Peptide-Functionalized Electrospun Meshes for the Physiological Cultivation of Pulmonary Alveolar Capillary Barrier Models in a 3D-Printed Micro-Bioreactor.

In vitro environments that realize biomimetic scaffolds, cellular composition, physiological shear, and strain are integral to developing tissue models of organ-specific functions. In this study, an in vitro pulmonary alveolar capillary barrier model is developed that closely mimics physiological functions by combining a synthetic biofunctionalized nanofibrous membrane system with a novel three-dimensional (3D)-printed bioreactor. The fiber meshes are fabricated from a mixture of polycaprolactone (PCL), 6-armed star-shaped isocyanate-terminated poly(ethylene glycol) (sPEG-NCO), and Arg-Gly-Asp (RGD) peptides by a one-step electrospinning process that offers full control over the fiber surface chemistry. The tunable meshes are mounted within the bioreactor where they support the co-cultivation of pulmonary epithelial (NCI-H441) and endothelial (HPMEC) cell monolayers at air-liquid interface under controlled stimulation by fluid shear stress and cyclic distention. This stimulation, which closely mimics blood circulation and breathing motion, is observed to impact alveolar endothelial cytoskeleton arrangement and improve epithelial tight junction formation as well as surfactant protein B production compared to static models. The results highlight the potential of PCL-sPEG-NCO:RGD nanofibrous scaffolds in combination with a 3D-printed bioreactor system as a platform to reconstruct and enhance in vitro models to bear a close resemblance to in vivo tissues.

Mimicking the Natural Basement Membrane for Advanced Tissue Engineering.

Advancements in the field of tissue engineering have led to the elucidation of physical and chemical characteristics of physiological basement membranes (BM) as specialized forms of the extracellular matrix. Efforts to recapitulate the intricate structure and biological composition of the BM have encountered various advancements due to its impact on cell fate, function, and regulation. More attention has been paid to synthesizing biocompatible and biofunctional fibrillar scaffolds that closely mimic the natural BM. Specific modifications in biomimetic BM have paved the way for the development of in vitro models like alveolar-capillary barrier, airway models, skin, blood-brain barrier, kidney barrier, and metastatic models, which can be used for personalized drug screening, understanding physiological and pathological pathways, and tissue implants. In this Review, we focus on the structure, composition, and functions of in vivo BM and the ongoing efforts to mimic it synthetically. Light has been shed on the advantages and limitations of various forms of biomimetic BM scaffolds including porous polymeric membranes, hydrogels, and electrospun membranes This Review further elaborates and justifies the significance of BM mimics in tissue engineering, in particular in the development of in vitro organ model systems.

Radiolabeled Nanocarriers as Theranostics-Advancement from Peptides to Nanocarriers.

Endogenous targeted radiotherapy is emerging as an integral modality to treat a variety of cancer entities. Nevertheless, despite the positive clinical outcome of the treatment using radiolabeled peptides, small molecules, antibodies, and nanobodies, a high degree of hepatotoxicity and nephrotoxicity still persist. This limits the amount of dose that can be injected. In an attempt to mitigate these side effects, the use of nanocarriers such as nanoparticles (NPs), dendrimers, micelles, liposomes, and nanogels (NGs) is currently being explored. Nanocarriers can prolong circulation time and tumor retention, maximize radiation dosage, and offer multifunctionality for different targeting strategies. In this review, the authors first provide a summary of radiation therapy and imaging and discuss the new radiotracers that are used preclinically and clinically. They then highlight and identify the advantages of radio-nanomedicine and its potential in overcoming the limitations of endogenous radiotherapy. Finally, the review points to the ongoing efforts to maximize the use of radio-nanomedicine for efficient clinical translation.

Tuning the Elasticity of Nanogels Improves Their Circulation Time by Evading Immune Cells.

