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A2673 - Glycoproteins in Decellularized Lung Tissues - Novel Insights into Their Role for Bioengineering of Transplantable Organs
Author Block: F. E. Uhl1, R. A. Pouliot1, J. J. Uriarte1, F. Zhang2, R. Linhardt2, D. J. Weiss1; 1College of Medicine, University of Vermont, Burlington, VT, United States, 2Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, United States.
Rationale: Bioengineering approaches to overcome donor organ shortage for end stage chronic lung disease patients involve the decellularization and recellularization of human lungs. Up to date, no fully functional lung has been created mainly due to a lack of complete recellularizaton and long term functionality. Glycosaminoglycans (GAG) play critical roles in cell growth, differentiation, and function but the remaining GAGs and the matrikines in decellularized lung scaffolds are largely understudied. Understanding the role of both matrikine-dependent and matrikine-independent actions of GAGs remaining in the decellularized scaffolds will provide critical new information that will increase the likelihood of successful functional recellularization and of eventual therapeutic use. Methods: We analyzed the remaining GAG and matrikine composition of 3 native and decellularized human lungs from COPD and non-COPD patients. 3 different regions per lung were analyzed. Lungs were decellularized via a previously established protocol using Triton and sodium deoxycholate, lyophilized, and digested by actinase E. The GAGs were purified by MiniQ spin columns, desalted, further digested, 2-aminoacridone (AMAC)-labeled, and analyzed via liquid chromatography on an Agilent1200LC. Further, we cultivated cells in hydrogels produced from decellularized COPD and non-COPD lungs and in contact with specific GAGs (e.g. heparan sulfate (HS), chondroitin sulfate (CS)) to analyze the individual importance for cell attachment, proliferation, and differentiation. In order to analyze their matrikine binding capacity HS and CS were extracted from decellularized lungs, immobilized on streptavidin chips, and incubated with either TGFβ1, FGF2, or HGF. Binding was assessed by surface plasmon resonance (SPR) sensorgrams. Results: Decellularization lead to a significant differential loss in specific sidechain classes of both HS and CS. The GAG composition of the 3 different regions of the human lungs was fairly consistent demonstrating low intra-lung variability. Key matrikines like TGFβ1, FGF2, or HGF did not bind to decellularized lung homogenates paralleling a decrease in GAG sulfation due to decellularization. Lung epithelial cells cultivated with the addition of specific GAGs showed enhanced cell growth depending on the substrate used (CS>HS). These data suggest a critical role for both matrikine-dependent and -independent roles of selected GAGs in lung recellularization. Conclusions: Our study establishes how different factors influence the remaining GAG composition of decellularized lungs used for lung tissue engineering. The elucidation of how those GAGs are influencing cellular behavior will be beneficial during the process of recellularization. Better outcomes are predicted if GAGs are added back to decellularized scaffolds before cell seeding.