain limited by the lack of in vitro models that recapitulate the full spectrum of fibrotic disease progression. Here, we leverage tyramine-modified silk fibroin (SF-TA) hydrogels to investigate normal human lung fibroblasts (NHLF) responses to progressively stiffening environments relevant to pulmonary fibrosis. Two hydrogel formulations with distinct stiffening profiles over 14 days were prepared: a gradual-stiffening 0% SF-TA formulation reaching [~]20 kPa, and a rapidly stiffening 50% SF-TA formulation reaching [~]60 kPa. NHLFs were cultured on both formulations, with and without TGF{beta} (5 ng/mL), for 14 days and assessed for viability, metabolic activity, cytokine and collagen secretion, cytoskeletal organization, and mechanotransductive gene expression. The 0% SF-TA hydrogels drove sustained fibroblast proliferation and elevated secretion of IL-6, IL-8, and MCP-1, consistent with early inflammatory fibrosis. The 50% SF-TA hydrogels induced a metabolic plateau without senescence, suppressed inflammatory cytokine secretion, and, in the presence of TGF{beta}, led to significant upregulation of ACTA2 and CTGF, alongside -SMA stress fiber incorporation, consistent with established myofibroblast persistence. Both conditions produced comparable secreted collagen output by day 14. Together, these findings establish dynamically stiffening SF-TA hydrogels as a tunable platform for investigating stage-dependent fibroblast activation and mechanobiological progression in fibrosis.

Curcumin is a naturally occurring polyphenol that demonstrates considerable anti-cancer activity, however the aqueous insolubility, rapid metabolism and relatively low bioavailability are limiting to its clinical application. As such, a curcumin-magnesium (Cur-Mg) coordination complex was synthesized and subsequently encapsulated within DNA hydrogels (Cur-Mg-Hgel). The Cur-Mg complex was fully characterized using UV-Vis spectroscopy, FTIR and X-ray diffraction (XRD). UV-Vis, FTIR and XRD all support the formation of a coordination complex and suggest a decreased level of crystallinity compared to free curcumin. DNA hydrogels were formed and characterized using atomic force microscopy, rheology and swelling kinetic studies. In vitro cytotoxicity studies utilizing an MTT assay demonstrate dose dependent inhibition of HeLa cell proliferation and a slightly better retention of RPE-1 viability at low concentrations (suggesting some difference in sensitivity) though significant cell death is seen at higher concentrations and both cells. Intracellular production of ROS was measured using the DCFH-DA assay and is seen to increase when HeLa cells are treated with Cur-Mg-Hgel in comparison to un-treated controls. Annexin V/PI staining demonstrates primarily late or early apoptotic activity with minimal necrosis following treatment with Cur-Mg-Hgel. The evidence presented strongly supports the notion that Cur-Mg-Hgel is a ROS-modulating, pro-apoptotic Hydrogel suitable for cancer treatment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/724072v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@18727aeorg.highwire.dtl.DTLVardef@3e20adorg.highwire.dtl.DTLVardef@d3703eorg.highwire.dtl.DTLVardef@16e260e_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

Granular extracellular matrix (gECM)-based biomaterials commonly contain polymer components to improve scaffold cohesion and handling during fabrication and use. However, these polymer hydrogel components may dilute ECM content and increase fabrication and regulatory complexity. This study evaluated whether particle-only gECM wafers could serve as a simplified alternative to hydrogel-based gECM scaffolds while maintaining structural, mechanical, and biological performance. Decellularized human cartilage and skin tissues were processed and fabricated into three scaffold formats: gECM hydrogels, freeze-dried gECM hydrogel wafers, and freeze-dried particle-only gECM wafers. Across fabrication methods, scaffold swelling, volume fraction, and stiffness were strongly influenced by both tissue type and fabrication approach. gECM hydrogels exhibited the greatest swelling and lowest stiffness, while gECM wafers displayed higher volume fractions and greater mechanical stiffness. Notably, gECM particle-only wafers achieved performance comparable to gECM hydrogel wafers despite the absence of a secondary polymer network. Particle-only wafers also maintained swelling behavior and structural properties over 3 months of dry storage at room temperature, with only modest decreases in stiffness. In vitro studies showed sustained cell viability over 14 days on particle-only wafers, with chondrocytes infiltrating cartilage wafers and fibroblasts remaining primarily surface-localized on skin wafers. In addition, particle-only wafers remained cohesive during implantation into a bovine cartilage defect model. These findings demonstrate that particle-only gECM wafers can achieve structural integrity, mechanical performance, and cytocompatibility without the need for an additional polymer network, highlighting a simplified and ECM-rich biomaterial platform. By eliminating polymer carriers and enabling dry storage with preserved function, this approach supports the development of off-the-shelf, translationally accessible gECM particle-only wafers for tissue engineering applications.

