Lung tissue engineering and preservation of alveolar microstructure using a novel casting method

Abstract

We used a rat model to decellularize and seed alveolar cells on a three-dimensional lung scaffold to preserve alveolar microarchitecture. We verified the preservation of terminal respiratory structure by casting and by scanning electron microscopy (SEM) of the casts after decellularization. Whole lungs were obtained from 12 healthy Sprague-Dawley rats, cannulated through the trachea under sterile conditions, and decellularized using a detergent-based method. Casting of both natural and decellularized lungs was performed to verify preservation of the inner microstructure of scaffolds for further cell seeding. Alveolar cell seeding was performed using green fluorescent protein (GFP) lung cells and non-GFP lung cells, and a peristaltic pump. We assessed cell seeding using histological and immunohistochemical staining, and enzymatic evaluation. All cellular components were removed completely from the scaffolds, and histological staining and SEM of casts were used to verify the preservation of tissue structure. Tensile tests verified conservation of biomechanical properties. The hydroxyproline content of decellularized lungs was similar to native lung. Histological and immunohistochemical evaluations showed effective cell seeding on decellularized matrices. Enzymatic measurement of trypsin and alpha 1 antitrypsin suggested the potential functional properties of the regenerated lungs. Casts produced by our method have satisfactory geometrical properties for further cell seeding of lung scaffolds. Preservation of micro-architecture and terminal alveoli that was confirmed by SEM of lung casts increases the probability of an effective cell seeding process.     

Multigrid Block Preconditioning for a Coupled System of Partial Differential Equations Modeling the Electrical Activity in the Heart

The electrical activity of the heart may be modeled with a system of partial differential equations (PDEs) known as the bidomain model. Computer simulations based on these equations may become a helpful tool to understand the relationship between changes in the electrical field and various heart diseases. Because of the rapid variations in the electrical field, sufficiently accurate simulations require a fine-scale discretization of the equations. For realistic geometries this leads to a large number of grid points and consequently large linear systems to be solved for each time step. In this paper, we present a fully coupled discretization of the bidomain model, leading to a block structured linear system. We take advantage of the block structure to construct an efficient preconditioner for the linear system, by combining multigrid with an operator splitting technique.     

In vitro and in vivo antimicrobial efficacy of essential oils and individual compounds against Phytophthora parasitica var. nicotianae

Aim

To evaluate the antimicrobial effects of essential oils (EOs) from cassia, basil, geranium, lemongrass, cumin and thyme, as well as their major components, against Phytophthora parasitica var. nicotianae; to investigate morphological changes in hyphae and sporangia in response to treatment with cinnamaldehyde; and to further evaluate potential biocontrol capacities against tobacco black shank under greenhouse conditions.

Methods and Results

The results revealed that the extent of mycelial growth inhibition was primarily dependent on the composition and concentration of the EOs and the structure of individual compounds. Cinnamaldehyde had a significantly higher inhibitory effect on mycelial growth, formation of sporangia, and production and germination of zoospores in P. parasitica var. nicotianae in vitro, achieving complete inhibition of these phenotypes at 72, 36, 36 and 18 mg l^?1, respectively. Scanning electron microscopic observations revealed that cinnamaldehyde can cause considerable morphological degenerations of hyphae and sporangia such as cytoplasmic coagulation, shrivelled mycelia and sporangia aggregates and swelling and lysis of mycelia and sporangia walls. In vivo assays with cinnamaldehyde demonstrated that this compound afforded protective effect against tobacco black shank under greenhouse conditions in susceptible tobacco plants.

Conclusions

The results of in vitro and in vivo bioassays, together with SEM imaging of the microstructure of P. parasitica var. nicotianae supported the possibility of using cinnamaldehyde as a potent natural biofungicide in the greenhouse.

Significance and Impact of the Study

This study provides a theoretical basis for the potential use of cinnamaldehyde as commercial agents or lead compounds that can be exploited as commercial biofungicides in the protection of tobacco plants from P. parasitica var. nicotianae infection.     

Elimination of Aspergillus fumigatus conidia from the airways of mice with allergic airway inflammation

Background

Aspergillus fumigatus conidia can exacerbate asthma symptoms. Phagocytosis of conidia is a principal component of the host antifungal defense. We investigated whether allergic airway inflammation (AAI) affects the ability of phagocytic cells in the airways to internalize the resting fungal spores.

Methods

Using BALB/c mice with experimentally induced AAI, we tested the ability of neutrophils, macrophages, and dendritic cells to internalize A. fumigatus conidia at various anatomical locations. We used light microscopy and differential cell and conidium counts to determine the ingestion potential of neutrophils and macrophages present in bronchoalveolar lavage (BAL). To identify phagocyte-conidia interactions in conducting airways, conidia labeled with tetramethylrhodamine-(5-(and-6))-isothiocyanate were administered to the oropharyngeal cavity of mice. Confocal microscopy was used to quantify the ingestion potential of Ly-6G⁺ neutrophils and MHC II⁺ antigen-presenting cells located in the intraepithelial and subepithelial areas of conducting airways.

