Jeffrey W. Kysar

The effect of preparation conditions on the properties of glassy polymers has been a subject of intense research, and these glassy polymers also age with deleterious consequences on properties. Here, we surprisingly find a similar preparation dependence for polymer-grafted nanoparticle melts (PGNP), even when the chains are in the melt. Specifically, we show that processing PGNPs by spin-casting vs slowly casting them from solutions yields temporally stable states with vastly different properties, including surface morphologies, mechanical properties, and gas transport. We propose that these differences arise because the end-grafted polymer brushes, especially for high grafting density and short chain lengths, are in an extremely long-lived colloidal glassy state, even though the chains themselves are mobile. Simulations suggest that there are strong variations in the chain interpenetration states between adjacent nanoparticles driven by solvent evaporation rates. With slower evaporation, there is evidently increased chain interpenetration leading to mechanical property improvements, while collapsed brushes dramatically increase gas permeability properties under fast evaporated conditions. These results strongly argue that processing protocols are an unappreciated control variable in determining the temporally stable properties of this class of materials.

mechanical balance. Compared to conventional skin constructs (CSCs) that have open boundaries on all sides, ESCs exhibited higher sensitivity to TGF-β1, leading to increased ECM deposition, myofibroblast activation, YAP signaling upregulation, matrix stiffness and reduced hydraulic permeability. Inhibiting YAP signaling with verteporfin (VTP) reduced collagen deposition, prevented tissue stiffening, and attenuated several fibrosis markers, confirming the role of mechanotransduction in fibrosis progression using human cells. Transcriptome analysis revealed upregulation of fibrosis-associated genes, including COL10A1, COL11A1, and ACTA2, counterbalanced by elevation of anti-fibrotic regulators such as DKK2, which suggests the activation of negative feedback mechanisms. These findings establish the ESC platform as a robust human-relevant mechanomimetic model for studying fibrosis and evaluating anti-fibrotic therapies, addressing a critical need for translational drug discovery.

Skin fibrosis results from excessive extracellular matrix (ECM) deposition and tissue remodeling due to persistent inflammation and mechanotransduction dysregulation. Current in vivo animal models lack human relevance, while conventional 2D and 3D in vitro models misrepresent physiological mechanical forces. To address this gap, we developed a miniaturized Edgeless-Skin Chip (ESC) platform with gravity-driven perfusion, enabling enhanced biomechanical mimicry for fibrosis modeling. ESCs present bioengineered skin grown around a 3D-printed scaffold, mimicking the continuous geometry of human skin and in vivo mechanical balance. Compared to conventional skin constructs (CSCs) that have open boundaries on all sides, ESCs exhibited higher sensitivity to TGF-β1, leading to increased ECM deposition, myofibroblast activation, YAP signaling upregulation, matrix stiffness and reduced hydraulic permeability. Inhibiting YAP signaling with verteporfin (VTP) reduced collagen deposition, prevented tissue stiffening, and attenuated several fibrosis markers, confirming the role of mechanotransduction in fibrosis progression using human cells. Transcriptome analysis revealed upregulation of fibrosis-associated genes, including COL10A1, COL11A1, and ACTA2, counterbalanced by elevation of anti-fibrotic regulators such as DKK2, which suggests the activation of negative feedback mechanisms. These findings establish the ESC platform as a robust human-relevant mechanomimetic model for studying fibrosis and evaluating anti-fibrotic therapies, addressing a critical need for translational drug discovery.

. Patient-derived induced pluripotent stem cells (iPSCs) enable the personalized study of RDEB pathogenesis and potential therapies. However, current skin cell differentiation protocols via 2D culture perform suboptimally when applied to engineered 3D skin constructs (ESC). Here, we present an approach to source fibroblasts (iFBs) and keratinocytes (iKCs) from iPSC-derived skin organoids using an optimized differentiation protocol, and utilize them to engineer ESCs modeling wild-type and RDEB phenotypes. The resulting iPSC-derived skin cells display marker expression consistent with primary counterparts and produce ESCs exhibiting significant extracellular matrix remodeling, protein deposition, and epidermal differentiation. RDEB constructs recapitulated hallmark disease features, including absence of collagen VII and reduced iFB proliferation. This work establishes a robust and scalable strategy for generating physiologically-relevant, iPSC-derived skin constructs, offering a powerful model for studying RDEB mechanisms and advancing personalized regenerative medicine.

