Anjani Ravindra1, William D'Angelo2, Li Zhang2, Janet Reing2, Scott Johnson2, Michael Myerburg3, Stephen F Badylak4. 1. Department of Pediatrics, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania. 2. McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania. 3. Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, UPMC Montefiore Hospital, Pittsburgh, Pennsylvania. 4. McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Surgery, University of Pittsburgh, UPMC Presbyterian Hospital F1281, Pittsburgh, Pennsylvania; Department of Bioengineering, University of Pittsburgh, Benedum Hall, Pittsburgh, Pennsylvania. Electronic address: thomasre@upmc.edu.
Abstract
BACKGROUND: Extracellular matrix (ECM) bioscaffolds produced by decellularization of source tissue have been effectively used for numerous clinical applications. However, decellularized tracheal constructs have been unsuccessful due to the immediate requirement of a functional airway epithelium on surgical implantation. ECM can be solubilized to form hydrogels that have been shown to support growth of many different cell types. The purpose of the present study is to compare the ability of airway epithelial cells to attach, form a confluent monolayer, and differentiate on homologous (trachea) and heterologous (urinary bladder) ECM substrates for potential application in full tracheal replacement. MATERIALS AND METHODS: Porcine tracheas and urinary bladders were decellularized. Human bronchial epithelial cells (HBECs) were cultured under differentiation conditions on acellular tracheal ECM and urinary bladder matrix (UBM) bioscaffolds and hydrogels and were assessed by histology and immunolabeling for markers of ciliation, goblet cell formation, and basement membrane deposition. RESULTS: Both trachea and urinary bladder tissues were successfully decellularized. HBEC formed a confluent layer on both trachea and UBM scaffolds and on hydrogels created from these bioscaffolds. Cells grown on tracheal and UBM hydrogels, but not on bioscaffolds, showed positive-acetylated tubulin staining and the presence of mucus-producing goblet cells. Collagen IV immunolabeling showed basement membrane deposition by these cells on the surface of the hydrogels. CONCLUSIONS: ECM hydrogels supported growth and differentiation of HBEC better than decellularized ECM bioscaffolds and show potential utility as substrates for promotion of a mature respiratory epithelium for regenerative medicine applications in the trachea.
BACKGROUND: Extracellular matrix (ECM) bioscaffolds produced by decellularization of source tissue have been effectively used for numerous clinical applications. However, decellularized tracheal constructs have been unsuccessful due to the immediate requirement of a functional airway epithelium on surgical implantation. ECM can be solubilized to form hydrogels that have been shown to support growth of many different cell types. The purpose of the present study is to compare the ability of airway epithelial cells to attach, form a confluent monolayer, and differentiate on homologous (trachea) and heterologous (urinary bladder) ECM substrates for potential application in full tracheal replacement. MATERIALS AND METHODS: Porcine tracheas and urinary bladders were decellularized. Human bronchial epithelial cells (HBECs) were cultured under differentiation conditions on acellular tracheal ECM and urinary bladder matrix (UBM) bioscaffolds and hydrogels and were assessed by histology and immunolabeling for markers of ciliation, goblet cell formation, and basement membrane deposition. RESULTS: Both trachea and urinary bladder tissues were successfully decellularized. HBEC formed a confluent layer on both trachea and UBM scaffolds and on hydrogels created from these bioscaffolds. Cells grown on tracheal and UBM hydrogels, but not on bioscaffolds, showed positive-acetylated tubulin staining and the presence of mucus-producing goblet cells. Collagen IV immunolabeling showed basement membrane deposition by these cells on the surface of the hydrogels. CONCLUSIONS: ECM hydrogels supported growth and differentiation of HBEC better than decellularized ECM bioscaffolds and show potential utility as substrates for promotion of a mature respiratory epithelium for regenerative medicine applications in the trachea.
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