ACS Biomaterials Science & Engineering

Cystic fibrosis (CF) is a muco-obstructive lung disease where inflammatory responses due to chronic infection result in the accumulation of neutrophil extracellular traps (NETs) in the airways. NETs are web-like complexes comprised mainly of decondensed chromatin that function to capture and kill bacteria. Prior studies have established excess release of NETs in CF airways increases viscoelasticity of mucus secretions and reduces mucociliary clearance. Despite the pivotal role of NETs in CF disease pathogenesis, current in vitro models of this disease do not account for their contribution. Motivated by this, we developed a new approach to study the pathobiological effects of NETs in CF by combining synthetic NET-like biomaterials, composed of DNA and histones, with an in vitro human airway epithelial cell culture model. To determine the impact of synthetic NETs on airway clearance function, we incorporated synthetic NETs into mucin hydrogels and cell culture derived airway mucus to assess their rheological and transport properties. We found that the addition of synthetic NETs significantly increases mucin hydrogel and native mucus viscoelasticity. As a result, mucociliary transport in vitro was significantly reduced with the addition of mucus containing synthetic NETs. Given the prevalence of bacterial infection in the CF lung, we also evaluated the growth of Pseudomonas aeruginosa in mucus with or without synthetic NETs. We found mucus containing synthetic NETs promoted microcolony growth and prolonged bacterial survival. Together, this work establishes a new biomaterial enabled approach to study innate immunity mediated airway dysfunction in CF.

Bacterial biofilms are often highly resistant to antimicrobials causing persistent infections which when not effectively managed can significantly worsen clinical outcomes. As such, alternatives to standard antibiotic therapies have been highly sought after to address difficult-to-treat biofilm-associated infections. We hypothesized a biomaterial-based approach using the innate functions of mucins to modulate bacterial surface attachment and virulence could provide a new therapeutic strategy against biofilms. Based on our testing in Pseudomonas aeruginosa biofilms, we found synthetic mucus biomaterials can inhibit biofilm formation and significantly reduce the thickness of mature biofilms. In addition, we evaluated if synthetic mucus biomaterials could work synergistically with DNase and/or -amylase for enhanced biofilm dispersal. Combination treatment with these antibiofilm agents and synthetic mucus biomaterials resulted in up to 3 log reductions in viability of mature P. aeruginosa biofilms. Overall, this work provides a new bio-inspired, combinatorial approach to address biofilms and antibiotic-resistant bacterial infections.

Lung cancer is the leading cause of global cancer death and prevention strategies are key to reducing mortality. Medical prevention may have a larger impact than treatment on mortality by targeting high-risk populations and reducing their lung cancer risk. Premalignant lesions (PMLs) that can be intercepted by prevention agents are difficult to study in humans but easily accessible in murine preclinical carcinogenesis studies. Precision-cut lung slices (PCLS) are underutilized as an ex vivo model for lung cancer studies due to limited culture time. Embedding PCLS within bioengineered hydrogels extends PCLS viability and functionality for up to six weeks. Here, we embedded PCLS generated from urethane-induced murine PMLs in cell-degradable and non-degradable hydrogels to study viability and activity of the tissues over six weeks. PMLs in hydrogel-embedded PCLS maintained viability, gene expression, and proliferation. Treatment of hydrogel-embedded PCLS containing urethane-induced PMLs with iloprost, a known lung cancer prevention agent, recapitulated in vivo gene expression and activity. These studies also showed that iloprost reduced proliferation and PML size in hydrogel-embedded PCLS, with some differences based on hydrogel formulation and suggested that hydrogel-embedded PCLS models may support long-term culture of in vivo generated PMLs to improve preclinical studies of lung cancer and prevention agents.

