Decellularisation and Characterisation of Porcine Pleura as Bioscaffolds in Tissue Engineering

Abstract

Persistent air leaks caused by thoracic surgery, physical trauma, or spontaneous pneumothoraces are a cause of patient morbidity with need for extended chest tube durations and surgical interventions. Current treatment measures involve mechanical closure of air leaks in the compromised pleura. Organ and membrane decellularisation offers a broad range of biomimetic scaffolds of allogeneic and xenogeneic origins, exhibiting innate tissue-specific characteristics. We explored a physicochemical method for decellularising porcine pleural membranes (PPM) as potential tissue-engineered surrogates for lung tissue repair. Decellularised PPM (dPPM) was characterised with histology, quantitative assays, mechanical testing, and sterility evaluation. Cytotoxicity and recellularisation assays assessed biocompatibility of decellularised PPM (dPPM). Haematoxylin and Eosin (H&E) staining showed an evident reduction in stained nuclei in the dPPM, confirmed with nuclear staining and analysis ( ^∗∗∗∗p < 0.0001). Sulphated glycosaminoglycans (sGAG) and collagen histology demonstrated minimal disruption to the gross structural assembly of core extracellular matrix (ECM) in dPPM. Confocal imaging demonstrated realignment of ECM fibres in dPPM against native control. Quantitative analysis defined a significant change in the angular distribution ( ^∗∗∗∗p < 0.0001) and coherence ( ^∗∗∗p < 0.001) of fibre orientations in dPPM versus native ECM. DNA quantification indicated ≥85% reduction in native nuclear dsDNA in dPPM ( ^∗∗p < 0.01). Collagen and sGAG quantification indicated reductions of both ( ^∗∗p < 0.01). dPPM displayed increased membrane thickness ( ^∗∗∗p < 0.001). However, Young’s modulus (459.67 ± 10.36 kPa) and ultimate tensile strength (4036.22 ± 155.1 kPa) of dPPM were comparable with those of native controls at (465.82 ± 10.51 kPa) and (3912.9 ± 247.42 kPa), respectively. In vitro cytotoxicity and scaffold biocompatibility assays demonstrated robust human mesothelial cell line (MeT-5A) attachment and viability. DNA quantification in reseeded dPPM with MeT-5A cells exhibited significant increase in DNA content at day 7 ( ^∗∗p < 0.01) and day 15 ( ^∗∗∗∗p < 0.0001) against unseeded dPPM. Here, we define a decellularisation protocol for porcine pleura that represents a step forward in their potential tissue engineering applications as bioscaffolds.