ACS Biomaterials Science & Engineering

The spinal cord has poor ability to regenerate after injury, which may be due to cell loss, cyst formation, inflammation, and scarring. A promising approach to treat spinal cord injury (SCI) is the use of biomaterials. We have developed a novel hydrogel scaffold fabricated from oligo(poly(ethylene glycol) fumarate) (OPF) as a 0.08 mm thick sheet containing polymer ridges and a cell-attractive surface chemistry on the other side. When the cells are cultured on OPF with the chemical patterning, the cells attach, align, and deposit ECM along the direction of the pattern. Animals implanted with the rolled scaffold sheets had greater hindlimb recovery compared to the multichannel scaffold control, likely due to the greater number of axons growing across. Inflammation, scarring, and ECM deposits were equal across conditions. Overall, the results suggest that the scaffold sheets promote axon outgrowth that can be guided across the scaffold, thereby promoting hindlimb recovery.

Stretch is an important biomechanical stimulus facilitating tissue development in the respiratory system by programming the epithelium, endothelium, and extracellular matrix (ECM). Lung tissue undergoes stretch induced lung differentiation under normal prenatal and postnatal development. Furthermore, supraphysiological and aberrant stretch responses are known mechanisms of acute lung injury and ECM disruption. Current in vitro human tissue cyclic mechanical stretch (CMS) models suffer from significant, well-recognized disadvantages and are poorly validated in vivo for longer-term study. In vitro precision-cut lung slice (PCLS) models are commonly used to study the complex structural arrangement and cellular interactions of human tissue, as well as various lung diseases, including BPD.3 PCLS maintain lung tissue architecture and the variety of cell types present in the lung, allowing for a more realistic imitation of the lung microenvironment.3 Existing agarose-inflated PCLS models are hindered by retention of agarose media in the tissue, affecting material properties and complicating stretch studies. Our novel PCLS approach utilizes several technical innovations including a removable hydrogel for inflation and uses supportive poly(ethylene glycol) (PEG) hydrogels enable improved viability and phenotype retention during cyclic mechanical stretch (CMS). This platform will induce PCLS CMS for biochemical assays (e.g. transcriptomics, proteomics) after exposure.

Primary mixed glial cultures are key tools to isolate and study astrocytes, microglia and oligodendrocytes. Cell-substrate adhesion is critical for neural cell survival and differentiation. Cationic polymers like poly-D-lysine (PDL) are widely used to promote cell adhesion to cell culture substrates, however, PDL is not stable long-term, with cultured cells often detaching (peeling) after 2-3 weeks. Dendritic polyglycerol amine (dPGA) is a synthetic polycationic non-protein polymer biomimetic of poly-lysine that is highly resistant to degradation by cellular proteases. Substrates coated with dPGA promote cell adhesion and improve survival in long-term neuronal cultures. Here we assessed dPGA as a substrate coating to provide long-term support for mixed glial cultures. Oligodendrocyte precursor cells (OPCs) were isolated weekly by differential adhesion from cultures grown in T75 flasks with PDL or dPGA-coated substrates. Following two "shake-off" isolations, the cell layer in most PDL-coated flasks fully detached, rendering these flasks unusable for further culture. In contrast, dPGA-coated flasks consistently yielded cells for six or more sequential isolations over seven weeks in culture. dPGA-coated flasks produced more cells, a greater percentage of O4+ cells, and maintained similar proportions of OPCs and MBP-positive cells as when isolated from a PDL-coated substrate. dPGA is cyto-compatible, functionally superior, easy to use, low cost and a stable alternative to conventional cell substrate coatings. The enhanced long-term stability of mixed glial cultures grown on a dPGA substrate has the capacity to increase cellular yield, reduce animal use, and facilitate studies of oligodendrocyte cell biology.