Tissue engineered constructs are increasingly used for both modeling organs and disease in vitro as well as for therapeutic intervention. In addition to collagen, these constructs commonly include native extracellular matrix proteins (ECM), such as fibronectin and laminin. Given the critical role of inflammatory pathways in disease and in response to implanted materials, it is important to understand the role these proteins play in regulating the inflammatory environment. Fibronectin and laminin influence neutrophil function and endothelial activation in 2D, but their regulation of the inflammatory environment in 3D engineered constructs is not clear. For this study, we used an inflammation-on-a-chip device that includes a model blood vessel surrounded by a collagen I hydrogel with fibronectin and/or laminin. We investigated the additive effects of both proteins and a range of concentrations for each protein to determine concentration dependence. Both fibronectin and laminin have concertation dependent effects on neutrophils and the endothelium. High concentrations (50 {micro}g/mL) of fibronectin reduced neutrophil migration, while 20 {micro}g/mL laminin reduced neutrophil extravasation and migration, potentially due to lower ICAM-1 expression by the endothelium. Interestingly, 50 {micro}g/mL of laminin significantly disrupted endothelial vessel formation and reduced ICAM-1 and VE-cadherin expression, likely due to significant changes in the collagen architecture. The inclusion of fibronectin and laminin, even at physiological levels, results in significant effects on neutrophil behavior, endothelial vessel formation, and collagen architecture. These proteins impact the inflammatory environment and thus need to be considered when modeling diseases and designing therapeutics, especially when neutrophils or an endothelium are involved. Translational Impact StatementThis work uses an inflammation-on-a-chip device to study how fibronectin and laminin impact neutrophil behavior and vascular inflammation as these proteins are commonly used in engineered constructs. We found that fibronectin impairs neutrophil migration, while laminin decreases neutrophil extravasation and migration and at higher concentrations also prevents endothelial vessel formation. Therefore, researchers should be aware that these proteins will alter the inflammatory environment when including them in engineered constructs.

Endometrial cancer is one of the most common gynecological cancers in the world, with an estimated 382,000 new cases and 90,000 deaths each year. There is no specific treatment, as the underlying causes of endometrial cancer neoplasia are poorly understood. This study focuses on the development and validation of a three-dimensional (3D) in vitro multilayer, multicellularhydrogel that facilitates drug screening analysis. We hypothesized that a specific combination of natural (collagen type I and IV, fibrinogen, fibronectin, Laminin) and synthetic (GELMA, PEGDA) polymers would maximize microvessel formation and cell invasion. The 3D model incorporates human microvascular endothelial cells (hMVEC) and endometrial cancer cells (HEC-1A) atop hydrogel formulations mimicking cell-specific extracellular matrix components. Using a D-optimal experimental design, 45 hydrogel combinations were generated. The predicted hydrogel formulation to maximize all cell responses enhanced higher microvessel formation and cancer invasion compared to the gold standard Matrigel. Subsequent validation emphasizes the importance of a disease-specific model and cell crosstalk in maximizing microvessel formation and cancer invasion. The optimized 3D model adeptly captures variances in cell responses among endometrial cancer cell lines from distinct stages. Finally, the platform is employed to compare cell viability, microvessel formation, and cancer invasion across Ishikawa, KLE, and HEC-1A cells after Paclitaxel exposure, delivered both as a free drug and loaded in poly(caprolactone) (PCL) nanoparticles. Overall, this study provides a valuable tool for exploring intricate interactions within the tumor microenvironment, offering a holistic understanding of cell responses and fostering the development of targeted therapeutic strategies for endometrial cancer.

