No abstract

Poly(-isopropyl acrylamide) (pNIPAM) is a stimulus-responsive polymer that has been of great interest to the bioengineering community. When the temperature is lowered below its lower critical solution temperature (∼32 °C), pNIPAM rapidly hydrates, and adherent cells detach as intact cell sheets. This cell-releasing behavior in a physiologically relevant temperature range has led to NIPAM's use for engineered tissues and other devices. In a previous study, however, the authors found that although most techniques used to polymerize NIPAM yield biocompatible films, some formulations from commercially-available NIPAM (cpNIPAM) can be cytotoxic. In this work, the authors investigate the reasons underlying this anomaly. The authors evaluated the response of a variety of cell types (e.g., bovine aortic endothelial cells, BAECs; monkey kidney epithelial cells, Vero cells; and mouse embryonic fibroblasts, 3T3s) after culture on substrates spin-coated with sol-gel (spNIPAM) and commercially-prepared (cpNIPAM). The relative biocompatibility of each cell type was evaluated using observations of its cell morphology and function (e.g., XTT and Live/Dead assays) after 48 and 96 h in culture. In addition, the substrates themselves were analyzed using NMR, goniometry, and XPS. The authors find that all the cell types were compromised by 96 h in culture with cpNIPAM, although the manner in which the cells are compromised differs; in particular, while Vero and 3T3 cells appear to be undergoing cytotoxic death, BAECs undergo apoptic death. The authors believe that this result is due to a combination of factors, including the presence of short chain oligomers of NIPAM in the commercially-available preparation. This work will provide valuable insights into the cytotoxicity of commercially-prepared polymer substrates for this type of bioengineering work and therefore into the applicability of cells grown on such surfaces for human subjects.

Fibrotic lung involvement contributes to sarcoidosis fatality in western countries, afflicting up to 20% of patients with this condition. To date, development of fibrosis in sarcoidosis is poorly understood and often unpredictable, with persistent progression in some patients and spontaneous remission in others. Previous studies suggest that fibrotic sarcoidosis is driven by immune cell activity; however, there have been no studies exploring the influence of sarcoidosis lung extracellular matrix (ECM) on immune cell phenotype. The use of decellularization techniques produces scaffolds that preserve the architecture, biochemical components, and mechanical properties of the lung and can be utilized to model the microenvironment of lungs in diseases such as IPF, scleroderma, and COPD. However, this approach has not yet been applied to the study of sarcoidosis lungs. Here we characterized the architectural and biochemical aspects of decellularized stage IV sarcoidosis ECM and developed a culture system utilizing these decellularized scaffolds to investigate the fibrotic progression of immune cells in response to the diseased lung microenvironment. Methods: Explanted lung tissue obtained at autopsy was used to generate control lung scaffolds (CLS) and stage IV sarcoidosis lung scaffolds (SLS) through well characterized detergent-based decellularization methods. Histologic evaluation, LC/MS-based proteomics, and immunofluorescence were used to assess the anatomic and biochemical composition of the scaffolds. Peripheral blood mononuclear cells (PBMCs) isolated from subjects with Stage IV sarcoidosis were cultured in the CLS and SLS for 24 hours, 7 days, and 14 days of culture at which point their ability to adopt a mesenchymal phenotype was assessed through CD45 and pro-collagen Iα1 (pro-coll Iα1) staining. Results: Relative to CLS, SLS showed altered tissue architecture and changes in biochemical composition consistent with accelerated ECM turnover including increased detection and activity of metalloproteinases, decreased detection of basement membrane components such as collagen IV and laminin, and increased expression of proteins related to adhesion such as fibronectin and periostin. Functionally, these attributes combined to augment detection of PBMCs-derived CD45+ pro-coll Iα1+ cells after 7 days of culture. Conclusions: SLS demonstrate differences in biochemical composition reflecting ongoing ECM remodeling and enhanced cellular adhesion that are associated with a mesenchymal phenotype in cultured leukocytes. These factors may contribute to the development of pulmonary fibrosis in stage IV sarcoidosis lungs.