The diverse electrical, chemical and structural properties of the functional derivatives of carbon nanotubes (CNTs) have shown biomedical possibilities for neuroprosthesis or neural interfaces. However, the studies have been generally confined to metallic CNTs that affect cell viability unless chemically functionalized for biocompatibility. Here, we explored the effects of semiconducting single-walled carbon nanotubes (ssw-CNT), on the active electrical properties of dissociated hippocampal neurons in-vitro using multielectrode array, calcium imaging and whole-cell patch clamp recordings. The findings show that ssw-CNT treatment regulates neural network excitability from burst to tonic firing by changing the calcium dynamics. However, at a single neuronal level, ssw-CNT increases neuronal excitability.

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.

The excised animal cornea is the gold standard for testing and evaluating drug permeability through a cornea. However, it has a concise shelf life and encounters ethical concerns. Also, ex-vivo models, which are incomplete replicas of the human cornea, may provide faulty results in the pre-clinical studies. To circumvent these problems, we have proposed an in-vitro biomimetic lipid-polymer composite membrane (BLCM) model as an artificial cornea to study drug permeability. We designed and fabricated a free-standing, electro-spun polystyrene (PS) nanofibrous membrane and impregnated its pores with phosphatidylcholine (PC). SEM, FTIR, and goniometer characterized the BLCM. Permeation data of the drug ganciclovir through the BLCM model in a Franz-diffusion cell corroborates with the excised goat corneal system. Also, owing to the simple and scalable fabrication method, BLCM can be used as an alternative to animal models for initial drug permeability screening and studies and accelerate drug development.

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.

Epithelial cells are well-known to be modulated by extracellular mechanical factors including substrate stiffness. However, the effect of substrate stiffness on an epithelial cells principal function -creating an effective barrier to protect the underlying tissue - cannot be directly measured using existing experimental techniques. We developed a strategy involving ethylenediamine aminolysis and glutaraldehyde crosslinking to chemically graft polyacrylamide hydrogels with tunable stiffness to PET Transwell membranes. Grafting success was evaluated using spectroscopic methods, scrape tests, and extended incubation in culture. By assessing apical to basolateral transfer of fluorescent tracers, we demonstrated that our model is permeable to biologically relevant molecules and usable for direct measurement of barrier function by calculating paracellular permeability.\n\nWe found that BEAS-2B epithelial cells form a more effective barrier on stiff substrates, likely attributable to increased cell spreading. We also observed barrier impairment after treatment with transforming growth factor beta, indicating loss of cell-cell junctions, validating our models ability to detect biologically relevant stimuli. Thus, we have created an experimental model that allows explicit measurement of epithelial barrier function for cells grown on different substrate stiffnesses. This model will be a valuable tool to study mechanical regulation of epithelial and endothelial barrier function in health and disease.

Lateral displacement of microparticles suspended in a viscoelastic fluid flowing through a microfluidic channel occurs due to an imbalance in the first (N1) and second (N2) normal stress differences. Here, we studied the lateral displacement of fluorescent microparticles suspended in a polyethylene glycol (PEG) solution in a two-phase flow with aqueous sodium alginate, flowing through a unique microfluidic device that manufactures microparticles seeded alginate-based hollow microfibers. Parameters such as concentration of the aqueous sodium alginate and flow rate ratios were optimized to enhance microparticle seeding density and minimize their loss to the collection bath. 4 % w/v aqueous sodium alginate was observed to confine the suspended microparticles within the hollow region of microfibers as compared to 2 % w/v. Moreover, the higher flow rate ratio of the core fluid, 250 L min-1 resulted in about 192 % increase in the microparticle seeding density as compared to its lower flow rate of 100 L min-1. The shear thinning index (m) was measured to be 0.91 for 2 % w/v and 0.75 for 4 % w/v sodium alginate solutions. These results help gain insights into understanding the microparticle displacement within a viscoelastic polymer solution flowing through a microfluidic channel and motivate further studies to investigate the cellular response with the optimized parameters.