Re-entrant arrhythmias--the leading cause of sudden cardiac death--are caused by diseased myocardial tissue and consequent delayed myocardial conduction. Access to the coronary veins that cross the "culprit" scar regions where re-entry originates can provide improved pacing to these delayed regions, offering a novel opportunity to prevent ventricular arrhythmias and circumvent the need for painful defibrillation, risky cardiac ablation, or toxic and often ineffective antiarrhythmic medications. However, there are no pacing electrodes which are small or focal enough to navigate these tributaries. To address this need, we have developed an injectable conductive hydrogel that can fill the epicardial coronary veins and their mid-myocardial tributaries. When connected to a standard pacing lead, these injected hydrogels can be converted into flexible electrodes that directly pace the previously inaccessible mid-myocardial tissue. In our two-component system, hydrogel precursor solutions can be injected through a dual lumen catheter in a minimally invasive deployment strategy to provide direct access to the diseased regions with precision and ease. Mixing of the two solutions upon injection into the vein activates redox-initiated crosslinking of the gel for rapid in situ cure without an external stimulus. An ex vivo porcine model was used to identify the requisite viscosity and cure rate for gel retention and homogeneity. Ionic species added to the hydrogel precursor solutions conferred conductivity above target myocardium values that was retained after implantation. Successful in vivo deployment demonstrated that the hydrogel electrode filled the anterior interventricular vein with extension into the septal (mid-myocardial) venous tributaries to depths far more distal and refined than any current technologies allow. In addition to successful capture and pacing of the heart, analysis of surface ECG tracings revealed a novel pacing observation highly specific for and suggestive of capture of extensive swaths of septal myocardial tissue. This is the first report of an injectable electrode used to successfully pace the mid-myocardium and mimic physiologic conduction. Furthermore, in vivo cardiac electroanatomical mapping studies in an ablation scar model showed uniform capture along the hydrogel in the vessels as well as increased capture area compared to point pacing. Collectively, these findings demonstrate that this injectable hydrogel electrode can be deployed to scarred regions of the heart to provide a reliable pacing modality that most closely resembles native conduction with the potential to eliminate delayed myocardial conduction and associated re-entry. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/467102v2_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1577526org.highwire.dtl.DTLVardef@cc510dorg.highwire.dtl.DTLVardef@1dd426eorg.highwire.dtl.DTLVardef@1346aab_HPS_FORMAT_FIGEXP M_FIG C_FIG One Sentence SummaryInjectable hydrogel electrodes achieve pacing that mimics physiologic conduction by capturing midmyocardial tissue

Microphysiological systems (MPSs) hold great potential for fundamental discovery and accelerating the drug discovery pipeline through simplifying complex tissues to their first principles and enabling real-time, high-resolution monitoring. Hydrophilic biomaterials, such as hydrogels, are important for MPS innovations due to their ability to emulate the native extracellular matrix and tunable mechanical properties. Furthermore, hydrogels can be tailored to improve tissue maturity as well as the efficacy of instrumentation. However, many biopolymers are non-conductive, presenting complications for modeling excitable tissue environments like the heart. In this work, we show that an 8% (w/v) Gelatin Methacryloyl (GelMA) + 3.5% (v/v) Choline Acrylate hydrogel, nicknamed Gel-Amin, can amplify extracellular voltage recordings from a culture of cardiomyocytes (CMs) from commercial microelectrode arrays. Our laser cut and assemble method for manufacturing 3D MPSs allowed direct comparisons of CM signal propagation in Gel-Amin compared to control GelMA cultures in a single system. This innovative material supported in vitro CM cultures with improved synchronicity and greater signal-to-noise ratios (SNRs), suggesting potential improvements over conventional biomaterial limitations. Here, we developed a cost-effective in vitro cardiac tissue model that allows real-time electrical activity monitoring.

Respiratory illnesses, like chronic obstructive pulmonary disease (COPD) and asthma, pose significant global health challenges due to their chronic nature and limited treatment options. Airway smooth muscle (ASM) plays a vital role in respiratory diseases, particularly in airway remodelling and obstruction. ASM, which encircles the bronchial tree and extends to the trachea, plays a vital yet not fully understood role in lung physiology. However, its dysfunction is strongly associated with asthma and COPD progression, leading to excessive contraction, increased inflammatory mediator release, and ASM hypertrophy. However, identifying its precise function is challenging due to limitations in existing research models for assessing ASM contraction. In vivo models offer a comprehensive physiological perspective but possess ethical concerns and they do not allow for the direct measurement of ASM contraction. Meanwhile, ex vivo and in vitro models provide a more direct assessment; however, they lack crucial physiological factors. Understanding how ASM cells interact with their surroundings is essential for gaining deeper insights into respiratory disorders. To address this gap, we aimed to mimic the human airway smooth muscle-on-a-chip model, incorporating ASM cells in a 3D microenvironment. This microfluidic platform provides a physiologically relevant environment, allowing for studying complex mechanisms that drive airway remodelling and dysfunction in respiratory diseases. The ASM-on-a-chip is designed for long-term 3D cell culture of ASM cells that reorient itself to form a smooth muscle fibre. The design provides side channels for manipulating the constituent of the hydrogel to study the effect of compounds on AMS remodelling.

