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.

Topographical influence of electrospun basement membrane mimics on formation of cellular monolayer.

Functional unit of many organs like lung, kidney, intestine, and eye have their endothelial and epithelial monolayers physically separated by a specialized extracellular matrix called the basement membrane. The intricate and complex topography of this matrix influences cell function, behavior and overall homeostasis. In vitro barrier function replication of such organs requires mimicking of these native features on an artificial scaffold system. Apart from chemical and mechanical features, the choice of nano-scale topography of the artificial scaffold is integral, however its influence on monolayer barrier formation is unclear. Though studies have reported improved single cell adhesion and proliferation in presence of pores or pitted topology, corresponding influence on confluent monolayer formation is not well reported. In this work, basement membrane mimic with secondary topographical cues is developed and its influence on single cells and their monolayers is investigated. We show that single cells cultured on fibers with secondary cues form stronger focal adhesions and undergo increased proliferation. Counterintuitively, absence of secondary cues promoted stronger cell-cell interaction in endothelial monolayers and promoted formation of integral tight barriers in alveolar epithelial monolayers. Overall, this work highlights the importance of choice of scaffold topology to develop basement barrier function in in vitro models.

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.

Enzyme mimetic microgel coating for endogenous nitric oxide mediated inhibition of platelet activation.

Nitric oxide (NO) continuously generated by healthy endothelium prevents platelet activation and maintains vascular homeostasis. However, when artificial surfaces, like of extracorporeal membrane oxygenator comes in contact with blood, protein adsorption and thereby platelet activation takes place, which eventually leads to thrombus formation. To overcome this, we present an antifouling microgel coating mimicking the function of enzyme glutathione peroxidase to endogenously generate NO in the blood plasma from endogenous NO-donors and maintain a physiological NO flux. Microgels are synthesized by copolymerization of highly hydrophilic N-(2-hydroxypropyl)methacrylamide (HPMA) and glycidyl methacrylate (GMA) with diselenide crosslinks. For immobilization of the microgels on hydrophobic poly(4-methylpentene) (TPX) membranes bioengineered amphiphilic anchor peptides with free thiols are used. The anchor peptide attaches to the TPX membranes by hydrophobic interactions while the free thiols are presented for crosslinking with the microgels. The hydrophilic nature of the microgel coating prevents protein adsorption while the reversible diselenide bridges make the microgels responsive to the reducing environment and lead to the formation of reactive selenols/selenolates. The generated selenols/selenolates provide an efficient and sustained NO-release from endogenous S-nitrosothiols (RSNOs) mimicking the enzymatic function of glutathione peroxidase. On exposure to the whole blood, the microgel coating inhibited platelet activation and prolonged the blood clotting time.

Reconstruction of Ultra-thin Alveolar-capillary Basement Membrane Mimics.

Alveolar-capillary basement membrane (BM) is ultra-thin (<2 µm) extracellular matrix that maintains integral epithelial-endothelial cell layers. In vitro reconstructions of alveolar-capillary barrier supported on synthetic scaffolds closely resembling the fibrous and ultra-thin natural BM are essential in mimicking the lung pathophysiology. Although BM topology and dimensions are well known to significantly influence cellular behavior, conventionally used BM mimics fail to recreate this natural niche. To overcome this, electrospun ultra-thin 2 µm poly(caprolactone) (PCL) nanofibrous mesh is used to establish an alveolar-capillary barrier model of lung endothelial/epithelial cells. Transepithelial electrical resistance (TEER) and permeability studies reveal integral tight junctions and improved mass transport through the highly porous PCL meshes compared to conventional dense membranes with etched pores. The chemotaxis of neutrophils is shown across the barrier in presence of inflammatory response that is naturally impeded in confined regions. Conventional requirement of 3 µm or larger pore size can lead to barrier disruption due to epithelial/endothelial cell invasion. Despite high porosity, the interconnected BM mimic prevents barrier disruption and allows neutrophil transmigration, thereby demonstrating the physiological relevance of the thin nanofibrous meshes. It is envisioned that these bipolar cultured barriers would contribute to an organ-level in vitro model for pathological disease, environmental pollutants, and nanotoxicology.

Dual-Degradable Biohybrid Microgels by Direct Cross-Linking of Chitosan and Dextran Using Azide-Alkyne Cycloaddition.