Peptide receptor radionuclide therapy is used to treat solid tumors by locally delivering radiation. However, due to nephro- and hepato-toxicity, it is limited by its dosage. To amplify radiation damage to tumor cells, radiolabeled nanogels can be used. We show that by tuning the mechanical properties of nanogels significant enhancement in circulation half-life of the gel could be achieved. We demonstrate why and how small changes in the mechanical properties of the nanogels influence its cellular fate. Nanogels with a storage modulus of 37 kPa were minimally phagocytosed by monocytes and macrophages compared to nanogels with 93 kPa modulus. Using PET/CT a significant difference in the blood circulation time of the nanogels was shown. Computer simulations affirmed the results and predicted the mechanism of cellular uptake of the nanogels. Altogether, this work emphasizes the important role of elasticity even for particles that are inherently soft such as nano- or microgels.

Protease Responsive Nanogels for Transcytosis across the Blood-Brain Barrier and Intracellular Delivery of Radiopharmaceuticals to Brain Tumor Cells.

Despite profound advances in treatment approaches, gliomas remain associated with very poor prognoses. The residual cells after incomplete resection often migrate and proliferate giving a seed for highly resistant gliomas. The efficacy of chemotherapeutic drugs is often strongly limited by their poor selectivity and the blood brain barrier (BBB). Therefore, the development of therapeutic carrier systems for efficient transport across the BBB and selective delivery to tumor cells remains one of the most complex problems facing molecular medicine and nano-biotechnology. To address this challenge, a stimuli sensitive nanogel is synthesized using pre-polymer approach for the effective delivery of nano-irradiation. The nanogels are cross-linked via matrix metalloproteinase (MMP-2,9) substrate and armed with Auger electron emitting drug 5-[125 I]Iodo-4"-thio-2"-deoxyuridine ([125 I]ITdU) which after release can be incorporated into the DNA of tumor cells. Functionalization with diphtheria toxin receptor ligand allows nanogel transcytosis across the BBB at tumor site. Functionalized nanogels efficiently and increasingly explore transcytosis via BBB co-cultured with glioblastoma cells. The subsequent nanogel degradation correlates with up-regulated MMP2/9. Released [125 I]ITdU follows the thymidine salvage pathway ending in its incorporation into the DNA of tumor cells. With this concept, a highly efficient strategy for intracellular delivery of radiopharmaceuticals across the challenging BBB is presented.

Tolerability of facial electrostimulation in healthy adults and patients with facial synkinesis.

PURPOSE: To evaluate optimal stimulation parameters with regard to discomfort and tolerability for transcutaneous electrostimulation of facial muscles in healthy participants and patients with postparetic facial synkinesis. METHODS: Two prospective studies were performed. First, single pulse monophasic stimulation with rectangular pulses was compared to triangular pulses in 48 healthy controls. Second, 30 healthy controls were compared to 30 patients with postparetic facial synkinesis with rectangular pulse form. Motor twitch threshold, tolerability threshold, and discomfort were assessed using a numeric rating scale at both thresholds. RESULTS: Discomfort at motor threshold was significantly lower for rectangular than for triangular pulses. Average motor and tolerability thresholds were higher for patients than for healthy participants. Discomfort at motor threshold was significantly lower for healthy controls compared to patients. Major side effects were not seen. CONCLUSIONS: Surface electrostimulation for selective functional and tolerable facial muscle contractions in patients with postparetic facial synkinesis is feasible.

Cochlear MRI Signal Change Following Vestibular Schwannoma Resection Depends on Surgical Approach.