Cervical cancer, the second leading cause of cancer-related death for women worldwide, remains a preventable yet persistent disease that disproportionately affects women in low and middle-income countries (LMICs). While existing therapies for treating cervical dysplasia are effective, they are often inaccessible in LMICs. Ethanol ablation is an alternative low-cost, accessible therapy that we previously enhanced into an ethyl cellulose (EC)-ethanol gel formulation to improve efficacy. When seeking to evaluate EC-ethanol for cervical dysplasia, we found a paucity of relevant animal models. Thus, in this study, we developed a 3D in vitro model of cervical dysplasia featuring a central lesion of cervical cancer cells surrounded by fibroblasts and keratinocytes to enable the evaluation of EC-ethanol and other novel therapeutics. Our GelMA-based 3D model successfully captured the architectural complexity of cervical dysplasia, showcasing cell response and high viability. The GelMA hydrogel formulation (8.7% w/v) exhibited viscoelastic properties akin to human cervical tissue. Using micro-CT imaging, we assessed EC-ethanol injection deposition in the hydrogel, revealing retention of virtually the entire injected volume near the injection site. Finally, we evaluated the EC-ethanol injections efficacy in eliminating cervical cancer cells. The EC-ethanol injection led to a significant decrease in cancer cell viability while preserving healthy cells in the 3D in vitro model. Taken together, our in vitro model mirrored the architecture of cervical dysplasia and demonstrated the potential of EC-ethanol for localized treatment of cervical dysplasia.

Neutrophils recruited to the airways are important for innate lung defense and can release neutrophil extracellular traps (NETs) to capture and eliminate microbes. While NETs are not abundant in healthy airways, uncontrolled NETosis is a known pathological feature and contributor to both chronic and acute respiratory diseases. Prior studies have shown that mucin glycoproteins secreted in the oral cavity and cervicovaginal tract can modulate NETosis, but it remains unknown whether mucins secreted in the respiratory tract influence NET formation. In these studies, we discovered that human airway mucus strongly inhibits NETosis in primary human neutrophils in a sialic acid dependent manner. In comparison, mucus produced by human airway epithelial cells genetically engineered to lack either MUC5B or MUC5AC secreted airway mucins showed a reduced ability to suppress NETosis. To assess how the lung microenvironment in obstructive lung diseases may influence mucus-dependent NET formation, we engineered a synthetic, mucin-laden hydrogel model with physical properties resembling that of mucus in a healthy lung and a disease-affected lung. When neutrophils were cultured on these gel substrates, we found that increasing gel stiffness led to a significantly greater extent of NETosis. Together these data demonstrate a new functional role of airway mucus in modulating neutrophil homeostasis in the respiratory tract and provide evidence that mucus dysfunction in disease can impair its ability to regulate NETosis.

Radiofrequency ablation is a mainstay of cardiac rhythm management despite high recurrence rates. Current radiofrequency ablation catheters are limited by poor contact with trabeculated cardiac tissue that promotes uneven heating with hot spots that cause collateral damage and regions of incomplete ablation that promote recurrence. Herein, we report on a conductive hydrogel coating of radiofrequency ablation catheters to improve tissue contact while promoting efficient energy transfer. A method was developed to graft a polyether urethane diacrylamide hydrogel to the distal tip of the catheter that maintained stable adhesion following drying, sterilization, and rehydration. The coating also remained intact after passage through an introducer sheath and 50 cycles of radiofrequency ablation at clinical power. The hydrogel-coated catheter demonstrated enhanced tissue contact that was dependent on hydrogel modulus. Hydrogel-mediated ablation prevented steam pop incidence and generated homogeneous lesions in an ex vivo ablation model; however, increased hydrogel conductivity is needed to achieve comparable lesion dimensions as the bare metal catheter and prevent coating damage at higher power. Collectively, these results establish a tunable hydrogel coating method that addresses limitations of conventional radiofrequency ablation and offers a promising approach to enhance the safety and efficacy of cardiac ablation therapies.