In this work, biocompatible and degradable biohybrid microgels based on chitosan and dextran were synthesized for drug delivery applications. Two kinds of bio-based building blocks, alkyne-modified chitosan and azide-modified dextran, were used to fabricate microgels via single-step cross-linking in water-in-oil emulsions. The cross-linking was initiated in the presence of copper(II) without the use of any extra cross-linkers. A series of pH-responsive and degradable microgels were successfully synthesized by varying the degree of cross-links. The microgels were characterized using 1H NMR and FTIR spectroscopy which proved the successful cross-linking of alkyne-modified chitosan and azide-modified dextran by copper(II)-mediated click reaction. The obtained microgels exhibit polyampholyte character and can carry positive or negative charges in aqueous solutions at different pH values. Biodegradability of microgels was shown at pH 9 or in the presence of Dextranase due to the hydrolysis of carbonate esters in the microgels or 1,6-α-glucosidic linkages in dextran structure, respectively. Furthermore, the microgels could encapsulate vancomycin hydrochloride (VM), an antibiotic, with a high loading of approximately 93.67% via electrostatic interactions. The payload could be released in the presence of Dextranase or under an alkaline environment, making the microgels potential candidates for drug delivery, such as colon-specific drug release.

Electroactive and degradable supramolecular microgels.

In this work, we synthesized electroactive and degradable microgels based on biomacromolecular building blocks, which enable the controlled release of therapeutic drugs. Functional chitosan-poly(hydroquinone) (Ch:PHQ) microgels exhibiting redox-active and pH-sensitive properties were synthesized by an oxidative polymerization in an inverse miniemulsion system. Physically crosslinked microgels were formed by polymerization of hydroquinone in the presence of chitosan through the formation of hydrogen bonds between PHQ and Ch. A series of microgel samples with variable Ch : PHQ ratios were synthesized. These obtained microgels exhibit pH-responsive properties due to the protonation/deprotonation of amino-groups of chitosan in the microgel system. Poly(hydroquinone) is a redox-active polymer exhibiting a two-electron/proton-transfer behavior and conveys this property to the microgels as confirmed by cyclic voltammetry. In addition, the microgels can be switched by electrochemical means: they swell in the oxidized state or shrink in the reduced state. In the presence of urea or lysozyme, the microgels undergo a fast degradation due to the disruption of hydrogen bonds acting as physical crosslinks in the microgel networks or due to the cleavage of glucosidic linkages of the incorporated chitosan scaffold, respectively. Doxorubicin (DOX), an anticancer drug, could be effectively encapsulated into the microgels and released in the presence of an enzyme, indicating that these biodegradable microgels could be used as drug delivery vehicles for tumor cells.

Hepatic involvement in dengue Fever in children.

OBJECTIVE: Hepatic dysfunction is common in dengue infection and the degree of liver dysfunction in children varies from mild injury with elevation of transaminases to severe injury with jaundice. This study was undertaken to assess the spectrum of hepatic involvement in dengue infection. METHODS: 110 children with serologically positive dengue fever aged between 2 months - 14 years were studied for their hepatic functions both clinically and biochemically after excluding malaria, enteric fever, Hepatitis A and Hepatitis B with relevant investigations. FINDINGS: All cases were grouped into DF (Dengue fever), DHF (Dengue hemorrhagic fever) and DSS (Dengue shock syndrome) according to WHO criteria. The spectrum of hepatic manifestations included hepatomegaly (79%), hepatic tenderness (56%), jaundice (4.5%), raised levels of aspartate transaminase (AST)(93%), alanine transaminase (ALT)(78%), alkaline phosphatase (AP) (57%), prolonged prothrombin time (PT) (20%), reduced levels of serum albumin (66%) and abnormal abdomen ultrasound (65%). CONCLUSION: Hepatic dysfunction was observed more in DHF and DSS group compared to DF group. About 17.27% of children had >10 fold increase in the liver enzymes. There was no correlation between the degree of hepatic enlargement or hepatic tenderness with the abnormalities of liver functions. Any child with fever, jaundice and tender hepatomegaly in geographical areas where dengue is endemic, the diagnosis of dengue infection should be strongly considered.

Combined hydrogen production and storage with subsequent carbon crystallization.

We provide evidence of low-temperature hydrogen evolution and possible hydrogen trapping in an anthracite coal derivative, formed via reactive ball milling with cyclohexene. No molecular hydrogen is added to the process. Raman-active molecular hydrogen vibrations are apparent in samples at atmospheric conditions (300 K, 1 bar) for samples prepared 1 year previously and stored in ambient air. Hydrogen evolves slowly at room temperature and is accelerated upon sample heating, with a first increase in hydrogen evolution occurring at approximately 60 degrees C. Subsequent chemical modification leads to the observation of crystalline carbons, including nanocrystalline diamond surrounded by graphene ribbons, other sp2-sp3 transition regions, purely graphitic regions, and a previously unidentified crystalline carbon form surrounded by amorphous carbon. The combined evidence for hydrogen trapping and carbon crystallization suggests hydrogen-induced crystallization of the amorphous carbon materials, as metastable hydrogenated carbons formed via the high-energy milling process rearrange into more thermodynamically stable carbon forms and molecular hydrogen.