The clinical need for minimally invasive inner ear diagnostics and therapeutics has grown rapidly in recent years, particularly with the development of gene therapies for treating hearing and balance disorders. These therapies often require delivery of large injectate volumes that can cause hearing damage. In response to this challenge, dual-lumen microneedles, with two separate fluidic pathways controlled independently by micropumps, were designed for simultaneous aspiration and delivery to the inner ear across the round window membrane (RWM) and were fabricated using 2-photon polymerization (2PP). To assess the proof of concept of the dual-lumen microneedle device, simultaneous injection of 5 µL of adeno-associated virus (AAV) expressing green fluorescent protein (GFP) and aspiration of 5 µL of perilymph was performed in guinea pigs in vivo. Hearing thresholds were measured using auditory brainstem response (ABR) at time points before and 1 week after the procedure. Confocal imaging of the cochlea, the utricle, and the contralateral inner ear was employed to quantify and characterize the spatial distribution of hair cells with AAV transduction. Dual-lumen microneedle devices were found to be functional in the surgical setting. There was hearing loss limited to higher frequencies of 24 kHz and 28 kHz with ABR mean threshold shifts of 13 dB sound pressure level (SPL) (p = 0.03) and 23 dB SPL (p < 0.01), respectively. Furthermore, cochlear AAV transduction with a stereotypical basoapical gradient was observed in all animals (n = 5). Thus, dual-lumen microneedles can facilitate delivery of large volumes of therapeutic material into the inner ear, overcoming the limitations of single-lumen microneedles.

Intracochlear injection through the round window membrane (RWM) has been proposed to overcome imprecise drug delivery into the inner ear. Using a novel ultrasharp microneedle, we compared the perilymphatic dexamethasone (DEX) concentration achieved after intratympanic vs. intracochlear injection at two different time points and assessed its safety in guinea pigs. For this purpose, DEX sodium phosphate (10 mg/mL) was administered either in the right middle ear space via continuous intratympanic injection or in the right scala tympani of the cochlea with microneedle-mediated injection (1 µL at 1 µL/min) across the RWM. Both groups were evaluated at 1-hour or 3-hour time points. Perilymph from both cochleae was sampled for liquid chromatography-mass spectrometry, and bilateral cochleae were harvested for immunofluorescence. Eighteen guinea pigs were included. The mean DEX concentration was higher in the intracochlear delivery group than in the intratympanic delivery group at 1-hour time point (mean difference 67,863 ng/mL, 95% CI (8,352–127,374 ng/mL), p = 0.03). No difference was found at 3-hour time point. In every animal on both cochleae, no disruption in hair and supportive cells of the organ of Corti and utricle was observed. Significant middle ear inflammation was observed with the intratympanic delivery method compared to intracochlear. In conclusion, microneedle-mediated intracochlear injection achieves higher perilymphatic DEX concentration than the intratympanic route by a factor of 7 while preserving the cochlear architecture and inducing significantly less middle ear inflammation. In this new era of inner ear therapeutics, the potential for translational application is tangible and promising.

