Dale, T. P. | bioRxiv Science

bioRxiv2023

Decellularisation and characterisation of porcine pleura for lung tissue engineering

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 [≥] 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.

bioengineering↗

bioRxiv2022

Hypoxic conditions promote a proliferative, poorly differentiated, and pro-secretory phenotype in COPD lung tissue progenitor cells in vitro

Chronic obstructive pulmonary disease patients experience variable symptoms dependent on the presence of an emphysematous versus a chronic bronchitis phenotype. Both presentations can be associated with lung tissue and systemic hypoxia, at its most severe leading to Cor pulmonale. Despite this, minimal attention has been given to the effects of hypoxia at the cellular disease level. We isolated and cultured progenitor cells from the distal lung tissue of a 64 year-old, male, emphysematous donor in ambient (21%) and hypoxic (2%) oxygen conditions. Proliferative capacity was determined on collagen coated culture plastic and growth-inactivated 3T3-J2 co-cultures. Epithelial (E-cadherin and pan-cytokeratin) and progenitor (TP63, cytokeratin 5) marker expression were examined. Expanded cells were differentiated at air-liquid interface and ciliated, mucous producing, and club cell populations identified. Isolated cells were positive for the epithelial, pan-cytokeratin and E-cadherin, and progenitor, TP63 and cytokeratin 5, cell markers at isolation and again at passage 5. Passage 5 expanded cells in hypoxia had increased the proportion of TP63 expressing cells by 10% from 51.6 {+/-} 1.2% to 62.6 {+/-} 2.3% (p [≤] 0.01). Proliferative capacity was greater in 3T3J2 co-cultured cells overall and in 2% oxygen this supported the emergence of a proliferation unrestricted population with a limited differentiation capacity. Cells expanded on collagen I in either oxygen underwent differentiation having been expanded with the production of ciliated cells positive for {beta}IV tubulin, and mucin 5ac, mucin 5b and CC10 positive secretory cells. Epithelial barrier formation was reduced significantly (p [≤] 0.0001) in hypoxia-expanded cells compared to normoxia. qRT-PCR showed higher expression of mucins in 2% expanded cells, significantly so with MUC5B (P [≤] 0.05) although mucin protein secretion was greater in 21% expanded cells. Concomitantly these results demonstrate that hypoxia promotes a proliferative phenotype while reducing the overall differentiation capacity of the cells. Further, the retained differentiation potential becomes skewed to a more secretory phenotype demonstrating that hypoxia may be contributing to disease symptom and severity in COPD patients.

physiology↗