Lung cancer is the leading global cause of cancer-related deaths. Although smoking cessation is the best preventive action, nearly 50% of all lung cancer diagnoses occur in people who have already quit smoking. Research into treatment options for these high-risk patients has been constrained to rodent models of chemical carcinogenesis, which are time-consuming, expensive, and require large numbers of animals. Here we show that embedding precision-cut lung slices within an engineered hydrogel and exposing this tissue to a carcinogen from cigarette smoke creates an in vitro model of lung cancer premalignancy. Hydrogel formulations were selected to promote early lung cancer cellular phenotypes and extend PCLS viability up to six weeks. In this study, hydrogel-embedded lung slices were exposed to the cigarette smoke derived carcinogen vinyl carbamate, which induces adenocarcinoma in mice. At six weeks, analysis of proliferation, gene expression, histology, tissue stiffness, and cellular content revealed that vinyl carbamate induced the formation of premalignant lesions with a mixed adenoma/squamous phenotype. Two putative chemoprevention agents were able to freely diffuse through the hydrogel and induce tissue-level changes. The design parameters selected using murine tissue were validated with hydrogel-embedded human PCLS and results showed increased proliferation and premalignant lesion gene expression patterns. This tissue-engineered model of human lung cancer premalignancy is the starting point for more sophisticated ex vivo models and a foundation for the study of carcinogenesis and chemoprevention strategies.

Modulation of pH is crucial to maintaining the chemical homeostasis of biological environments. The irregular metabolic pathways exhibited by cancer cells result in the production of acidic byproducts that are excreted and accumulate in the extracellular tumor microenvironment, reducing its pH. As a consequence of the lower pH in tumors, cancer cells increase the expression of metastatic phenotypes and chemotherapeutic resistance. A significant limitation in current cancer therapies is the inability to locally deliver the chemotherapy, leading to significant damage to healthy cells in systemic administration. To overcome these challenges, we present an injectable chitosan-polyethylene glycol hydrogel that is dual-loaded with doxorubicin and sodium bicarbonate providing alkaline buffering of extracellular acidity and simultaneous chemotherapeutic delivery to increase chemotherapeutic efficacy. We conducted in vitro studies of weak base chemotherapeutic and alkaline buffer release from the hydrogel. The release of doxorubicin from hydrogels increased in a low pH environment and was dependent on the encapsulated sodium bicarbonate concentration. We investigated the influence of pH on doxorubicin efficacy and viability of MCF-7 and MDA-MB-231 breast cancer cell lines. The results show a 2 to 3 fold increase in IC50 values from neutral pH to low pH, showing decreased cancer cell viability at neutral pH as compared to acidic pH. The IC50 results were shown to correlate with a decrease in intracellular uptake of doxorubicin at low pH. The proposed hydrogels were confirmed to be non-toxic to healthy MCF-10A mammary epithelial cells. Rheological studies were performed to verify that the dual loaded hydrogels were injectable. The mechanical and release properties of the hydrogels were maintained after extended storage. The chemotherapeutic activity of doxorubicin was evaluated in the presence of the proposed pH regulating hydrogels. The findings suggest a promising non-toxic, biodegradable hydrogel buffer delivery system that can achieve two simultaneous important goals of local acidosis neutralization and chemotherapeutic release.

Idiopathic pulmonary fibrosis is a devastating chronic lung disease characterized by progressive scarring of the lung, which leads to impaired gas exchange and ultimately death. While research has provided us with extensive understanding on end-stage disease, the factors that lead to forward-feedback loops of fibrotic progression are still not fully known. Cell intrinsic activation, pathological extracellular matrix (ECM) composition, and increased tissue stiffness are all hallmarks of advanced fibrosis, but the relative contribution of these factors to disease has been difficult to disentangle using classic in vivo models. In this study we created biomaterials-based 3D lung models that incorporate geometrically relevant co-culture of lung epithelial cells and fibroblasts with tunable stiffness, ECM-containing hybrid-hydrogels. Using this model system, we demonstrated that environmental stiffness has the strongest effect on overall fibroblast activation. RNAseq analysis revealed unique gene-level changes in both fibroblasts and epithelial cells due to both composition and stiffness, highlighting the importance of incorporating both factors into any in vivo disease models. Overall, these results reinforce the value of biomaterials-based models in understanding disease pathogenesis, and their potential for screening of treatment responses.