RATIONALE AND OBJECTIVES The potential of contrast-enhanced MRI for diagnosing endolymphatic hydrops is limited by long wait times following intravenous (IV) or intratympanic (IT) delivery, high contrast dosages, and inconsistent signal intensity enhancements. This study investigates microneedle-mediated intracochlear (IC) gadodiamide injection for consistent and efficient contrast delivery with minimal contrast dosage. MATERIALS AND METHODS A 100 µm diameter microneedle with 35 µm lumen was used to inject 1 µL of diluted gadodiamide (17.4 mM) into a guinea pig cochlea via the round window membrane. Serial MRI imaging was performed in a post-mortem animal using a 9.4 T small-animal MRI. Maximum intensity projections of MRI scans were generated to visualize diffusion of contrast within cochlea over time; mean intensities in defined regions of interest (ROIs) were calculated. Contrast diffusion time and intensity enhancements were determined. RESULTS Contrast was observed in the basal turn of scala tympani (ST) and scala vestibuli (SV) in the first MRI scan for all subjects which was acquired as early as 35 min after injection. Two-tailed paired t-tests confirmed that contrast reached the first two turns of ST and SV within 60 min, and the second half of third turns and apical turns of ST and SV within 90 min (p < 0.05). Intensity enhancements, defined as the percentage increase of the ROI mean intensity in the injection side compared to the contralateral side, exceeded 100% in the first turn and ranged from 12% to 32% in the third and apical turns of ST and SV at 90 min after injection. CONCLUSIONS IC gadodiamide enables controllable and efficient contrast delivery with significantly lower contrast dosage, making it a viable alternative for contrast-enhanced cochlear MRI.

HYPOTHESIS: Microneedle-mediated intracochlear injection of siRNA-Lipofectamine through the round window membrane (RWM) can be used to transfect cells within the cochlea. BACKGROUND: Our laboratory has developed 100-μm diameter hollow microneedles for intracochlear injection through the guinea pig RWM. In this study, we test the feasibility of microneedle-mediated injection of siRNA and Lipofectamine, a commonly used reagent with known cellular toxicity, through the RWM for cochlear transfection. METHODS: Fluorescently labeled scramble siRNA was diluted into Lipofectamine RNAiMax and OptiMEM. One microliter of 5 μM siRNA was injected through the RWM of Hartley guinea pigs at a rate of 1 μl/min (n = 22). In a control group, 1.0 μl of Lipofectamine, with no siRNA, was diluted into OptiMEM and injected in a similar fashion (n = 5). Hearing tests were performed before and either at 24 hours, 48 hours, or 5 days after injection. Afterward, animals were euthanized, and cochleae were harvested for imaging. Control cochleae were processed in parallel to untreated guinea pigs. RESULTS: Fluorescence, indicating successful transfection, was observed within the basal and middle turns of the cochlea with limited distribution in the apex at 24 and 48 hours. Signal was most intense in the organ of Corti, spiral ligament, and spiral ganglion. Little to no fluorescence was observed at 5 days post-injection. No significant changes in auditory brainstem response (ABR) were noted post-perforation at 5 days, suggesting that siRNA-Lipofectamine at low doses does not cause cochlear toxicity. CONCLUSIONS: Small volumes of siRNA and Lipofectamine can be effectively delivered to cochlear structures using microneedles, paving the way for atraumatic cochlear gene therapy.

Recent evidence shows that it is possible to identify the elements responsible for sensorineural hearing loss, such as pro-inflammatory cytokines and macrophages, by performing perilymph sampling. However, current studies have only focused on the diagnosis of such as otologic conditions. Hearing loss is a feature of certain neuroinflammatory disorders such as multiple sclerosis, and sensorineural hearing loss (SNHL) is widely detected in Alzheimer's disease. Although the environment of the inner ear is highly regulated, there are several communication pathways between the perilymph of the inner ear and cerebrospinal fluid (CSF). Thus, examination of the perilymph may help understand the mechanism behind the hearing loss observed in certain neuroinflammatory and neurodegenerative diseases. Herein, we review the constituents of CSF and perilymph, the anatomy of the inner ear and its connection with the brain. Then, we discuss the relevance of perilymph sampling in neurology. Currently, perilymph sampling is only performed during surgical procedures, but we hypothesize a simplified and low-invasive technique that could allow sampling in a clinical setting with the same ease as performing an intratympanic injection under direct visual check. The use of this modified technique could allow for perilymph sampling in people with hearing loss and neuroinflammatory/neurodegenerative disorders and clarify the relationship between these conditions; in fact, by measuring the concentration of neuroinflammatory and/or neurodegenerative biomarkers and those typically expressed in the inner ear in aging SNHL, it could be possible to understand if SNHL is caused by aging or neuroinflammation.