Results

Allergen challenge induced transient neutrophil recruitment to the airways. Application of A. fumigatus conidia at the acute phase of AAI provoked recurrent neutrophil infiltration, and consequently increased the number and the ingestion potential of the airway neutrophils. In the absence of recurrent allergen or conidia provocation, both the ingestion potential and the number of BAL neutrophils decreased. As a result, conidia were primarily internalized by alveolar macrophages in both AAI and control mice at 24 hours post-inhalation. Transient influx of neutrophils to conducting airways shortly after conidial application was observed in mice with AAI. In addition, the ingestion potential of conducting airway neutrophils in mice with induced asthma exceeded that of control mice. Although the number of neutrophils subsequently decreased, the ingestion capacity remained elevated in AAI mice, even at 24 hours post-conidia application.

Conclusions

Aspiration of allergen to sensitized mice enhanced the ingestion potential of conducting airway neutrophils. Such activation primes neutrophils so that they are sufficient to control dissemination of non-germinating A. fumigatus conidia. At the same time, it can be a reason for the development of sensitivity to fungi and subsequent asthma exacerbation.     

Fabrication of Nano-engineered Transparent Conducting Oxides by Pulsed Laser Deposition

Nanosecond Pulsed Laser Deposition (PLD) in the presence of a background gas allows the deposition of metal oxides with tunable morphology, structure, density and stoichiometry by a proper control of the plasma plume expansion dynamics. Such versatility can be exploited to produce nanostructured films from compact and dense to nanoporous characterized by a hierarchical assembly of nano-sized clusters. In particular we describe the detailed methodology to fabricate two types of Al-doped ZnO (AZO) films as transparent electrodes in photovoltaic devices: 1) at low O₂ pressure, compact films with electrical conductivity and optical transparency close to the state of the art transparent conducting oxides (TCO) can be deposited at room temperature, to be compatible with thermally sensitive materials such as polymers used in organic photovoltaics (OPVs); 2) highly light scattering hierarchical structures resembling a forest of nano-trees are produced at higher pressures. Such structures show high Haze factor (>80%) and may be exploited to enhance the light trapping capability. The method here described for AZO films can be applied to other metal oxides relevant for technological applications such as TiO₂, Al₂O₃, WO₃ and Ag₄O₄.     

Surface-bound basement membrane components accelerate amyloid-β peptide nucleation in air-free wells: An in vitro model of cerebral amyloid angiopathy

Cerebral amyloid angiopathy is caused by deposition of the amyloid β-peptide which consists of mainly 39–40 residues to the cortical and leptomeningeal vessel walls. There are no definite in vitro systems to support the hypothesis that the vascular basement membrane may act as a scaffold of amyloid β-peptide carried by perivascular drainage flow and accelerate its amyloid fibril formation in vivo. We previously reported the critical roles of interfaces and agitation on the nucleation of amyloid fibrils at low concentrations of amyloid β-peptide monomers. Here, we reproduced the perivascular drainage flow in vitro by using N-hydroxysuccinimide-Sepharose 4 Fast flow beads as an inert stirrer in air-free wells rotated at 1 rpm. We then reproduced the basement membranes in the media of cerebral arteries in vitro by conjugating Matrigel and other proteins on the surface of Sepharose beads. These beads were incubated with 5 μM amyloid β(1–40) at 37 °C without air, where amyloid β(1–40) alone does not form amyloid fibrils. Using the initiation time of fibril growth kinetics (i.e., the lag time of fibril growth during which nuclei, on-pathway oligomers and protofibrils are successively formed) as a parameter of the efficiency of biological molecules to induce amyloid fibril formation, we found that basement membrane components including Matrigel, laminin, fibronectin, collagen type IV and fibrinogen accelerate the initiation of amyloid β-peptide fibril growth in vitro. These data support the essential role of vascular basement membranes in the development of cerebral amyloid angiopathy.     

Host response to intravenous injection of epsilon toxin in mouse model: A proteomic view

Epsilon toxin (ETX) is an extremely potent pore‐forming toxin and a category B biological agent. ETX is a major virulence determinant of Clostridium perfringens toxinotypes B and D, and is implicated in pathogenesis of rapidly fatal economically important pulpy kidney disease in lambs caused by toxinotype D. Despite being a toxin, ETX can be utilized as a tool to target glutamatergic neurons and for drug delivery into the CNS. 2DE‐MS approach was employed to elucidate the host response to ETX following intravenous injection in mouse model. In total, 136 proteins were identified either differentially expressed in brain (18) and kidney (33); showing specific interaction with ETX from lysates of brain (4), kidney (21), or from plasma (42); and urine markers (18) of intoxication. Differentially expressed proteins in kidney included those involved in calcium homeostasis and cytoskeletal organization. Proteins involved in ER and oxidative stress and energy metabolism also showed differential levels in the target tissue after ETX treatment. The known functions of the proteins differentially expressed and those interacting with ETX indicate involvement of interlinked pathways. This study provides first proteomic account of host response to ETX exposure providing clues to mechanism of toxicity and potential therapeutic targets.