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.

Pathological collagen degradation is a central feature of impaired wound healing, dermal aging, periodontal breakdown, intestinal barrier injury and connective tissue degeneration. Current strategies often focus on direct inhibition of matrix metalloproteinases or collagenases; however, complete blockade of collagen remodeling may interfere with normal repair. UT-018, a bioactive formulation that acts as a tissue-protective and regenerative agent, was evaluated as a collagenous extracellular matrix modulator. Across in vitro kinetic assays, endpoint signal analysis, integrated area-under-curve (AUC) analysis and substrate preincubation studies, UT-018 produced concentration-dependent preservation of collagen against collagenase challenge. Importantly, collagen protection persisted after substrate preincubation with UT-018, with approximately 33%, 60% and 65% protection at 5, 10 and 25 mM UT-018 concentrations, respectively. Exploratory kinetic transformations did not support a simple competitive collagenase inhibitor model. Instead, the collective pattern supports a substrate-directed mechanism involving collagen shielding, reduced cleavage susceptibility and indirect modulation of collagenase activity. These findings position UT-018 as a potential first-in-class collagen resilience modulator for wound healing, gastrointestinal barrier protection, oral care, dermal preservation and regenerative medicine applications. Highlights- UT-018 preserves collagen content in in vitro collagenase challenge assays. - The Protection is UT-018 concentration-dependent across kinetic, endpoint and AUC readouts. - Preincubation of substrate with UT-018 retains protection after collagenase challenge. - The data support matrix-directed stabilization by UT-018 rather than classical active-site collagenase inhibition.

Post-traumatic osteoarthritis (PTOA) is a progressive joint disease for which no disease-modifying osteoarthritis drugs (DMOADs) have been approved. Although injectable drug delivery systems can prolong therapeutic retention within the joint, it remains unclear whether local drug release kinetics influence disease-modifying efficacy. Here, we developed a modular platform of injectable supramolecular hydrogels using biocompatible, generally recognized as safe (GRAS) amphiphilic molecules and systematically engineered a range of degradation and drug release profiles. Using the cathepsin-K inhibitor L-006235 as a model DMOAD, we generated hydrogels with distinct release kinetics and evaluated their therapeutic performance in PTOA. Hydrogels exhibiting slower degradation and more sustained drug release like Sucrose Stearate (SS hydrogel) showed prolonged intra-articular retention and improved therapeutic outcomes. In a destabilization of the medial meniscus (DMM) mouse model, sustained-release formulations significantly reduced cartilage degeneration, preserved aggrecan expression, improved joint histopathology, and enabled effective monthly dosing. In contrast, formulations with faster degradation and release kinetics required more frequent administration to achieve comparable benefits. To our knowledge, this is the first study to establish local drug release kinetics as a critical determinant of disease-modifying efficacy in PTOA. This work provides one of the clearest demonstrations to date that engineering intra-articular release kinetics, rather than merely prolonging residence time, can improve disease-modifying outcomes. Our findings establish local release kinetics as a key design parameter for osteoarthritis therapeutics and highlight the potential of tunable supramolecular hydrogels for long-acting drug delivery.