The reinforcing and addictive properties of nicotine result from concentration- and time-dependent activation, desensitization, and upregulation of nicotinic acetylcholine receptors. However, time-resolved [nicotine] measurement in people who consume nicotine is challenging, as current approaches are expensive, invasive, tedious, and discontinuous. To address the challenge of continuous nicotine monitoring in human biofluids, we report the encapsulation of a purified, previously developed fluorescent biosensor protein, iNicSnFR12, into acrylamide hydrogels and polyethylene glycol diacrylate (PEGDA) hydrogels. We optimized the hydrogels for optical clarity and straightforward slicing. With fluorescence photometry of the hydrogels in a microscope and an integrated miniscope, [nicotine] is detected within a few min at the smoking- and vaping-relevant level of 10 - 100 nM (1.62 - 16.2 ng/ml), even in a 250 {micro}m thick hydrogel at the end of 400 {micro}m dia multimode fiber optic. Concentration-response relations are consistent with previous measurements on isolated iNicSnFR12. Leaching of iN-icSnFR12 from the hydrogel and inactivation of iNicSnFR12 are minimal for several days, and nicotine can be detected for at least 10 months after casting. This work provides the molecular, photophysical, and mechanical bases for personal, wearable continuous [nicotine] monitoring, with straightforward extensions to existing, homologous "iDrugSnFR" proteins for other abused and prescribed drugs.

Nearly 80% of cystic fibrosis patients are affected by persistent lung infections, with Pseudomonas aeruginosa being one of the major culprits. Treatment of P. aeruginosa is further complicated by its ability to form biofilms. Anionic compounds within the biofilm and thick cystic fibrosis mucus interact with cationic antimicrobials, hindering treatment efficacy. In this study, we investigated the treatment of lung infections by delivering antimicrobials via polyelectrolyte surfactants that are composed of an anionic poly(alkylacrylic acid) backbone with grafted polyetheramine pendent chains. When combined with cationic antimicrobials, they self-assemble into nanoparticles via electrostatic interactions. We assessed the role of backbone chemistry and graft density on nanoparticle physical properties and evaluated the antimicrobial activity of these formulations against planktonic and biofilm cultures of P. aeruginosa strains derived from clinical isolates. All synthesized polyelectrolyte surfactants demonstrated high levels of antimicrobial encapsulation, with the extent of drug bound corresponding to the calculated hydrophilic-lipophilic balance values. We observed significantly increased antimicrobial activity against planktonic cultures using nanoformulations containing one of the polyelectrolyte surfactants, PMAA-g-10%J. In contrast, all tested nanoformulations retained, but did not increase, activity against biofilms. By monitoring membrane potentials and nanoparticle uptake, it was found that the nanoparticles directly associate with the bacterial cell membranes, which may enhance drug delivery and underlie the improved activity against the planktonic bacteria. In conclusion, we provide a proof of concept for the design of polyelectrolyte surfactants for the nanoencapsulation and delivery of cationic drug cargoes against P. aeruginosa infections.

Opioid use disorder (OUD) has become a growing concern in the U.S. and has been a dominant presence among pregnant women, resulting in an unprecedented amount of prescription medications, particularly naltrexone (NTX), prescribed for pregnant women. Because of unknown potential harm that NTX can impose on the fetus and its premature brain, the needs for safety and regulation of NTX are still undetermined. To address this issue, a microfluidic device is fabricated to mimic structural phenotypes and physiological characteristic of an in vivo placental barrier to evaluate near-transport simulations of NTX and its primary metabolite, 6{beta}-naltrexol, across the placental barrier. Following transport analysis, cell layers are evaluated for possible gene-expressions released by an in vivo human placenta during NTX and 6{beta}-naltrexol placental exposure. When a 100 ng/mL dose of NTX and 6{beta}-naltrexol (1:1) is administered to the maternal channel, the mean fetal concentration for co-culture models exhibited ~2.5 % of NTX and ~2.2% of 6{beta}-naltrexol of the initial maternal concentration. To prototype and simulate fetal-brain exposure, perfusate from a fetal channel is directed to cultured N27 cells that are then evaluated for gene-expression.

