ACS Biomaterials Science & Engineering

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to establish a stable endothelial monolayer, whereas the influence of mechanical history is transient and ultimately superseded by the current mechanical environment.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.

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.

Synthetic grafts remain ineffective for small-caliber vascular applications due to thrombosis and intimal hyperplasia. To address these limitations, our lab designed a multilayer graft consisting of a hydrogel coating that promotes post-implantation endothelialization and an electrospun mesh that matches arterial mechanical properties. Damage-resistant hydrogels were engineered using a double-network system composed of polyether urethane diacrylamide and N-acryloyl glycinamide to enhance fracture resistance through hydrogen bonding. In this study, we utilized redox initiation to apply conformal, durable hydrogels to electrospun grafts. Bioactivity wa introduced using streptococcal collagen-like proteins containing 1{beta}1 and 2{beta}1 integrin-binding motifs, enabling selective cell-material interactions that support endothelialization while preserving acute thromboresistance. To establish the feasibility of these grafts as off-the-shelf devices, we evaluated coating integrity and bioactivity retention following sterilization and dynamic physiological loading. Sterilized composites exhibited surgically-associated damage resistance, indicating that sterilization did not compromise hydrogel durability. Coating integrity and bioactivity were also preserved after six weeks of physiological loading. Acut thromboresistance was supported by both static platelet adhesion assays and dynamic whole-blood bioreactor studies using heparinized blood, with low platelet adhesion observed relative to ePTFE. Finally, a pilot ovine carotid model demonstrated successful surgical handling and sustained graft patency. Collectively, these results highlight the promise of multilayer vascular grafts as durable, thromboresistant conduits for small-diameter vascular applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/741222v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@14ec910org.highwire.dtl.DTLVardef@17259e8org.highwire.dtl.DTLVardef@6cb611org.highwire.dtl.DTLVardef@1251d7d_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

Inflammation-driven increases in nociception are prominent in pain pathologies associated with intervertebral disc (IVD) degeneration yet are difficult to model in vitro. Since neurons are exposed to multi-modal stimuli in vivo, it is critical for these exposures to be conserved in an in vitro test system. We developed a polydimethylsiloxane (PDMS)-based microfluidic platform to interrogate peripheral sensory neurons (SNs) in the presence of conditioned media from nucleus pulposus cells from the degenerated IVD, to model a potential impact of IVD cells secretome on pain sensing. Our platform enables controlled perfusion of cell-derived biochemical cues alongside a defined homogeneous electric field (EF) and supports real-time optical analysis. Computational modeling, fluid perfusion experiments, and conductivity measurements confirmed stable fluid transport and tunable homogeneous EF generation within the device. As proof of concept for neuronal stimulation, neuroblastoma (N2a) cells loaded with a fluorescent Ca2+ indicator exhibited a 56% increase in Ca2+ transient activity when exposed to media from degenerated IVDs, concomitant with increased IVD-derived IL-1{beta} production. Importantly, EF-stimulated Ca2+ transients increased in SNs derived from human induced pluripotent stem cells when exposed to conditioned media from primary human IVD cells, demonstrating the translation of this model system to human cells. Together, these results establish a versatile platform that enables controlled and simultaneous exposure to biochemical and electrical stimuli to quantify inflammation-driven peripheral neuronal hyperexcitability in tissue-neuron crosstalk.

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.

Hydrogels are widely used in sensing, delivery, and tissue engineering because their transport properties can be tuned through material design. However, while hydrogel permeability is often characterized using small molecules, many practical applications depend on the uptake and retention of much larger species, including protein conjugates and nanoparticles. Here, we systematically investigate how polyethylene glycol (PEG)-acrylate hydrogel microparticle formulation influences accumulation of signal-generating probes spanning a broad size range. We fabricated particles across a 36-condition design space varying nominal PEG-acrylate molecular weight, polymer weight percent, and UV crosslinking dose, and related formulation-dependent probe accumulation to particle swelling behavior. Increasing nominal PEG-acrylate molecular weight and decreasing polymer weight percent produced more highly swollen particles and strongly enhanced accumulation of fluorescent streptavidin conjugates, with the largest effects observed for bulky labels such as allophycocyanin and phycoerythrin. Gold nanoparticle accumulation was even more formulation-restricted, with detectable colorimetric signal observed primarily in the most permissive formulations. These findings establish design rules linking PEG hydrogel formulation to size-dependent accumulation and show that formulations suitable for small probes may be inadequate for larger reporters. More broadly, this framework may inform the design of hydrogels for particle-based assays as well as other applications where transport of macromolecules or nanoscale materials is important.

