Poster BB14 - Moderate Mechanical Ventilation Results in Enhanced Re-epithelialization of Decellularized Lung Scaffolds

Introduction: Lung transplantation, the current treatment for advanced lung diseases, is hindered by the scarcity of suitable donor organs. To address this challenge, bioengineered lungs, created via re-epithelialization of acellular scaffolds with universal cell lines, have emerged as a promising alternative. However, the existing recellularization protocols are not adequate and do not fully recapitulate functional epithelium. While protocols predominantly rely on chemical stimuli, the mechanical environment, especially the influence of biophysical cues such as mechanical ventilation, remain inadequately explored. This study aims to investigate the effects of cyclical stretch in bioengineered lung scaffolds on distal lung epithelial cells, with a focus on enhancing attachment, proliferation, and function of the reconstituted epithelium.

Materials and

Methods: We utilized our previously established Negative Pressure Wet Ventilation System (NPWVS) for in order to investigate cyclical stretch in a mouse lung model. Acellular mouse scaffolds (n=6 per group) were subjected to perfusion and ventilation at different ventilation conditions (16 and 32 breaths per minutes (BPM) at a tidal volume of 300μL and perfusion at 1.5mL/min). These were compared to control groups including, decellularized but non-perfused/non-ventilated group (n=6), and perfused-only group (n=6). We also included a group of acellular scaffolds perfused and ventilated at 16 BPM with an unphysiological tidal volume of 1500μL (5x the physiological tidal volume of a mouse; n=6) tidal volume and group of mouse lungs which were not decellularized (native; n=6). Structural changes of the scaffolds were assessed through histological analysis on alveolar integrity (mean-linear-intercept), and compositional changes in the scaffolds were analyzed using extra-cellular matrix (ECM) component assays (e.g. sulfated glycosaminoglycan (sGAG) assay). Mechanical properties were evaluated using uniaxial tensile strength testing and atomic force microscopy (AFM). Additionally, we examined the cellular response of human lung epithelial cells (A549 cells) to cyclically stretched scaffolds and cyclical stretch, using Precision Cut Lung Slices (PCLSs) and whole-organ bioreactor-based recellularization. 40 PCLSs were created by sectioning nonventilated/ventilated decellularized lungs and were repopulated with A549 cells for 5 days. Similarly, A549 cells were seeded into each of 15 decellularized lungs (5 different ventilation conditions) for a 5-day whole-lung recellularization. The assessment of surface coverage and cell viability were conducted with Live/Dead Imaging, and proliferation was assessed by Ki67 immuno-staining.

Results, Conclusions, and Discussions: The NPWVS set up was illustrated in Figure 1. The mean-linear-intercept measurement of the decellularized scaffolds showed that all ventilation and perfusion groups did not experience alveolar structural changes except for the 1500μL/16BPM ventilation group, which had a 42%±25% (p < 0.001) intercept increment compared to the perfusion-only control. sGAG quantification also revealed that lung scaffold composition was unaffected under all the ventilation and perfusion conditions. Furthermore, the stress-strain relationship showed that moderate ventilation parameters did not alter the macroscale mechanical property (stiffness) of decellularized mouse lung scaffolds compared to the perfusion-only control. In contrast, we observed significantly reduced stiffness (58%±42% loading stress decrement and 69%±35% tangential modulus decrement at 0.20 strain, p< 0.05) in the positive control of excessively ventilated lungs (with 1500μL/16BPM ventilation). AFM measurement of alveolar septum stiffness indicated no significant differences between microscale mechanical properties of ventilated and perfused decellularized scaffolds. These structural and mechanical analysis indicated expected changes, which are suggesting damage to the scaffold under excessive tidal volume. Cellular response on PCLS showed significantly improved outcomes in A549 cell adhesion (increased by 89%±29%), viability (increased by 16%±9%), and proliferation (increased by 14%±8%) for A549 cells seeded on PCLS from moderately ventilated conditions (with 300μL /16BPM ventilation) at Day 5 (all p< 0.05). When ventilation conditions were incorporated to our whole organ recellularization protocols, the results showed that 5-day moderate ventilation at the two different rates (300μL/16BPM and 300μL/32BPM ventilation) enhanced A549 recellularization of whole lungs, increasing the cell coverage rate by 28%±21% (p < 0.05) and 55%±29% (p < 0.01) compared to the perfusion-only control. These results indicated the potential of moderate ventilation in enhancing cell spreading, survival, and proliferation in decellularized lungs.
We show that ventilation with a moderate tidal volume enhanced the coverage, survival, and proliferation of human lung epithelial cells within mouse lung scaffolds, without causing structural damage or changes in mechanical properties. Future research will focus on expanding this work for further optimization of recellularization protocols and deciphering the effects of ventilation on primary human lung epithelial cell function and associated mechano-transduction signaling pathways.

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