Decellularised Pleural Membrane Patches In Pulmonary Regenerative Medicine

Abstract

*Purpose/Objectives: Prolonged alveolar air leaks as post-surgical complications to routine lung resections and biopsies are a significant cause for 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 the incidence and severity of sustained air leaks. As a treatment modality, it can provide the relevant physiological cues that stimulate endogenous tissue regeneration. Adopting the classic tissue engineering paradigm of conditioning cells in a bio-instructive microenvironment niche, we focused on optimising a decellularisation protocol for porcine pleural membranes (PPM) and characterising the biological scaffolds for their retention of the innate mechanical strength, biochemical composition, microarchitecture, and bioactivity of the native pleural membranes. *Methodology: PPM decellularisation was carried out using a combinatorial approach of physical (freeze-thaw cycles) and chemical (0.5% sodium deoxycholate and 1% Triton-X 100 in 10mMTris buffer) treatments. Protocol efficiency was determined with histological analysis (Hematoxylin & Eosin, Alcian blue, and Picrosirius red staining), nuclear membrane integrity study (DAPI staining), and quantitative bioassays (Picogreen assay for nuclear DNA quantification, Sircol™ insoluble collagen assay, and dimethylmethylene blue (DMMB) glycosaminoglycan assay). Decellularised PPM were also assessed for their cytotoxicity (Live-Dead cytotoxicity kit, Invitrogen™, and Trypan blue exclusion assay) and biocompatibility (MeT-5A cell-line seeding and culture). *Results: H&E staining of decellularised PPM showed the absence of stained nuclei, consistent with the significant reduction (p?<?0.0001) in DAPI stained nuclei counts against native controls. PicoGreen assay confirmed efficient decellularisation as the quantified nuclear DNA in the decellularised PPM was less than 50?ng/mg. of dry weight of tissue. Staining for sulphated glycosaminoglycans (sGAG) and collagen with alcian blue and picrosirius red respectively, exhibited minimal disruption to the innate structural alignment of the native ECM fibers. However, quantifying collagen and GAG content per mg. of dry weight of tissue, in the decellularised PPM showed a significant reduction in comparison with the native controls. Mechanical characterisation studies revealed a significant increase in decellularised membrane thickness but not affecting the innate 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 assay carried out on seeded MeT-5A cells in contact with decellularised PPM for five days, exhibited minimal effect on cell proliferation and viability. Preliminary scaffold biocompatibility studies revealed promising results with the decellularised PPM seeded with MeT-5A cells promoting cellular attachment, proliferation, and viability for two weeks under standard culture conditions. *Conclusion/Significance: Our pilot study represents a step forward in deriving bioactive ECM scaffolds in the form of decellularised PPM. Future work entails expanding the characterisation regime to include proteomics and ultrastructural studies. 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 scaffold-based therapeutic approaches.