In breast cancer, collagen fiber alignment and the second harmonic generation forward-to-backward ratio (F/B), an optical measurement sensitive to fibril-scale organization within collagen fibers, have each been associated with metastatic outcome. Although both are measured at the tumor-stromal interface, in vitro studies have largely examined them separately, because experimental systems have not allowed the two to be varied independently. To address this technical gap, we used a microfluidic biofabrication approach to generate paired aligned and unaligned regions within 3D collagen hydrogels via extensional strain and varied measured F/B across gels by changing the pre-gel pH. The resulting library covered alignment values reported in breast tumors and F/B values associated with poorer metastatic outcome in invasive ductal carcinoma at matched matrix stiffness, pore diameter, and fiber fraction. In MDA-MB-231 cells, alignment biased migration along the fiber axis without changing migration speed, as expected, but the magnitude of the contact guidance response varied with measured F/B. In contrast, MCF-7 cells showed little overall contact guidance, yet migrated ~30% faster in high F/B than low F/B gels in both aligned and unaligned regions. Together, these results show that cells migrated differently in matrices with similar fiber alignment. Thus, including measured F/B may provide additional information to help anticipate how cells migrate in collagen environments that appear similar based on alignment alone.

Parkinsons Disease (PD) is the second-most prevalent neurodegenerative disease, often characterized by neural motor dysfunction, oxidative stress, and dopamine receptor malfunction leading to improper dopamine levels in the system. DNA tetrahedron nanostructures are a promising drug delivery agent due to their biocompatibility and properties of controlled and sustained release. In this study we evaluated the potential of using TD-mediated Levodopa delivery for a MPTP induced Parkinsons Disease model in Zebrafish larvae. The induction of Parkinsonism led to morphological behaviour changes like the presence of tremors, erratic swimming behaviour, latency, and reduced locomotor activity, even elevated reactive oxygen species (ROS) and apoptosis was observed. These effects and symptoms were alleviated when the larvae were treated using TD:Levodopa conjugates, particularly at the 1:100 ratio. At the molecular level, genes like TH, DAT, SOX2, PARKIN and apoptotic genes like BCL2 and caspases showed alteration in expression in the Parkinsonism model and post treatment was induced. This highlights the potential of using DNA nanocages as a novel drug delivery agent as therapeutic strategy for Parkinsons disease. TOCDopamine loaded DNA nanocages with the capacity to overcome biological barriers for release of dopamine with neuroprotection activity in Parkinsons disease model of zebrafish. O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/742731v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@40d78forg.highwire.dtl.DTLVardef@107f59forg.highwire.dtl.DTLVardef@18ea275org.highwire.dtl.DTLVardef@14bc089_HPS_FORMAT_FIGEXP M_FIG C_FIG

The outer blood-retinal barrier (OBRB) is the primary interface through which systemically circulating drugs reach the retina. A tool that measures delivery across this barrier would support the development of targeted therapies as alternatives to repeated intravitreal injection, and the screening of drugs that reach the retina as an off-target toxicity. Such a tool should deliver drugs fluidically through a vascular compartment, measure transport across the retinal pigment epithelium (RPE), and display disease phenotypes relevant to efficacy. Here we adapt the SiM platform, which places epithelium and endothelium in direct juxtaposition across a permeable, optically transparent silicon nitride nanomembrane. ARPE-19 and human umbilical vein endothelial cells (HUVECs) were used as development cell sources. ARPE-19 monocultures reached a transepithelial electrical resistance of 68 {+/-} 26 {Omega} cm2 by 28 days, and ARPE-19 + HUVEC co-cultures reached a small-molecule permeability of 6.34 {+/-} 1.3 x 10-4 cm min-1 within 14 days, a state reported elsewhere only after longer culture. The barriers developed an intervening basement membrane. Drugs perfused through the basal vascular channel crossed into an open apical well, where sampling and mass spectrometry showed transport correlating with lipophilicity, as reported in vivo. The device also displayed two clinically relevant phenotypes. Digoxin at a clinically toxic concentration reduced viability in the co-barrier by about half and doubled permeability. In a vascularized configuration, VEGF drove endothelial invasion of the RPE layer, as seen in neovascular AMD. The SiM-OBRB therefore satisfies basic design criteria for measurement of drug bioavailability, toxicity, and efficacy.