OBJECTIVE: Information on cochlear MRI signal change following vestibular schwannoma (VS) surgery by the retrolabyrinthine approach (RLA) is nonexisting, and information using the translabyrinthine approach (TLA) is scarce. We aimed to evaluate cochlear MRI fluid signal in patients with a unilateral VS, before and after surgery by the RLA or the TLA, that can have clinical importance for subsequent cochlear implantation feasibility. STUDY DESIGN: Retrospective cohort study. SETTING: University hospital. PATIENTS: One hundred one patients with a unilateral VS. INTERVENTION: VS resection by the TLA or the RLA. Pre- and postoperative T2-weighted MRI. MAIN OUTCOME MEASURE: Cochlear signal change using a semiquantitative system for grading cochlear asymmetry, with grades ranging from 1 (normal fluid signal both sides) to 4 (no fluid signal one side). RESULTS: Seventy-four patients were operated by the TLA and 27 by the RLA. The number of cochleas with grade 3 and 4 asymmetries postoperative was significantly higher than preoperative. The postoperative proportions of grade 1 (TLA 20%, RLA 56%) and grade 2-4 asymmetry (TLA 80%, RLA 44%) were significantly different between the two groups. In the TLA group, 46 patients (62%) demonstrated an increased asymmetry postoperatively, as compared with three patients (11%) in the RLA group. CONCLUSIONS: Postoperative decrease of cochlear MRI fluid signal is more likely to occur after translabyrinthine surgery (occurring in 62%), as compared with retrolabyrinthine surgery (occurring in 11%). The decrease of cochlear signal may be due to compromised vascularity or fibrosis.

Cellular responses to beating hydrogels to investigate mechanotransduction.

Cells feel the forces exerted on them by the surrounding extracellular matrix (ECM) environment and respond to them. While many cell fate processes are dictated by these forces, which are highly synchronized in space and time, abnormal force transduction is implicated in the progression of many diseases (muscular dystrophy, cancer). However, material platforms that enable transient, cyclic forces in vitro to recreate an in vivo-like scenario remain a challenge. Here, we report a hydrogel system that rapidly beats (actuates) with spatio-temporal control using a near infra-red light trigger. Small, user-defined mechanical forces (~nN) are exerted on cells growing on the hydrogel surface at frequencies up to 10 Hz, revealing insights into the effect of actuation on cell migration and the kinetics of reversible nuclear translocation of the mechanosensor protein myocardin related transcription factor A, depending on the actuation amplitude, duration and frequency.

Microgels Sopping Up Toxins-GM1a-Functionalized Microgels as Scavengers for Cholera Toxin.

Vibrio cholerae is a Gram-negative bacterium that causes secretory diarrhea and constitutes a major health threat in the industrialized world and even more in developing countries. Its main virulence factor is the cholera toxin, which is internalized by intestinal epithelial cells after binding to the glycosphingolipid receptor GM1a on their apical surface. A potential future solution to dampen complications of cholera infection is by scavenging the cholera toxin by presenting competitive binding motifs to diminish the in vivo toxicity of V. cholerae. Here, we generate GM1a-functionalized and biocompatible microgels with diameters of 20 µm using drop-based microfluidics. The microgels are designed to exhibit a mesoporous and widely meshed network structure, allowing diffusion of the toxin protein deep into the microgel scavengers. Flow cytometry demonstrates strong and multivalent binding at high capacity of these microgels to the binding domain of the cholera toxin. Cell culture-based assays reveal the ability of these microgels to scavenge and retain the cholera toxin in direct binding competition to colorectal cells. This ability is evidenced by suppressed cyclic adenosine monophosphate production as well as reduced vacuole formation in mucus-forming colorectal HT-29 cells. Therefore, glycan-functionalized microgels show great potential as a non-antibiotic treatment for toxin-mediated infectious disorders.

Extended Retrolabyrinthine Approach: Results of Hearing Preservation Surgery Using a New System for Continuous Near Real-time Neuromonitoring in Patients With Growing Vestibular Schwannomas.