Mechanical ventilation is often required in patients with pulmonary disease to maintain adequate gas exchange. Despite improved knowledge regarding the risks of over ventilating the lung, ventilator induced lung injury (VILI) remains a major clinical problem due to inhomogeneities within the diseased lung itself as well as the need to increase pressure or volume of oxygen to the lung as a life-saving measure. VILI is characterized by increased physical forces exerted within the lung, which results in cell death, inflammation and long-term fibrotic remodeling. Animal models can be used to study VILI, but it is challenging to distinguish the contributions of individual cell types in such a setup. In vitro models, which allow for controlled stretching of specific lung cell types have emerged as a potential option, but these models and the membranes used in them are unable to recapitulate some key features of the lung such as the 3D nanofibrous structure of the alveolar basement membrane while also allowing for cells to be cultured at an air liquid interface (ALI) and undergo increased mechanical stretch that mimics VILI. Here we develop a lung on a chip device with a nanofibrous synthetic membrane to provide ALI conditions and controllable stretching, including injurious stretching mimicking VILI. The lung on a chip device consists of a thin (i.e. [~]20 {micro}m) stretchable poly(caprolactone) (PCL) nanofibrous membrane placed between two channels fabricated in polydimethylsiloxane (PDMS) using 3D printed molds. We demonstrate that this lung on a chip device can be used to induce mechanotrauma in lung epithelial cells due to cyclic pathophysiologic stretch ([~]25%) that mimics clinical VILI. Pathophysiologic stretch induces cell injury and subsequently cell death, which results in loss of the epithelial monolayer, a feature mimicking the early stages of VILI. We also validate the potential of our lung on a chip device to be used to explore cellular pathways known to be altered with mechanical stretch and show that pathophysiologic stretch of lung epithelial cells causes nuclear translocation of the mechanotransducers YAP/TAZ. In conclusion, we show that a breathable lung on a chip device with a nanofibrous membrane can be easily fabricated using 3D printing of the lung on a chip molds and that this model can be used to explore pathomechanisms in mechanically induced lung injury.

Polydimethylsiloxane (PDMS), often assumed to be biocompatible, is widely used in microfluidic devices and biomedical research. Here, we systematically assess the organismal effects of PDMS network components and their leachates using Caenorhabditis elegans as a whole-animal model. We demonstrate that uncrosslinked vinyl-terminated PDMS (v-PDMS) chains, which comprise the majority of a PDMS network and are known to diffuse into aqueous environments, exhibit acute, environmentally-dependent toxicity. Low-molecular-weight v-PDMS (6 kDa) caused mild lethality in nutrient-rich S-Medium but significantly higher mortality in minimal S-Buffer, showing that media composition strongly influences toxic effects. Adding cholesterol, calcium, or magnesium notably reduced v-PDMS-induced lethality, whereas trace metals increased it. Using a DAF-16::GFP reporter strain, we show that cholesterol influences organismal stress responses to v-PDMS exposures. Progeny from starved parents showed full resistance to v-PDMS, suggesting transgenerational stress memory plays a role in reducing PDMS toxicity. We also find that linear siloxanes cause modest but significant lethality, whereas cyclic siloxanes do not. The PDMS crosslinker TDSS, however, provides partial protection when present with v-PDMS, revealing diverse biological effects among PDMS network precursors. Overall, these results show that PDMS-derived components are not universally harmless and that susceptibility depends greatly on environmental conditions, sterol levels, and physiological history. Our findings emphasize the importance of carefully evaluating PDMS formulations for biomedical use and offer a framework for assessing polymer leachate toxicity in living organisms.

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.

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.