Metastatic dissemination is initiated by tumor cells interpreting spatially organized biochemical and biophysical cues that remain difficult to reproduce using conventional migration assays. Here, we developed a computationally guided metastasis-on-a-chip (MET-on-a-chip) platform based on the concept of the Minimally Functional Unit (MFU), in which only the biological components required to answer a defined experimental question are incorporated. The platform consists of two independent culture chambers connected through an array of confined microchannels that permits diffusion of soluble factors while constraining tumor cell migration. Rather than relying on empirical optimization, finite-element COMSOL simulations were first used to predict molecular transport, define growth factor loading conditions, identify biologically relevant exposure regions, and guide the rational design of the microfluidic assay. Computational predictions were experimentally validated using 70-kDa FITC-dextran diffusion and VEGF release studies, confirming the formation of stable spatial concentration gradients across the microfluidic platform. The simulations further demonstrated that both growth factor loading and cell positioning relative to the predicted gradients critically influenced assay performance, leading to the optimization of the platform through spatial reconfiguration of the tumor compartment. Using the optimized configuration, we compared the migratory responses of neuroblastoma, Ewing sarcoma, and osteosarcoma cells to vascular (VEGF-A165) and lymphatic (VEGF-C) chemotactic cues. VEGF-C significantly increased migration through the microchannel array in Ewing sarcoma and osteosarcoma cells, whereas VEGF-A165 produced no significant effect. In contrast, neuroblastoma cells exhibited minimal migration under either condition, revealing tumor-specific differences in responsiveness to VEGF signaling. Together, these findings establish a computationally guided workflow for the rational design of metastasis-on-a-chip assays, in which predictive modeling informs experimental design before biological validation. By substantially reducing empirical trial-and-error while enabling quantitative control over growth factor exposure, this strategy provides a robust framework for developing minimally functional microphysiological systems capable of dissecting individual steps of the metastatic cascade under experimentally defined conditions. Translational Impact StatementMetastatic dissemination remains one of the greatest clinical challenges in pediatric oncology, yet experimental models capable of quantitatively evaluating early migratory events remain limited. The computationally guided MET-on-a-Chip workflow presented here provides a human-relevant platform in which soluble microenvironmental cues can be systematically investigated under controlled and predictive conditions. Although demonstrated here using VEGF-A165 and VEGF-C, the platform can be readily adapted to study virtually any chemotactic factor, cytokine, extracellular vesicle population, or therapeutic candidate involved in metastatic dissemination. The modular MFU design allows biological complexity to be incorporated progressively as dictated by the scientific question, providing a flexible framework for future applications. In the longer term, this workflow could be combined with patient-derived tumor cells, organoids, or biopsy material to investigate patient-specific metastatic behavior and evaluate anti-metastatic therapeutic strategies in a personalized setting. Beyond identifying pro-migratory signaling pathways, the platform may serve as a preclinical tool to prioritize compounds capable of preventing tumor cell dissemination before evaluation in more complex animal models or clinical studies.

Chronic wounds such as diabetic foot ulcers are typically more alkaline than healing wounds, making wound pH a valuable diagnostic and prognostic marker. However, point-of-care pH monitoring remains limited by the availability of point-of-care wound pH sensing systems that offer quantitative pH determination, low toxicity, and small portable detectors. Here, we report a colorimetric pH-sensing wound dressing that enables in situ pH detection using a conventional smartphone camera. The anionic pH-sensitive dye HPTS was loaded onto cationic microparticles and embedded within a calcium-crosslinked alginate hydrogel. Across the clinically relevant range of pH 6.0-9.0, increasing pH produced a progressively more intense yellow coloration, quantified through the blue channel of smartphone-acquired RGB images. The dressing displayed a strong, rapid, and reversible signal in vitro with low dye release. In a full-thickness excisional wound model in mice, wound pH changes were detected in vivo. The combination of a pH-sensitive colorimetric hydrogel with a conventional smartphone detector offers accessible wound pH monitoring at the point-of-care.

FluPep is a 16 amino-acid peptide with potent anti-viral activity against influenza A virus, including the H1N1 subtype. To understand how FluPep exerts its effects, gold nanoparticles were functionalised with FluPep, since gold nanoparticles provide a very sensitive probe that can be detected optically. Our results demonstrate that nanoparticle-FluPep conjugates did not bind to influenza virus particles in vitro. However, the nanoparticle-FluPep conjugates bound to polyanions on the cell surface, and after washing cells they remained cell-associated and were able to inhibit flu virus infectivity. Nanoparticle-FluPep conjugates bound to heparin affinity columns and their binding to cells could be reduced by pre-treatment of the cells with heparinase and neuraminidase, but not with chondroitinase ABC. These data indicated that FluPep bound to heparan sulfate and sialic acid-containing glycans in the pericellular matrix and in doing so prevented viral infectivity. The importance of the interaction of FluPep with heparan sulfate (HS) was examined by extending FluPep at its C-terminus with amino acid sequences responsible for the interactions of growth factors with heparin. The resultant two new engineered peptides, termed superFluPep1 and superFluPep2, were more potent than parental FluPep. The results demonstrate that FluPep likely exerts its antiviral activity by interfering with virus-cell interactions and that enhancing the binding of such peptides to heparan sulfate is an effective strategy to increase their anti viral potency.