OBJECTIVE: To report hearing preservation results after retrolabyrinthine vestibular schwannoma surgery, using a new system for continuous near real-time monitoring of cochlear nerve function. STUDY DESIGN: Retrospective chart review. SETTING: Tertiary referral center, University Hospital. PATIENTS: Thirty-one consecutive patients with growing vestibular schwannomas and opting for hearing preservation surgery. INTERVENTIONS: Tumor removal by a modified, extended retrolabyrinthine approach, using a new system for continuous near real-time monitoring of cochlear nerve function. MAIN OUTCOME MEASURES: Pure-tone average and speech discrimination (SD) 1-year postoperative. Preservation of word recognition score class. Preservation of serviceable hearing (SD>50%). RESULTS: Any hearing was preserved in 83 and 69% had preserved word recognition score class or better. Serviceable hearing was preserved in 77%. SD was unchanged in 48%, improved in 21%, and poorer in 31%. Of 18 patients with potential for improvement (SD 90% or worse preoperatively), 33% improved (SD increase 10% or more). CONCLUSION: The hearing preservation rate is favorable using the modified, extended retrolabyrinthine approach and a new system for continuous near real-time monitoring of cochlear nerve function for removal of growing vestibular schwannomas, as 77% preserved serviceable hearing 1 year after surgery. Hearing improved after surgery in 33%. Using the new neuromonitoring system, serviceable hearing preservation rate improved from 53 to 77% at our center.

Design-functionality relationships for adhesion/growth-regulatory galectins.

Glycan-lectin recognition is assumed to elicit its broad range of (patho)physiological functions via a combination of specific contact formation with generation of complexes of distinct signal-triggering topology on biomembranes. Faced with the challenge to understand why evolution has led to three particular modes of modular architecture for adhesion/growth-regulatory galectins in vertebrates, here we introduce protein engineering to enable design switches. The impact of changes is measured in assays on cell growth and on bridging fully synthetic nanovesicles (glycodendrimersomes) with a chemically programmable surface. Using the example of homodimeric galectin-1 and monomeric galectin-3, the mutual design conversion caused qualitative differences, i.e., from bridging effector to antagonist/from antagonist to growth inhibitor and vice versa. In addition to attaining proof-of-principle evidence for the hypothesis that chimera-type galectin-3 design makes functional antagonism possible, we underscore the value of versatile surface programming with a derivative of the pan-galectin ligand lactose. Aggregation assays with N,N'-diacetyllactosamine establishing a parasite-like surface signature revealed marked selectivity among the family of galectins and bridging potency of homodimers. These findings provide fundamental insights into design-functionality relationships of galectins. Moreover, our strategy generates the tools to identify biofunctional lattice formation on biomembranes and galectin-reagents with therapeutic potential.

Facial nerve schwannomas presenting with vestibular dysfunction: a case series.

Facial nerve schwannomas (FS) can symptomatically mimic vestibular schwannomas (VS). In addition, FS can be difficult to distinguish from VS on magnetic resonance imaging (MRI). Although disequilibrium is not uncommon in patients with FS, no previous studies have investigated the vestibular function in such patients. Three cases of FS presented vestibular dysfunction as measured with caloric test, video head impulse test (VHIT), and vestibular evoked myogenic potentials (VEMPs). All patients in this study had a considerable affection of the vestibular function as assessed by the vestibular test panel. Audiovestibular evaluation of FS provides important information that may influence treatment strategy. As VS, FS should be evaluated with vestibular tests prior to intervention.

[Workup and treatment of vestibular schwannomas].

Vestibular schwannomas are benign tumours originating from the eighth cranial nerve. The incidence in Denmark is rising and is around 200 per million per year. Pure tone audiometry, discrimination score and tinnitus anamnesis determine, whether a diagnostic MRI is merited. Around one fourth of newly diagnosed patients receive surgery due to the size of the tumour, whereas the remaining three fourths go into a "wait-and-scan" regime. 30-40% of the patients in this regime experience growth and are offered either surgery or radiotherapy.

Human Co- and Triple-Culture Model of the Alveolar-Capillary Barrier on a Basement Membrane Mimic.