Stroke remains a leading cause of adult disability, driven in part by the formation of a non-permissive extracellular matrix environment that limits endogenous repair. Injectable biomaterials that can modulate this microenvironment while enabling localised therapeutic delivery, represent a promising strategy for post-stroke brain regeneration. Here, we report the development of a thermoresponsive hybrid hydrogel composed of chitosan, {beta}-glycerophosphate, silk fibroin, polyvinyl alcohol and polyvinyl pyrrolidone, engineered to provide a tuneable physicochemical properties and enhanced biological functionalities for intracerebral delivery. Systemic optimisation identified a formulation (F6) that exhibited rapid gelation at physiological temperature, appropriate viscoelastic properties, a microporous architecture, and controlled biodegradation, conducive to cellular infiltration and molecular transport. In a mouse model of photothrombotic stroke, intracerebral delivery of the F6 hydrogel attenuated reactive astrogliosis and microglial activation in the peri-infarct region, while enhancing neurogenesis in the subventricular zone. Notably, incorporation of brain-derived neurotrophic factor within the hydrogel significantly improved functional recovery over 8 weeks, demonstrating the capacity of this system to act as a localised delivery platform for neuro-regenerative therapeutics. Together, this study establishes a tuneable thermoresponsive hydrogel platform that integrates structural support with controlled therapeutic delivery, highlighting its potential as a minimally invasive strategy for modulating the post-stroke microenvironment and promoting functional recovery.

PurposeExtracellular vesicles (EVs) are increasingly explored as natural vehicles for drug delivery and gene therapy approaches. However, reproducible yield and scalability of EV production still pose major challenges in the clinical translation of EV-based therapies. In this study, we sought to quantify and characterize EVs released by suspension-cultured HEK293 cells (Expi293F cells) grown in shaker flasks or small-scale bioreactors, to investigate how the culturing environment affects EV production yield. MethodsExpi293F cells were cultivated (N=3) in either shaker flasks or a bioreactor system, and total cell density, viability, and size were monitored. Supernatants were drawn daily post-cell seeding and were analyzed for EV quantity, size, morphology, and CD63 expression. ResultsNo significant differences were observed in terms of total cell density, viability, and cell size between both cultivation settings. However, cultivation of Expi293F cells in the bioreactor environment significantly increased EV yield by 3-fold compared to shaker flask cultivation (p < 0.01). Other parameters such as average nanoparticle size, EV morphology, and CD63 expression remained comparable between both cultivation methods. ConclusionThese results demonstrate that Expi293F-derived EV yield can be increased by culturing cells in a scalable bioreactor system. These findings pave the way towards the production of therapeutic-based EVs in a scalable and reproducible manner suitable for future (pre-)clinical applications.

Ex vivo airway engineering approaches such as 3D bioprinting offer a promising strategy for generating functional airway replacements, but the fabrication of hollow, patient-specific proximal airway constructs using translationally relevant bioinks remains challenging. This study describes the development of biocompatible, polymer-blended human airway-derived decellularized extracellular matrix (AW-dECM) bioinks for engineering structurally and mechanically relevant airway tissues. An optimal formulation consisting of 30 mg/mL AW-dECM and nanofibrillar cellulose alginate conjugated to RGD supported the bioprinting of simple and complex hollow airway structures with mechanical properties comparable to native airways ([~]8-10 kPa). The bioinks also promoted primary human airway epithelial cell viability, adhesion, and differentiation into mucociliary and secretory phenotypes during 28 days of air-liquid interface culture. Furthermore, subcutaneous implantation in immunocompetent rats demonstrated excellent biodegradative stability and overall biocompatibility over 30 days. Collectively, these findings establish a foundation for improved physiological airway models and future tissue-engineered airway replacements.