Four-dimensional (4D) materials incorporating functional gradient designs offer a powerful platform for engineering dynamic structures capable of programmed shape transformations in response to environmental stimuli. However, most gradient-based 4D systems rely on uniaxial gradients, which typically generate simple, symmetric deformations with uniform curvature, limiting their ability to recreate biomimetic architectures that require spatially coordinated morphogenesis. Here, we report a biaxial gradient-engineered 4D hydrogel system capable of programmable, non-uniform shape morphing within a single construct. A one-step photocrosslinking strategy integrates vertical light attenuation and horizontal grayscale photomask patterning to establish orthogonal crosslinking gradients along two directions, producing spatially heterogeneous swelling stresses that drive controlled multi-directional deformation. The resulting hydrogels exhibit tunable swelling and mechanical properties, enabling precise regulation of curvature distribution and shape transformation. This biaxial gradient platform generates diverse biomimetic architectures, including swan-neck, fiddlehead fern, sea star, and Euonymus europaeus-like structures. Importantly, the system supports cell-laden biofabrication, where human mesenchymal stem cell-encapsulated constructs maintain high viability and undergo chondrogenic differentiation while preserving programmed morphologies. This work establishes biaxial gradient-programmed 4D hydrogels as a robust strategy for integrating morphogenesis with tissue formation, advancing biomimetic biofabrication and morphogenetic tissue engineering.

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.

Synthetic optical skin models offer reproducible, tunable optical properties but lack biological responsiveness, while tissue engineered skin models provide cellular authenticity but suffer from optical variability and limited controllability. The growing demand for alternatives to animal models in the development and validation of optical biomedical technologies highlights the need for a new class of test system that combines the strengths of both approaches while addressing their respective limitations. Here, we introduce the concept of a modular biohybrid skin model, a new testing concept that integrates an optically defined artificial epidermal layer, incorporating polydopamine nanoparticles for changes in skin tone, with living human keratinocytes in two and three-dimensional configurations. In the Optical Protection Model, UV-B-induced apoptosis in primary keratinocytes is quantitatively modulated by model pigmentation level, demonstrating a relationship between optical attenuation and caspase 3/7 activity across three artificial skin tone conditions. In a Structured Dermal Model, keratinocytes seeded onto a hydrogel scaffold localize within follicle-like microcavities, as confirmed by live/dead staining and confocal z-stack imaging. Together, these experiments lead to a new category of test system in the space between inert optical models and variable tissue models that may contribute to reducing the reliance on animal models in biomedical optics.

Musculoskeletal symptoms are frequently reported following silicone breast implantation. However, the biological mechanisms linking implant-derived silicone exposure to skeletal muscle alterations remain poorly understood, partly because the biological effects of silicone have long been debated in the context of its biocompatibility. Here, we chemically characterized the low-molecular-weight fraction of the breast implant silicone exposome, readily released from implant gel through gel bleed, and investigated its potential biological consequences using an integrated approach combining analytical chemistry, clinical transcriptomics and histology, and controlled in vitro muscle experiments. Transcriptomic analyses of periprosthetic tissues associated with silicone implant rupture revealed unexpected myogenic and neuromuscular signatures in tissue conventionally regarded as predominantly fibrous, together with alterations in lipid metabolism and transport. These findings were supported by histological evidence of close interactions between periprosthetic tissue and skeletal muscle. Chemical analysis of the implant-gel extract detected linear siloxane L2 and cyclic siloxanes D3-D8, with tentative assignment of D9. In vitro, C2C12 cells exposed to the implant-gel extract showed up to 30% reduced viability and decreased expression of key neuromyogenic genes. Together, these findings provide convergent chemical, clinical, and experimental evidence that low-molecular-weight constituents of the breast implant silicone exposome may constitute a biologically active exposure capable of affecting skeletal muscle. The associated alterations in lipid metabolism and transport further provide a mechanistic framework for investigating the cellular handling and potential tissue distribution of hydrophobic silicone-derived species. These findings position silicone gel bleed as a biologically relevant source of chemical exposure rather than solely a material-integrity phenomenon.