Front Cover: Currently available heart valve prostheses have no growth potential, requiring children with heart valve diseases to endure multiple valve replacement surgeries. In article number 2300011, David Kalfa, Jeffrey W. Kysar, Richard L. Li and co-workers develop the proof of concept of a polymeric valved conduit that can be expanded by transcatheter balloon dilation to accommodate patient growth.

OBJECTIVES: Oral or intratympanic corticosteroids are commonly used to treat sudden sensorineural hearing loss (SSHL), tinnitus, and Meniere disease. Direct intracochlear delivery has been proposed to overcome the variability in bioavailability and efficacy of systemic or middle ear delivery. In this study, we aim to characterize the physiologic consequences of microneedle-mediated direct intracochlear injection of dexamethasone through the round window membrane (RWM). METHODS: In Hartley guinea pigs (n = 5), a post-auricular incision followed by bullostomy was made to access the round window membrane. Using 100 μm diameter hollow microneedles, 1.0 μl of 10 mg/ml dexamethasone was injected through the RWM over 1 min. Compound action potential (CAP) and distortion product otoacoustic action emissions (DPOAE) were measured before perforation, at 1 h, and at 5 h following injection. CAP hearing thresholds were measured from 0.5 to 40 kHz, and DPOAE f2 frequencies ranged from 1.0 and 32 kHz. Repeated measures ANOVA followed by pairwise t-tests were used for statistical analysis. RESULTS: ANOVA identified significant CAP threshold shifts at four frequencies (4, 16, 36, and 40 kHz) and differences in DPOAE at 1 frequency (6 kHz). Paired t-tests revealed differences between the pre-perforation and 1 h time point. By 5 h post injection, both CAP hearing thresholds and DPOAE recover and are not significantly different from baseline thresholds. CONCLUSION: Direct intracochlear delivery of dexamethasone via microneedles results in temporary shifts in hearing thresholds that resolve by 5 hours, thus supporting microneedle technology for the treatment of inner ear disorders. LEVEL OF EVIDENCE: NA Laryngoscope, 134:388-392, 2024.

HYPOTHESIS: Microneedle-mediated intracochlear injection through the round window membrane (RWM) will facilitate intracochlear delivery, not affect hearing, and allow for full reconstitution of the RWM within 48 hours. BACKGROUND: We have developed polymeric microneedles that allow for in vivo perforation of the guinea pig RWM and aspiration of perilymph for diagnostic analysis, with full reconstitution of the RWM within 48 to 72 hours. In this study, we investigate the ability of microneedles to deliver precise volumes of therapeutics into the cochlea and assess the subsequent consequences on hearing. METHODS: Volumes of 1.0, 2.5, or 5.0 μL of artificial perilymph were injected into the cochlea at a rate of 1 μL/min. Compound action potential (CAP) and distortion product otoacoustic emission were performed to assess for hearing loss (HL), and confocal microscopy was used to evaluate the RWM for residual scarring or inflammation. To evaluate the distribution of agents within the cochlea after microneedle-mediated injection, 1.0 μL of FM 1-43 FX was injected into the cochlea, followed by whole mount cochlear dissection and confocal microscopy. RESULTS: Direct intracochlear injection of 1.0 μL of artificial perilymph in vivo , corresponding to about 20% of the scala tympani volume, was safe and did not result in HL. However, injection of 2.5 or 5.0 μL of artificial perilymph into the cochlea produced statistically significant high-frequency HL persisting 48 hours postperforation. Assessment of RWMs 48 hours after perforation revealed no inflammatory changes or residual scarring. FM 1-43 FX injection resulted in distribution of the agent predominantly in the basal and middle turns. CONCLUSION: Microneedle-mediated intracochlear delivery of small volumes relative to the volume of the scala tympani is feasible, safe, and does not cause HL in guinea pigs; however, injection of large volumes induces high-frequency HL. Injection of small volumes of a fluorescent agent across the RWM resulted in significant distribution within the basal turn, less distribution in the middle turn, and almost none in the apical turn. Microneedle-mediated intracochlear injection, along with our previously developed intracochlear aspiration, opens the pathway for precision inner ear medicine.