Controlled release systems for subcutaneous peptide delivery often exhibit a pronounced initial burst release followed by inadequate maintenance of therapeutic exposure, limiting depot lifetime and increasing pharmacokinetic variability. Here, we engineer a dynamic, injectable hydrogel depot technology for months-long delivery of lipidated peptides. Using semaglutide as a model, we establish a modular formulation framework integrating: (i) formulation-driven tuning of depot mechanics to control release kinetics, (ii) cargo complexation strategies leveraging hydrophobic and multivalent ion-mediated interactions, and (iii) oxidative stabilization through sacrificial antioxidant excipients. We evaluated depot performance by rheology, in vitro cargo release, and in vivo pharmacokinetic and pharmacodynamic studies in rodents. Optimized formulations sustained semaglutide exposure for over six weeks from a single administration with two-fold reduction in peak-to-trough exposure and comparable total bioavailability relative to daily dosing, resulting in improved glucose control, weight regulation, and preservation of pancreatic islet content. These results suggest potential for quarterly dosing in humans. Together, this work establishes integrated and generalizable structure-property-performance relationships that account for cargo-matrix and cargo-excipient interactions across burst, diffusion, and erosion regimes to inform a practical formulation framework for engineering long-acting depots for sustained peptide delivery.

The transplantation of stem cell-derived extracellular vesicles (EVs) holds promise for tissue repair and regeneration, but scalable production and effective delivery to target tissue remain major challenges. Here, we present a biomaterial platform that combines high-yield, scalable nanovesicles (NVs) - EV mimetics derived from human induced pluripotent stem cells - with an adhesive silk hydrogel patch for localized and sustained delivery. We show that this platform enables efficient NV encapsulation via visible light crosslinking and supports controlled release over short (2 days), intermediate (7 days), and extended (up to 28 days) periods, while maintaining adhesion to heart tissue. Importantly, the sustained delivery of NVs for 3 days in vitro results in promoting anti-fibrotic cell remodeling and significant functional recovery of primary myofibroblast activation, modulating integrin signaling, actomyosin organization, and cell-matrix adhesion networks. Finally, we demonstrate biocompatibility, retention, and anti-fibrotic function of the patch in a murine ischemia-reperfusion injury model. Thus, we establish the proof-of-principle that di-tyrosine silk hydrogels can be used as a strategy to encapsulate and deliver NVs to the heart, thus offering an innovative delivery platform for NVs. Statement of significanceExtracellular vesicles (EVs) represent an emerging frontier in tissue engineering. Their cell-specific cargo contains biological information capable of repairing and regenerating injured tissues. However, their clinical translation is hindered by limited manufacturing scalability, undefined dosing and modes of administration, and low organ retention, particularly in the heart. This study addresses these challenges by combining stem cell-derived nanovesicles (NVs), which mimic biological EVs, with an adhesive hydrogel patch for localized and sustained delivery to the heart. We provide proof-of-principle that di-tyrosine photo-crosslinked silk hydrogels are a suitable delivery platform for cell-derived NVs, preserving NV bioactivity and their ability to remodel recipient cells following delivery both in vitro and in vivo. This study integrates three key advantages: (i) the use of scalable iPSC-derived nanovesicles as an EV-mimetic platform, addressing limitations in EV manufacturing; (ii) a mechanically robust and tunable silk fibroin hydrogel formed via visible light-induced di-tyrosine crosslinking without chemical modification; and (iii) an injection-free, adhesive patch-based delivery strategy enabling localized and sustained therapeutic administration to the heart. This innovative platform represents a significant advancement in the fields of nanomedicine and biomedical engineering. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/722555v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@fed253org.highwire.dtl.DTLVardef@1a270b0org.highwire.dtl.DTLVardef@19437c1org.highwire.dtl.DTLVardef@1d863ca_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

