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bioRxiv · 10.1101/2023.06.20.545830

Decellularisation and characterisation of porcine pleura for lung tissue engineering

Vikranth, T.·Dale, T. P.·Forsyth, N. R.
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Abstract

Decellularisation offers a broad range of biomimetic scaffolds of allogeneic and xenogeneic origins, exhibiting innate tissue-specific characteristics. We explored a physico-chemical 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 the biocompatibility of dPPM. Haematoxylin and Eosin staining showed an evident reduction in nuclei in dPPM, confirmed with nuclear staining and analysis (****p < 0.0001). Sulphated glycosaminoglycans (sGAG) and collagen histology demonstrated minimal disruption to the structural assembly of the 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 of fibre orientations (***p < 0.001) in dPPM versus native ECM. DNA quantification indicated [&ge;] 85% reduction in native nuclear dsDNA in dPPM (**p < 0.001). Collagen and sGAG quantification indicated reductions of both (**p < 0.001). dPPM displayed increased membrane thickness (*p < 0.05). However, the Youngs modulus (447.8 {+/-} 41.9 kPa) and ultimate tensile strength (5080 {+/-} 2034.5 kPa) of dPPM were comparable with that of native controls at (411.3 {+/-} 8.1 kPa) and (3933.3 {+/-} 1734.5), respectively. In vitro cytotoxicity and scaffold biocompatibility assays demonstrated robust human mesothelial cell line (MeT-5A) attachment and viability. Here, we define a decellularisation protocol for porcine pleura that represents a step forward in their potential application as bioscaffolds in lung tissue engineering. Impact statementDesign and development of off the shelf tissue engineered products can rely on decellularisation of native tissue that can be functionalised with cell ingress from surrounding host microenvironment. We describe a reproducible decellularisation method for porcine pleural membranes. Histology, mechanical testing, and biocompatibility studies demonstrated protocol efficiency in adequate removal of native tissue cellularity and retention of gross microarchitecture and bioactivity in the decellularised pleura. The study represents a step forward in establishing an off the shelf potential of decellularised pleura as site-specific mechanical barriers in curtailing prolonged air leaks and promoting spontaneous tissue regeneration with relevant physiological cues.

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Vikranth, T., Dale, T. P., Forsyth, N. R.. 2023-06-23. Decellularisation and characterisation of porcine pleura for lung tissue engineering. https://doi.org/10.1101/2023.06.20.545830

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