BACKGROUND We have developed 3D-printed microneedle technology for diagnostic aspiration of perilymph and intracochlear delivery of therapeutic agents. Single microneedle-mediated round window membrane (RWM) perforation does not cause hearing loss, heals within 48-72 h, and yields sufficient perilymph for proteomic analysis. In this study, we investigate the anatomic, physiologic, and proteomic consequences of repeated microneedle-mediated perforations of the same RWM at different timepoints. METHODS 100-μm-diameter hollow microneedles were fabricated using two-photon polymerization (2PP) lithography. The tympanic bullae of Hartley guinea pigs (n = 8) were opened with adequate exposure of the RWM. Distortion product otoacoustic emissions (DPOAE) and compound action potential (CAP) were recorded to assess hearing. The hollow microneedle was introduced into the bulla and the RWM was perforated; 1 μL of perilymph was aspirated from the cochlea over the course of 45 s. 72 h later, the above procedure was repeated with aspiration of an additional 1 μL of perilymph. 72 h after the second perforation, RWMs were harvested for confocal imaging. Perilymph proteomic analysis was completed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS Two perforations and aspirations were performed in 8 guinea pigs. In six, CAP, DPOAE, and proteomic analysis were obtained; in one, only CAP and DPOAE results were obtained; and in one, only proteomics results were obtained. Hearing tests demonstrated mild hearing loss at 1-4 kHz and 28 kHz, most consistent with conductive hearing loss. Confocal microscopy demonstrated complete healing of all perforations with full reconstitution of the RWM. Perilymph proteomic analysis identified 1855 proteins across 14 samples. The inner ear protein cochlin was observed in all samples, indicating successful aspiration of perilymph. Non-adjusted paired t-tests with p < 0.01 revealed significant changes in 13 of 1855 identified proteins (0.7%) between the first and second aspirations. CONCLUSIONS We demonstrate that repeated microneedle perforation of the RWM is feasible, allows for complete healing of the RWM, and minimally changes the proteomic expression profile. Thus, microneedle-mediated repeated aspirations in a single animal can be used to monitor the response to inner ear treatments over time.

Currently available heart valve prostheses have no growth potential, requiring children with heart valve diseases to endure multiple valve replacement surgeries with compounding risks. This study demonstrates the in vitro proof of concept of a biostable polymeric trileaflet valved conduit designed for surgical implantation and subsequent expansion via transcatheter balloon dilation to accommodate the growth of pediatric patients and delay or avoid repeated open-heart surgeries. The valved conduit is formed via dip molding using a polydimethylsiloxane-based polyurethane, a biocompatible material shown here to be capable of permanent stretching under mechanical loading. The valve leaflets are designed with an increased coaptation area to preserve valve competence at expanded diameters. Four 22 mm diameter valved conduits are tested in vitro for hydrodynamics, balloon dilated to new permanent diameters of 23.26 ± 0.38 mm, and then tested again. Upon further dilation, two valved conduits sustain leaflet tears, while the two surviving devices reach final diameters of 24.38 ± 0.19 mm. After each successful dilation, the valved conduits show increased effective orifice areas and decreased transvalvular pressure differentials while maintaining low regurgitation. These results demonstrate concept feasibility and motivate further development of a polymeric balloon-expandable device to replace valves in children and avoid reoperations.

Despite the advancements in skin bioengineering, 3D skin constructs are still produced as flat tissues with open edges, disregarding the fully enclosed geometry of human skin. Therefore, they do not effectively cover anatomically complex body sites, e.g., hands. Here, we challenge the prevailing paradigm by engineering the skin as a fully enclosed 3D tissue that can be shaped after a body part and seamlessly transplanted as a biological clothing. Our wearable edgeless skin constructs (WESCs) show enhanced dermal extracellular matrix (ECM) deposition and mechanical properties compared to conventional constructs. WESCs display region-specific cell/ECM alignment, as well as physiologic anisotropic mechanical properties. WESCs replace the skin in full-thickness wounds of challenging body sites (e.g., mouse hindlimbs) with minimal suturing and shorter surgery time. This study provides a compelling technology that may substantially improve wound care and suggests that the recapitulation of the tissue macroanatomy can lead to enhanced biological function.