Class B1 G-protein-coupled receptors (GPCRs), such as the calcitonin gene-related peptide (CGRP) receptor and parathyroid hormone 1 (PTH1) receptor, require native lipid interactions to maintain signalling-competent conformations. However, conventional detergents disrupt these environments. Amphipathic copolymers offer a detergent-free alternative, yet the field still lacks a clear understanding of which polymer architectures best preserve active-state GPCR pharmacology, limiting their broader translational utility. Here, we examine how distinct copolymer chemistries influence the functional integrity of class B1 GPCRs by comparing SMA 2000, DIBMA-12, and the electroneutral sulfo-DIBMA. Using NanoLuciferase bioluminescence resonance energy transfer (NanoBRET) ligand-binding, competition, and mini-G-protein recruitment assays on nanodisc-encapsulated receptors, we show that all three copolymers maintain high-affinity extracellular ligand binding but differ markedly in their ability to preserve intracellular signalling. Despite lower receptor extraction efficiency, only sulfo-DIBMA support mini-Gs engagement at the CGRP receptor and enable G-protein-dependent allosteric modulation at the PTH1 receptor, including conserved ligand affinity and prolonged residence time. These data reveal that polymer charge and backbone chemistry, rather than extraction yield, determine whether native-like nanodiscs retain the conformational landscape required for active-state signalling. Controlling non-specific ligand binding to the copolymer is a key requirement for a successful assay. Our findings identify sulfo-DIBMALP as a particularly superior environment for preserving native signalling behaviour in class B1 GPCRs, highlighting copolymer chemistry as an important determinant in detergent-free membrane protein studies. HIGHLIGHTSO_LISulfo-DIBMA encapsulated nanodiscs preserve active-state conformation of human calcitonin gene-related peptide receptor and parathyroid hormone 1 receptor. C_LIO_LIAll three copolymers (SMA 2000, DIBMA-12 and sulfo-DIBMA) preserve extracellular ligand binding but only sulfo-DIBMA preserves intracellular functional competence, including mini-Gs recruitment and G-protein-dependent allosteric modulation. C_LIO_LICopolymer chemistry, particularly the electroneutral, aliphatic nature of sulfo-DIBMA, may influence the preservation of signalling-competent states in two class B1 GPCRs by minimising charge-driven perturbations during solubilisation. C_LIO_LISulfo-DIBMALP provides a novel platform for studying dynamic membrane proteins with potential to provide mechanistic insights and facilitate drug discovery programmes in the future. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/724797v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@12db163org.highwire.dtl.DTLVardef@d8efb3org.highwire.dtl.DTLVardef@610dbaorg.highwire.dtl.DTLVardef@1cc3ce4_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cell migration in three-dimensional (3D) environments is highly plastic and regulated by extracellular matrix (ECM) cues. Engineered biomaterials provide controllable platforms to investigate how specific matrix signals regulate cell behavior in 3D, yet how defined biochemical signals control migration modes remain unclear. Here, we present tunable fibrous polyisocyanide (PIC) hydrogels functionalized with integrin-binding RGD peptides, cadherin-mimetic HAVDI peptides, or no ligands to direct mesenchymal, hybrid, or amoeboid-like migration of human adipose-derived stem cells without altering matrix mechanics. Using live-cell tracking, 3D displacement microscopy, matrix remodeling analysis, and YAP nuclear localization, we show that ligand identity governs adhesion organization, force transmission, and mechanotransduction. RGD-functionalized matrices promote {beta}1-integrin clustering, extensive matrix remodeling, strong YAP activation and upregulation of migration-related genes. In contrast, non-adhesive matrices limit adhesion formation, resulting in weak force transmission and amoeboid-like behavior. HAVDI-functionalized matrices induce cadherin clustering and heterogeneous cellular responses, indicating that a hybrid migration mode arises from adhesion organization rather than a distinct transcriptional program. Together, these findings demonstrate that ligand identity alone is sufficient to program migration mode in a force-responsive 3D matrix and provide a versatile platform to dissect cell-matrix interactions in complex environments. Statement of significanceO_LICell migration in tissues is highly adaptable, yet precise control of migration modes in defined 3D biomaterials remains challenging. C_LIO_LIWe introduce fibrous PIC hydrogels presenting RGD, HAVDI, or no adhesive ligands to bias human stem cells toward mesenchymal-like, hybrid, or amoeboid-like migration states. C_LIO_LIBy linking ligand identity to adhesion organization, matrix remodeling, YAP mechanotransduction, and gene expression, this work provides a minimal platform to dissect and engineer 3D cell-matrix interactions C_LI

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.