Prolonged alveolar air leaks are post-surgical complications to routine lung resections and biopsies that are a significant cause of patient morbidity. Extended duration of chest tube drainage and emergency revision surgeries are the standard approaches for its clinical management. Transplantable decellularised pleural membrane patches as adjuncts to traditional intraoperative closure techniques could reinforce the mechanical barrier, reducing incidence and severity of sustained air leaks. As a treatment modality, it can provide the physiological cues that stimulate endogenous tissue regeneration. We aimed to optimise a decellularisation and characterisation protocol for porcine pleural membranes (PPM), with minimal disruption to the microarchitecture, biochemical composition, and mechanical integrity of the native tissue.
PPM decellularisation was performed with physical (freeze-thaw cycles) and chemical (0.5% sodium deoxycholate and 1% Triton-X100 in 10mM Tris buffer) treatments. Protocol efficiency was determined via histological analysis (Hematoxylin and Eosin, Alcian blue and Picrosirius red), nuclear membrane integrity (DAPI staining), and quantitative bioassays (Picogreen dsDNA quantification and dimethyl methylene blue (DMMB) glycosaminoglycan assay). Decellularised PPM were assessed for their cytotoxicity (Live-Dead cytotoxicity kit, Invitrogen™, and Trypan blue exclusion assay) and biocompatibility (MeT-5A cell-line seeding and culture). Proteomics was carried out using antibody microarray technology (scioDiscover™, Sciomics GmbH)
H&E staining of decellularised PPM showed absence of stained nuclei, consistent with significant reduction (p < 0.0001) in DAPI stained nuclei counts against native controls. Residual DNA quantification in the decellularized PPM reflected over 90% reduction in native nuclear dsDNA (p < 0.001). Staining for sulphated glycosaminoglycans (sGAG) and collagen exhibited minimal disruption to the structural alignment of the native ECM. sGAG content in the decellularised PPM showed a significant reduction in comparison with native controls (p < 0.01). Mechanical characterisation studies showed that increased decellularised membrane thickness (p < 0.05) did not affect the inherent membrane stiffness, as the estimated Youngs modulus in the decellularised PPM (12782.7 kPa ± 3874) was comparable with the native controls (9259.5 kPa ± 2079). In vitro cytotoxicity and scaffold biocompatibility studies exhibited minimal inhibitory effect on MeT-5A cell line attachment, proliferation, and viability. Proteomics provided molecular readouts of the native and decellularised PPM proteome, reflecting differential protein expressions and enabling decoding of our decellularised PPM matrisome.
Our pilot study represents a step forward in deriving bioactive ECM scaffolds in the form of decellularised PPM. Studying the recellularisation dynamics of the cell-seeded scaffolds using primary mesothelial cultures will underpin our research towards developing proof of concept for the application of the relatively unexplored decellularised pleural membranes in biological ECM scaffold-based therapeutic approaches."