Cold Atmospheric Helium Plasma in the Post-COVID-19 Era: A Promising Tool for the Disinfection of Silicone Endotracheal Prostheses.

Despite the excellent properties of silicone endotracheal prostheses, their main limitation is the formation of a polymicrobial biofilm on their surfaces. It can cause local inflammation, interfering with the local healing process and leading to further complications in the clinical scenario. The present study evaluated the inhibitory effect of cold atmospheric plasma (CAP) on multispecies biofilms grown on the silicone protheses' surfaces. In addition to silicone characterization before and after CAP exposure, CAP cytotoxicity on immortalized human bronchial epithelium cell line (BEAS-2B) was evaluated. The aging time test reported that CAP could temporarily change the silicone surface wetting characteristics from hydrophilic (80.5°) to highly hydrophilic (<5°). ATR-FTIR showed no significant alterations in the silicone surficial chemical composition after CAP exposure for 5 min. A significant log reduction in viable cells in monospecies biofilms (log CFU/mL) of C. albicans, S. aureus, and P. aeruginosa (0.636, 0.738, and 1.445, respectively) was detected after CAP exposure. Multispecies biofilms exposed to CAP showed significant viability reduction for C. albicans and S. aureus (1.385 and 0.831, respectively). The protocol was not cytotoxic to BEAS-2B. CAP can be a simple and effective method to delay multispecies biofilm formation inside the endotracheal prosthesis.

Use of High-Rate Ventilation Results in Enhanced Recellularization of Bioengineered Lung Scaffolds.

While transplantation is a viable treatment option for end-stage lung diseases, this option is highly constrained by the availability of organs and postoperative complications. A potential solution is the use of bioengineered lungs generated from repopulated acellular scaffolds. Effective recellularization, however, remains a challenge. In this proof-of-concept study, mice lung scaffolds were decellurized and recellurized using human bronchial epithelial cells (BEAS2B). We present a novel liquid ventilation protocol enabling control over tidal volume and high rates of ventilation. The use of a physiological tidal volume (300 µL) for mice and a higher ventilation rate (40 breaths per minute vs. 1 breath per minute) resulted in higher cell numbers and enhanced cell surface coverage in mouse lung scaffolds as determined via histological evaluation, genomic polymerase chain reaction (PCR) analysis, and immunohistochemistry. A biomimetic lung bioreactor system was designed to include the new ventilation protocol and allow for simultaneous vascular perfusion. We compared the lungs cultured in our dual system to lungs cultured with a bioreactor allowing vascular perfusion only and showed that our system significantly enhances cell numbers and surface coverage. In summary, our results demonstrate the importance of the physical environment and forces for lung recellularization. Impact statement New bioreactor systems are required to further enhance the regeneration process of bioengineered lungs. This proof-of-concept study describes a novel ventilation protocol that allows for control over ventilation parameters such as rate and tidal volume. Our data show that a higher rate of ventilation is correlated with higher cell numbers and increased surface coverage. We designed a new biomimetic bioreactor system that allows for ventilation and simultaneous perfusion. Compared to a traditional perfusion only system, recellularization was enhanced in lungs recellularized with our new biomimetic bioreactor.

MRI method for labeling and imaging decellularized extracellular matrix scaffolds for tissue engineering.

PURPOSE: To develop a facile method for labeling and imaging decellularized extracellular matrix (dECM) scaffolds intended for regenerating 3D tissues. METHODS: A small molecule manganese porphyrin, MnPNH2 , was synthesized and used to label dECM scaffolds made from porcine bladder and trachea and murine whole lungs. The labeling protocol was optimized on bladder dECM, and imaging on a 3T clinical scanner was performed to assess reductions in T1 and T2 relaxation times. In vivo MRI was performed on dECM injected in the rat dorsum to verify sensitivity of detection. Toxicity assays for cell viability, metabolism, and proliferation were performed on human umbilical vein endothelial cells. The incorporation of MnPNH2 and its long-term retention in dECM were assessed on transmission electron microscopy and ultraviolet absorbance of eluted MnPNH2 over time. RESULTS: All tissues, including thick whole 3D organs, were uniformly labeled and demonstrated high signal-to-noise on MRI. A nearly 10-fold reduction in T1 was consistently obtained at a labeling dose of 0.4 mM, and even 0.2 mM provided sufficient contrast in vivo and ex vivo. No toxicity was observed up to 0.4 mM, the maximum tested. Binding studies suggested nonspecific association, and retention studies in the labeled whole decellularized lungs revealed less than 20% MnPNH2 loss over 30 days, the majority occurring in the first 3 days after labeling. CONCLUSION: The proposed labeling method is the first report for visualizing dECM on MRI and has the potential for long-term monitoring and optimization of dECM-based organ tissue engineering.

An in vivo image acquisition system for the evaluation of tracheal mechanics in rats.

