RESEARCH SUMMARY
This study tackles a major cause of early graft failure in bioengineered lungs—acute thrombosis and vascular leakage driven by incomplete, dysfunctional endothelial coverage of the decellularized pulmonary vasculature. Using decellularized mouse whole-lung scaffolds, the authors screened multiple vascular luminal surface preconditioning strategies on precision-cut lung slices (PCLS), including fibronectin, REDV peptide, anti-CD31 antibody, angiopoietin-1 + VEGF, and heparin + gelatin. Endothelial cell adhesion, metabolic activity/proliferation, migration, and endothelial function gene expression were quantified, identifying anti-CD31 antibody as the most robust coating, with consistent improvements in adhesion and growth and increased expression of barrier- and function-associated genes (including CLDN, CD34, and eNOS). Translating to whole-lung recellularization, anti-CD31–coated scaffolds seeded via pulmonary artery with endothelial cells and cultured in a perfusion bioreactor showed markedly improved vascular lining, reduced apoptosis (TUNEL/caspase-3), increased proliferation (Ki-67), improved tight junction organization (ZO-1), and superior vascular network reconstitution (µCT angiography). Functional vascular barrier performance improved substantially, demonstrated by reduced dextran extravasation (higher fraction recovered intravascularly vs uncoated). Critically, anti-CD31 preconditioning reduced thrombogenicity during 24 h ex vivo perfusion with whole blood, lowering platelet aggregation (integrin αIIb staining), preserving circulating platelet counts, and decreasing expression of thrombogenic genes (PLSCR1, TBXAS1, THBS1). In a short-term orthotopic mouse left lung transplant model (30 min reperfusion), anti-CD31–coated bioengineered lungs exhibited improved perfusion with significantly less thrombus formation than uncoated controls. Overall, the work establishes anti-CD31 antibody preconditioning as a practical, single-step strategy to enhance re-endothelialization, vascular barrier function, and antithrombotic performance of bioengineered lung scaffolds.
Uniaxial tensile mechanical testing was performed using a CellScale BioTester 5000 to assess whether decellularization preserved extracellular matrix mechanical integrity in mouse lung scaffolds prior to recellularization experiments. Rectangular tissue strips (approximately 0.6 mm × 1 mm × 4.5 mm) were excised from the left lobe of native and decellularized lungs (n=3/group), mounted along the long axis in the BioTester uniaxial tensile setup, and stretched at a constant displacement rate of 0.2 mm/s until failure. BioTester force–displacement output was used to generate stress–strain curves, from which elastic behavior and stiffness parameters were derived in MATLAB. These tensile results were used as a foundational quality-control checkpoint demonstrating that the decellularization process preserved bulk scaffold mechanical behavior, supporting the validity of subsequent vascular coating, endothelial seeding, perfusion bioreactor culture, and thrombogenicity assessments performed on mechanically representative lung matrices.