The development of an in vitro model resembling the alveolar-capillary barrier might be a highly beneficial tool to study lung physiology as well as the immune response of the lung to infection or after exposure to nanoparticles. This study is based on an in vitro alveolar barrier developed on a basement membrane mimic, composed of ultrathin nanofiber meshes generated via electrospinning using bioresorbable poly(ɛ-caprolactone). As cellular components, NCI H441, resembling the alveolar epithelial cells, and ISO-HAS-1, an endothelial cell line, were used to perform bipolar coculture experiments for a total cultivation period of 14 days. In addition to immunohistochemical and immunofluorescent studies, transepithelial electrical resistance (TER) and transport capabilities of the in vitro model system were investigated. Alveolar barrier function could be clearly determined for the postulated bipolar coculture system on the basement membrane mimic, since TER increased during the course of bipolar cultivation. Furthermore, to gain first insights into possible lung inflammatory reactions in vitro, this coculture model was further expanded by a human leukemia monocyte cell line (THP-1). This triple-culture system was able to maintain adequately the barrier properties of the bipolar coculture, thus making this in vitro model consisting of epithelial, endothelial, and immune cells on a basement membrane mimic a promising basis for further studies in tissue engineering.

Audiovestibular Loss of Function Correlates in Vestibular Schwannomas.

OBJECTIVES: The aim of the present study was to investigate the relationships between tumor size, hearing, and vestibular outcomes in patients with vestibular schwannomas (VSs). MATERIALS AND METHODS: Adult patients (n=124) with unilateral extrameatal VS prior to surgery were included in the study. This was a retrospective cohort study of preoperative audiovestibular investigations including audiometry, discrimination test, caloric test, cervical vestibular evoked myogenic potential (c-VEMP), and ocular vestibular evoked myogenic potential (o-VEMP). RESULTS: The difference between lesioned and non-lesioned ear was significant for all audiovestibular outcomes. The mean caloric deficit was 74%. No tumor sided o-VEMPs were elicited. Caloric deficit correlated with hearing loss measured with pure tone average and discrimination score. c-VEMP deficit was significantly associated with severe hearing loss and larger tumors. CONCLUSION: The presence of VS leads to a significant deterioration of audiovestibular function in all objective measures. Caloric test and o-VEMPS are sensitive though unspecific measures of VSs. Increasing tumor size is not directly associated with hearing loss and only somewhat to vestibular deficit. However, audiovestibular findings are correlated.

Impact of Glutathione Modulation on Stability and Pharmacokinetic Profile of Redox-Sensitive Nanogels.

Nanoparticles degradable upon external stimuli combine pharmacokinetic features of both small molecules as well as large nanoparticles. However, despite promising preclinical results, several redox responsive disulphide-linked nanoparticles failed in clinical translation, mainly due to their unexpected in vivo behavior. Glutathione (GSH) is one of the most evaluated antioxidants responsible for disulfide degradation. Herein, the impact of GSH on the in vivo behavior of redox-sensitive nanogels under physiological and modulated conditions is investigated. Labelling of nanogels with a DNA-intercalating dye and a radioisotope allows visualization of the redox responsiveness at the cellular and the systemic levels, respectively. In vitro, efficient cleavage of disulphide bonds of nanogels is achieved by manipulation of intracellular GSH concentration. While in vivo, the redox-sensitive nanogels undergo, to a certain extent, premature degradation in circulation leading to rapid renal elimination. This instability is modulated by transient inhibition of GSH synthesis with buthioninsulfoximin. Altered GSH concentration significantly changes the in vivo pharmacokinetics. Lower GSH results in higher elimination half-life and altered biodistribution of the nanogels with a different metabolite profile. These data provide strong evidence that decreased nanogel degradation in blood circulation can limit the risk of premature drug release and enhance circulation half-life of the nanogel.

Multistage Passive and Active Delivery of Radiolabeled Nanogels for Superior Tumor Penetration Efficiency.