Mechanical evaluation of tracheal grafts is of great relevance for transplant research. Although there are some publications demonstrating different techniques of tracheal mechanical evaluation, there is still no definitive or preferred protocol available. Here, we present a simple image processing acquisition system that can be used for in vivo experiments. Six male Wistar rats were submitted to orotracheal intubation and a longitudinal incision was made to expose the trachea. Images of tracheae were acquired from a video camera in different scenarios of bronchoconstriction using methacholine (MCh) (Basal, PBS, MCh 30 µg/kg, MCh 300 µg/kg, and postmetabolized) during imposed-inspiration and imposed-expiration. The area variation ratio (the ratio between areas during expiration vs. inspiration) was 1.1× for the Basal group, while the ratio for MCh 300 µg/kg was 6.5×. The area variation of imaged tracheae was statistically significant at the dose of MCh 300 µg/kg for imposed-inspiration versus imposed-expiration (P = .002). Likewise, elastance data of respiratory mechanics indicated a statistically significant difference at the dose of MCh 300 µg/kg for imposed-inspiration versus imposed-expiration (P = .026). Our image processing analysis protocol presented corresponding behavior when compared to mechanical parameters of the respiratory system. In addition, our image acquisition system was able to highlight the differences between imposed-inspiration and imposed-expiration. Image analysis of the tracheal area variation seems to be in agreement with the elastance of the respiratory system. Taken together, these observations may help future studies of tracheal transplantation for in situ assessment of graft patency.

Interrupted reprogramming of alveolar type II cells induces progenitor-like cells that ameliorate pulmonary fibrosis.

We describe here an interrupted reprogramming strategy to generate "induced progenitor-like (iPL) cells" from alveolar epithelial type II (AEC-II) cells. A carefully defined period of transient expression of reprogramming factors (Oct4, Sox2, Klf4, and c-Myc (OSKM)) is able to rescue the limited in vitro clonogenic capacity of AEC-II cells, potentially by activation of a bipotential progenitor-like state. Importantly, our results demonstrate that interrupted reprogramming results in controlled expansion of cell numbers yet preservation of the differentiation pathway to the alveolar epithelial lineage. When transplanted to the injured lungs, AEC-II-iPL cells are retained in the lung and ameliorate bleomycin-induced pulmonary fibrosis. Interrupted reprogramming can be used as an alternative approach to produce highly specified functional therapeutic cell populations and may lead to significant advances in regenerative medicine.

Mechanical Evaluation of Tracheal Grafts on Different Scales.

Tissue engineered (or bioengineered) tracheas are alternative options under investigation when the resection with end-to-end anastomosis cannot be performed. One approach to develop bioengineered tracheas is a complex process that involves the use of decellularized tissue scaffolds, followed by recellularization in custom-made tracheal bioreactors. Tracheas withstand pressure variations and their biomechanics are of great importance so that they do not collapse during respiration, although there has been no preferred method of mechanical assay of tracheas among several laboratories over the years. These methods have been performed in segments or whole tracheas and in different species of mammals. This article aims to present some methods used by different research laboratories to evaluate the mechanics of tracheal grafts and presents the importance of the tracheal biomechanics in both macro and micro scales. If bioengineered tracheas become a reality in hospitals in the next few years, the standardization of biomechanical parameters will be necessary for greater consistency of results before transplantations.

Assessment of the mechanics of a tissue-engineered rat trachea in an image-processing environment.

OBJECTIVES: Despite the recent success regarding the transplantation of tissue-engineered airways, the mechanical properties of these grafts are not well understood. Mechanical assessment of a tissue-engineered airway graft before implantation may be used in the future as a predictor of function. The aim of this preliminary work was to develop a noninvasive image-processing environment for the assessment of airway mechanics. METHOD: Decellularized, recellularized and normal tracheas (groups DECEL, RECEL, and CONTROL, respectively) immersed in Krebs-Henseleit solution were ventilated by a small-animal ventilator connected to a Fleisch pneumotachograph and two pressure transducers (differential and gauge). A camera connected to a stereomicroscope captured images of the pulsation of the trachea before instillation of saline solution and after instillation of Krebs-Henseleit solution, followed by instillation with Krebs-Henseleit with methacholine 0.1 M (protocols A, K and KMCh, respectively). The data were post-processed with computer software and statistical comparisons between groups and protocols were performed. RESULTS: There were statistically significant variations in the image measurements of the medial region of the trachea between the groups (two-way analysis of variance [ANOVA], p<0.01) and of the proximal region between the groups and protocols (two-way ANOVA, p<0.01). CONCLUSIONS: The technique developed in this study is an innovative method for performing a mechanical assessment of engineered tracheal grafts that will enable evaluation of the viscoelastic properties of neo-tracheas prior to transplantation.