The vagina is a fibromuscular tube-shaped organ spanning from the hymenal ring to the cervix that plays critical roles in menstruation, pregnancy, and female sexual health. Vaginal tissue constituents, including cells and extracellular matrix components, contribute to tissue structure, function, and prevention of injury. However, much microstructural function remains unknown, including how the fiber-cell and cell-cell interactions influence macromechanical properties. A deeper understanding of these interactions will provide critical information needed to reduce and prevent vaginal injuries. Our objectives for this work herein are to first engineer a suite of biomaterials for vaginal tissue engineering and second to characterize the performance of these biomaterials in the vaginal microenvironment. We successfully created fiber-reinforced hydrogels of gelatin-elastin electrospun fibers infiltrated with gelatin methacryloyl hydrogels. These composites recapitulate vaginal material properties, including stiffness, and are compatible with the vaginal microenvironment: biocompatible with primary vaginal epithelial cells and in acidic conditions. This work significantly advances progress in vaginal tissue engineering by developing novel materials and developing a state-of-the-art tissue engineered vagina.

Mechanical properties of the extracellular matrix (ECM) modulate cell-substrate interactions and influence cellular behaviors such as contractility, migrations, and proliferation. Although the effects of substrate stiffness on mechanobiology have been well studied, the role of ECM viscoelasticity in fibrotic progression remains less understood. To examine how viscoelasticity affects the biophysical properties and regulates the signaling of human mammary fibroblasts, we engineered elastic (E) and viscoelastic (VE) polyacrylamide hydrogels with comparable storage moduli ([~]14.52 {+/-} 1.03 kPa) but distinctly different loss moduli. Fibroblasts cultured on E hydrogels spread extensively (2428.93 {+/-} 864.71 m{superscript 2}), developed prominent stress fibers with higher zyxin intensity, and generated higher traction stresses (2931.57 {+/-} 1732.61 Pa). In contrast, fibroblasts on VE substrates formed smaller focal adhesion areas (54.2% reduction), exhibited lower critical adhesion strengths (51.8%), and generated 21% lower traction stresses (p < 0.001), indicating weaker adhesions. These substrates also promoted migrations and showed enhanced proliferation accompanied by reduced YAP activity, suggesting a mechanotransduction shift that may involve alternative signaling pathways. In contrast, E substrates showed YAP nuclear translocation, consistent with greater cytoskeletal tension and contractility. These findings highlight the importance of energy dissipation mechanisms in regulating fibroblast function on substrates mimicking the fibrotic milieu. Our results demonstrate that tuning the ECM viscoelasticity is a useful strategy to regulate cell behaviors in tissue engineered scaffolds, and develop better disease modeling for regenerative medicine.

Early skin inflammation requires coordinated immune regulation, with neutrophils acting as first-line responders. While the blood vasculature and its role in neutrophil recruitment during infection has been extensively studied, the lymphatic system remains comparatively understudied despite its known role in immune cell trafficking. Growing evidence suggests lymphatic vessels actively participate in regulating inflammatory responses, yet whether they coordinate neutrophil behavior during skin infection remains unclear. Staphylococcus aureus is particularly problematic in this context, employing multiple immune evasion strategies and representing a major driver of antibiotic-resistant skin and soft tissue infections worldwide. To address this gap, we developed a human-based 3D microphysiological system incorporating luminal lymphatic endothelial vessels, a collagen matrix and bacteria to model an infected microenvironment. We evaluated neutrophil migration, phagocytosis and NETosis in response to Escherichia coli and S. aureus. Lymphatic endothelium amplified neutrophil migration in a bacterial-dependent manner, with E. coli promoting directional migration toward the vessel while S. aureus suppressed migration and directionality despite increased phagocytic uptake. S. aureus also induced myeloperoxidase-positive NETs with nuclear morphology consistent with vital NETosis, rescued by DNase treatment. To our knowledge, this is the first demonstration that lymphatic endothelium directly drives neutrophil behavior during skin infection.