According to the National Cancer Institute, of the more than 10 million cancer survivors alive in the United States at least 270,000 were originally diagnosed under the age of 21. While the 5-year survival rates for most childhood cancers appear very promising, the long-term survival rates are still very dismal. There is significant long-term morbidity and mortality associated with treatment of childhood cancer, and the risk of these effects continues to increase years after completion of therapy. Among childhood cancer survivors the cumulative incidence of a chronic health condition is 73.4% 30 years after the original cancer diagnosis, with a cumulative incidence of 42.4% for severe, disabling, life-threatening, or death due to a chronic condition caused by the chemotherapy used to treat the initial malignancy. Brain tumors are the most prevalent solid tumor diagnosed in children, and account for 20 percent of all childhood cancer deaths. The efficacy of all chemotherapy agents can be limited by their toxicity, their instability, and their ability to be formulated into practical drug products for use in the clinical setting To address this gap, our group has developed a novel carbohydrate-based hydrogel, Amygel, that is capable of being loaded with drugs and injected directly into the site of disease. Local drug delivery using Amygel has potential to improve childhood cancer treatment outcomes and prevent the devastating effects of systemic chemotherapy exposure. Development of Amygel for clinical use has three focus areas including: increasing drug concentration at the target site; improving chemotherapy penetration through tumor tissue, and; establishing chemotherapy dosage forms for pediatric use. For this study, we formulated Amygel with dimethyl sulfoxide and integrated the chemotherapy doxorubicin (DOX). High-performance liquid chromatography (HPLC) was used to confirm the quality of DOX after hydrogel synthesis, rheology and syringability tests to characterize the mechanical properties, and performed an in vitro cytotoxicity test against the pediatric medulloblastoma cell line DAOY. On HPLC, we found that after integrating DOX into the Amygel matrix the drug maintained a strong band on the chromatograph at the same point with the same intensity as the control free drug, indicating there were no changes in the structural properties of DOX. The mechanical tests showed that there was a proportionate increase in the storage modulus of the drug-loaded hydrogels as the concentration of amylopectin increased from 3 wt% to 20 wt%, but even at 20 wt% the hydrogel remained below the medical standard for injectables that the burst force should not exceed 40 N and the sliding force below 20 N. Correlating with the rheology findings, as the concentration of amylopectin increased, and therefore the strength of the hydrogel, there was an increase in the magnitude of force required for gel injection. These mechanical studies additionally provide evidence that the mechanical stability of the gel is not dampened by the incorporation of DOX. Drug release and cytotoxicity studies demonstrated a sufficient release of DOX from the hydrogels, and that the DOX released was able to achieve significant (p<0.01) cell death.

Mucus is an impregnable barrier for drug delivery across the epithelia for treatment of mucosal-associated diseases. While current carriers are promising for mucus penetration, their surface chemistries do not possess chemical complexity to probe and identify optimal physicochemical properties desired for mucus penetration. As initial study, we use M13 phage display presenting random peptides to select peptides that can facilitate permeation through hyperconcentrated mucin. Here, a net-neutral charge, hydrophilic peptide was identified to facilitate transport of phage and fluorophore conjugates through mucin barrier compared to controls. This initial finding warrants further study to understand how composition and spatial distribution of physicochemical properties of peptides can be optimized to improve transport across the mucus barrier.

We report the design of a mucin hydrogel created using a thiol-based cross-linking strategy. By using a cross-linking reagent capable of forming disulfide linkages between mucins, the mucin-based hydrogels possess viscoelastic properties comparable to native mucus as measured by bulk rheology. We confirmed disulfide cross-links mediate gel formation in our system using chemical treatments to block and reduce cysteines where we found mucin hydrogel network formation was inhibited and disrupted, respectively. Particle tracking microrheology was used to investigate the kinetics and evolution of microstructure and viscoelasticity within the hydrogel as it formed. We found that the rate of gel formation could be tuned by varying the mucin to crosslinker ratio, producing network pore sizes in the range measured previously in human mucus. The results of this work provide a new, simple method for creating mucin hydrogels with physiologically relevant properties using readily available reagents.

Sarcoidosis is a multi-system disorder of granulomatous inflammation which most commonly affects the lungs. Its etiology and pathogenesis are not well defined in part due to the lack of reliable modeling. This article presents the development of a novel in vitro three-dimensional lung-on-chip organoid designed to mimic granuloma formation. A lung on chip fluidic macrodevice with three channels for cell culture insertion was developed and added to the previously developed a lung-on-membrane model. Granulomas were cultured from blood samples of patients with sarcoidosis and then inserted in the air-lung-interface (ALI) of the microchip to create a three-dimensional organoid sarcoidosis model (OSGM). The model was tested for cell viability with fibroblasts. We measured the cytokine profiles in medium of OSGM and compared with lung model without granuloma. Concentration of IL-1beta, Il-6, GM-CSF, and IFN-gamma were found significantly higher in OSGM group. The current model represents the first 3D OSGM created by adding a microfluidics system to a dual-chambered lung on membrane model and introducing developed sarcoid-granuloma to its ALI.