Development of nanosized drug delivery systems in cancer therapy is directed toward improving tumor selectivity and minimizing damages of healthy tissue. We introduce a delivery system with synergistic optimization and combination of passive and active targeting strategies. The approach is based on radiopeptide labeled redox sensitive hydrophilic nanogels, which exploit passive targeting by the enhanced permeability and retention effect while avoiding elimination by the mononuclear phagocyte system and fast hepatic and renal clearance. The targeting peptide promotes endocytotic uptake of the nanogels by cancer cells. Specific to this delivery system, tumor-specific degradation by the antioxidant glutathione enhances penetration and retention within the tumor tissue. Using in vivo molecular imaging we demonstrate the superiority of combined passive and active targeting with down-sizable nanogels over exclusive passive targeting. Furthermore, the homogeneous tumor distribution of functionalized nanogels compared to the clinically used mere radiopeptide supports the potentially high impact of our targeting concept.

Neuronal Fibers and Neurotransmitter Receptor Expression in the Human Endolymphatic Sac.

INTRODUCTION: Recent studies suggest that the human endolymphatic sac (ES) may have multiple functions, including an ion-transport capacity comparable to the kidney, an immunological capacity and a possible natriuretic capacity. Further, there have been speculations of a yet undefined role in intracranial pressure homeostasis. The anatomical location towards the sigmoid sinus would suggest a possible endo- and/or paracrine signaling. However, neuronal connections may also apply, but it remains very scarcely explored in the human ES. STUDY DESIGN: DNA micro-arrays and immunohistochemistry were used for analyses of fresh human ES tissue samples. METHODS: A total of 30 tissue samples from the human ES were obtained during translabyrinthine surgery for vestibular schwannoma. Microarray technology was used to investigate tissue sample gene expression, using adjacent dura mater as control. The expression of genes specific for neuronal signaling was determined and results for selected key molecules verified by immunohistochemistry. Transmission electron microscopy was used for ultrastructural analysis. RESULTS: For the transmission electron microscopy analysis, a direct innervation of the ES was observed with unmyelinated fibers imbedded in the ES epithelial lining. The microarrays confirmed, that several molecules involved in neuronal signaling were found expressed significantly in the ES DNA profile, such as the Cholecystokinin peptide and related receptors, Dopamine receptors 2 and 5, vesicular monoamine transporter 2 (VMAT2), plasma monoamine transporter (PMAT), and Serotonin 1D. All peptides were verified by immunohistochemistry. CONCLUSIONS: Based on global gene expression profiling and immuno-histochemical labeling, we conclude that the human ES expresses neuropeptide receptors and monoamine transporters. Combined with the ultrastructural demonstration of unmyelinated axons imbedded within the epithelial lining, the findings suggest that neuro-signaling mechanisms are involved in functions exerted by the ES.

Nerve Cells Decide to Orient inside an Injectable Hydrogel with Minimal Structural Guidance.

Injectable biomaterials provide the advantage of a minimally invasive application but mostly lack the required structural complexity to regenerate aligned tissues. Here, we report a new class of tissue regenerative materials that can be injected and form an anisotropic matrix with controlled dimensions using rod-shaped, magnetoceptive microgel objects. Microgels are doped with small quantities of superparamagnetic iron oxide nanoparticles (0.0046 vol %), allowing alignment by external magnetic fields in the millitesla order. The microgels are dispersed in a biocompatible gel precursor and after injection and orientation are fixed inside the matrix hydrogel. Regardless of the low volume concentration of the microgels below 3%, at which the geometrical constrain for orientation is still minimum, the generated macroscopic unidirectional orientation is strongly sensed by the cells resulting in parallel nerve extension. This finding opens a new, minimal invasive route for therapy after spinal cord injury.

Protecting patches in colloidal synthesis of Au semishells.

Protecting groups are commonly applied in multi-step molecular syntheses to protect one or multiple functional groups from reacting. After the reaction, they are removed from the molecule. In full analogy to this concept, we report the practical and scalable colloidal synthesis of Au semishells using polyphenylsiloxane protecting patches to prevent part of the surface of polystyrene nanoparticles from being covered with Au. After Au deposition, the patches are removed yielding Au semishells. We anticipate that this strategy can be extended to the synthesis of other types of non-centrosymmetric nanoparticles.