Aqueous two-phase deposition and fibrinolysis of fibroblast-laden fibrin micro-scaffolds

• Type of Material: Article
• Authors: Stephen Robinson, Georgia Institute of Technology; Jonathan Chang, Georgia Institute of Technology; Eric Parigoris, Georgia Institute of Technology; Louise Hecker, Emory University; Shuichi Takayama, Emory University
• Language: English
• Date: 2021-07-01
• Publisher: IOP PUBLISHING LTD
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2021 IOP Publishing Ltd
• License: Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1088/1758-5090/abdb85
• Title of Journal or Parent Work: BIOFABRICATION
• Volume: 13
• Issue: 3
• Grant/Funding Information: National Science Foundation Graduate Research Fellowship Program to J.C. and E.P. (DGE-1650044).; NIH (R21 AG061687 and R01 HL136141); Defense Threat Reduction Agency (DTRA) under Space and Naval Warfare Systems Center Pacific (SSC PACIFIC) Contract No. N66001-13-C-2027.
• Keywords: aqueous two-phase system; fibrinolysis; phenotypic assay; Materials Science; wound healing; Materials Science, Biomaterials; Science & Technology; Engineering; fibrin; fibrosis; Engineering, Biomedical; Technology

Abstract

This paper describes printing of microscale fibroblast-laden matrices using an aqueous two-phase approach that controls thrombin-mediated enzymatic crosslinking of fibrin. Optimization of aqueous two-phase formulations enabled polymerization of consistent sub-microliter volumes of cell-laden fibrin. When plasminogen was added to these micro-scaffolds, the primary normal human lung fibroblasts converted it to plasmin, triggering gradual degradation of the fibrin. Time-lapse live-cell imaging and automated image analysis provided readouts of time to degradation of 50% of the scaffold as well as maximum degradation rate. The time required for degradation decreased linearly with cell number while it increased in a dose-dependent manner upon addition of TGF-β1. Fibroblasts isolated from idiopathic pulmonary fibrosis patients showed similar trends with regards to response to TGF-β1 stimulation. Addition of reactive oxygen species (ROS) slowed fibrinolysis but only in the absence of TGF-β1, consistent with published studies demonstrating that pro-fibrotic cellular phenotypes induced by TGF-β1 are mediated, at least in part, through increased production of ROS. FDA-approved and experimental anti-fibrosis drugs were also tested for their effects on fibrinolysis rates. Given the central role of fibrinolysis in both normal and pathogenic wound healing of various tissues, the high-throughput cell-mediated fibrinolysis assay described has broad applicability in the study of many different cell types and diseases. Furthermore, aqueous two-phase printing of fibrin addresses several current limitations of fibrin bio-inks, potentially enabling future applications in tissue engineering and in vitro models.