Replacement of large tracheal defects remains an unmet clinical need. While recellularization of acellular tracheal grafts appeared to be a viable pathway, evidence from the clinic suggests otherwise. In hindsight, complete removal of chondrocytes and repopulation of the tracheal chondroid matrix to achieve functional tracheal cartilage may have been unrealistic. In contrast, the concept of a hybrid graft whereby the epithelium is removed and the immune-privileged cartilage is preserved is a radically different path with initial reports indicating potential clinical success. Here, we present a novel approach using a double-chamber bioreactor to de-epithelialize tracheal grafts and subsequently repopulate the grafts with exogenous cells. A 3 h treatment with sodium dodecyl sulfate perfused through the inner chamber efficiently removes the majority of the tracheal epithelium while the outer chamber, perfused with growth media, keeps most (68.6 ± 7.3%) of the chondrocyte population viable. De-epithelialized grafts support human bronchial epithelial cell (BEAS-2B) attachment, viability and growth over 7 days. While not without limitations, our approach suggests value in the ultimate use of a chimeric allograft with intact donor cartilage re-epithelialized with recipient-derived epithelium. By adopting a brief and partial decellularization approach, specifically removing the epithelium, we avoid the need for cartilage regeneration.

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.

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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.Electronic supplementary materialSupplementary information accompanies the paper on the npj Regenerative Medicine website (10.1038/s41536-018-0052-5).

Successful re-epithelialization of de-epithelialized tracheal scaffolds remains a challenge for tracheal graft success. Currently, the lack of understanding of the bioreactor hydrodynamic environment, and its relation to cell seeding outcomes, serve as major obstacles to obtaining viable tracheal grafts. In this work, we used computational fluid dynamics to (a) re-design the fluid delivery system of a trachea bioreactor to promote a spatially uniform hydrodynamic environment, and (b) improve the perfusion cell seeding protocol to promote homogeneous cell deposition. Lagrangian particle-tracking simulations showed that low rates of rotation provide more uniform circumferential and longitudinal patterns of cell deposition, while higher rates of rotation only improve circumferential uniformity but bias cell deposition proximally. Validation experiments with human bronchial epithelial cells confirm that the model accurately predicts cell deposition in low shear stress environments. We used the acquired knowledge from our particle tracking model, as a guide for long-term tracheal repopulation studies. Cell repopulation using conditions resulting in low wall shear stress enabled enhanced re-epithelialization of long segment tracheal grafts. While our work focuses on tracheal regeneration, lessons learned in this study, can be applied to culturing of any tissue engineered tubular scaffold.

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.

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.

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