The human airways are complex structures with important interactions between cells, extracellular matrix (ECM) proteins and the biomechanical microenvironment. A robust, well-differentiated in vitro culture system that accurately models these interactions would provide a useful tool for studying normal and pathological airway biology. Here, we report the feasibility and analysis of a physiologically relevant air-liquid interface (ALI) 3D airway organ tissue equivalent (OTE) model with three novel features: native pulmonary fibroblasts, solubilized lung ECM, and hydrogel substrate with tunable stiffness and porosity. We demonstrate the versatility of the OTE model by evaluating the impact of these features on human bronchial epithelial (HBE) cell phenotype. Variations of this model were analyzed during 28 days of ALI culture by evaluating epithelial confluence, trans-epithelial resistance, and epithelial phenotype via multispectral immuno-histochemistry and next-generation sequencing. Cultures that included both solubilized lung ECM and native pulmonary fibroblasts within the hydrogel substrate formed well-differentiated ALI cultures that maintained a barrier function and expressed mature epithelial markers relating to goblet, club and ciliated cells. Modulation of hydrogel stiffness did not negatively impact HBE differentiation and could be a valuable variable to alter epithelial phenotype. This study highlights the feasibility and versatility of a 3D airway OTE model to model the multiple components of the human airway 3D microenvironment.

This study investigates the perception of tactile wetness, a complex sensation experienced by humans. Previous research has primarily focused on either thermal or mechanical cues separately, or has used textiles as stimuli whose parameters are difficult to control. Here, we employed polyacrylamide hydrogels with varying stiffness levels soaked in liquids of distinct thermal conductivities. By psychophysically evaluating participants perception of wetness, we showed that the wetness judgments for the samples exhibit a transitive relationship based on the mechanical and thermal cues from an intrinsically tunable organic material. We developed a prediction model of human wetness judgment with an accuracy of 90% and found that the best metrics for the most accurate model were those that were the most human-adjacent: change in temperature at the skin-sample interface (thermal) and compressive force from 2 mm indentation of the sample (mechanical). Given these parameters, we developed a perceptual space capable of recreating 7 distinct levels of wetness perception with the physical parameters used in this study. The results provide insights into the relative contributions of mechanical and thermal stimulus properties in wetness perception. Most notably, this work highlights that the physical characteristics of the skin-stimulus interface can provide ample information for creating a wetness perceptual space, as opposed to the chemical composition of the hydrogels.

TetraStat is a novel synthetic sealant created with a tetra-armed polyethylene glycol (PEG) hydrogel. It has no risk of infection from biological pathogens and has a hemostatic mechanism independent of the blood coagulation pathway and controllable gelation. We evaluated the hemostatic effect of TetraStat in ex vivo and in vivo experiments for future clinical application. In ex vivo experiments using a circulatory system filled with phosphate-buffered saline under high pressure, needle punctures were astricted with TetraStat and two commercially available hemostatic agents (SURGICEL and TachoSil). For in vivo experiments, rat vena cavae were punctured with 14, 18, and 20 gauge needles, and hemorrhage occurred for several seconds. A porous PEG sponge soaked with TetraStat was applied as a hemostatic system for the massive hemorrhage. In the ex vivo experiment, punctures were sealed completely after 1 min astriction with TetraStat gel; in contrast, SURGICEL and TachoSil failed to seal the hole. In vivo experiments demonstrated that TetraStat successfully caused hemostasis in the punctured vena cava within 1 min of application in a dose-dependent manner. For SURGICEL and TachoSil, successful hemostasis occurred after 5 min astriction but was less frequent after 1 min astriction. Ex vivo and in vivo experiments revealed TetraStats high hemostatic ability under high pressure and in rat vena cava injuries under massive hemorrhage. A porous PEG sponge soaked with TetraStat is a promising advancement in hemostatic systems.