Despite their promise, lipid nanoparticle gene delivery systems have repeatedly failed clinical trials and struggle to achieve efficient, localized transfection in target tissues. The majority of endocytosed nanoparticles are degraded before nucleic acid release, and an inability to track particle distribution in vivo prevents validation of successful delivery. Alternatively, nanobubbles (NBs) are lipid-shelled, gas-core preclinical ultrasound contrast agents and stimuli-responsive drug delivery vehicles. Under varying acoustic pressures, NBs expand, contract, and burst, releasing cargo in an externally controlled, site-specific manner while scattering unique echoes for simultaneous ultrasound visualization. Here, we introduce a cationic nanobubble (CNB) formulation with a +42.3 mV zeta potential, 265 nm diameter, and 2.43x1011 NBs/mL concentration. CNBs produce stable ultrasound contrast, electrostatically load plasmid DNA onto their surface, and internalize into >99% of human prostate cancer cells within 15 minutes in vitro. CNBs remain brightly echogenic intracellularly and induce sonication-dependent expression of green fluorescent protein (GFP). In vivo, CNBs generate contrast in mouse livers for 50 minutes after intravenous administration. Therapeutic ultrasound stimulation over the liver causes a sharp reduction in ultrasound contrast, visualizing localized cavitation in the target organ and inducing a 2.5-fold increase in anti-GFP mean fluorescence intensity relative to the untransfected control. Importantly, no GFP expression is observed without ultrasound stimulation, supporting a mechanism for selective and site-specific gene delivery. This study presents a highly stable CNB capable of efficient DNA loading and ultrasound-dependent gene expression. These results provide a foundation for the future development of CNB platforms to advance image-guided, ultrasound-triggered gene therapy.

Biochemical gradients and mechanical microenvironments synergistically regulate tumor cell proliferation, migration, and phenotypic evolution, which are critical for recapitulating in vitro tumor physiological microenvironments. In this work, a multilayer-assembled microfluidic chip integrated with Tesla-valve flow channels was developed for 3D SKOV3 cell culture under combined biochemical gradients and mechanical stiffness interfaces. The reconfigurable multilayer PMMA structure enables flexible construction of diverse concentration gradient fields, while the embedded Tesla-valve geometry effectively stabilizes fluid flow, reduces flow velocity fluctuation, and generates smoother, more stable biochemical gradients compared with conventional curved channel designs. Multiphysics simulations were performed to systematically verify the flow field distribution and gradient formation performance of the optimized channel structure. Furthermore, by constructing heterogeneous hydrogel microenvironments consisting of rigid Pluronic F127 and soft dextran hydrogel inside microfluidic chambers, stable mechanical stiffness interfaces were successfully fabricated. Combined with TGF {beta}1 biochemical stimulation, the platform was applied to explore the durotactic migration and morphological changes of 3D-cultured SKOV3 cells at stiffness interfaces. Fluorescence staining results demonstrated that mechanical boundary conditions significantly affected cell migration behavior and cell aggregation phenotypes under consistent biochemical induction. With the advantages of low cost, simple assembly, good biocompatibility, and controllable dual physicochemical microenvironments, this multilayer microfluidic platform provides a reliable and efficient strategy for in vitro tumor microenvironment simulation and tumor cell mechanobiology research.

Three-dimensional (3D) spheroid culture provides a physiologically relevant alternative to conventional two-dimensional culture, but reliable spheroid formation in microwells depends on limiting cell-substrate adhesion. Pluronic F127 is an amphiphilic triblock copolymer that forms a hydrated surface layer, reducing protein adsorption. Here, we evaluated whether this intrinsic anti-fouling property could restore an anti-adhesive surface in used microwell plates to promote spheroid formation. Using chondrogenic ATDC5 and pancreatic {beta}-cell INS-1 cells, we characterized spheroid assembly kinetics, F127 cytotoxicity, surface hydrophilicity, protein adsorption, and spheroid morphology including size and shape factor. Both cell types formed compact spheroids within 24 hours on commercial anti-adhesive microwells. F127 coating markedly reduced water contact angle and protein adsorption, confirming increased surface hydrophilicity and reduced protein fouling. In microwells stripped of their original surface coating, F127 coating amounts of approximately 0.011-0.045 mg/cm2 consistently promoted spheroid formation across both cell types. Soluble F127 concentrations were confirmed to be non-cytotoxic up to 0.625% (w/v), while even complete dissolution of the highest tested coating amount would correspond to only 0.025% (w/v) F127. This simple, reproducible, and low-cost surface-modification strategy may provide an accessible approach for re-functionalizing microwell platforms for 3D cell culture.