Encoding Stem-Cell-Secreted Extracellular Matrix Protein Capture in Two and Three Dimensions Using Protein Binding Peptides Hadi Hezaveh, †,§ Steffen Cosson, †,§ Ellen A. Otte, †,§ Guannan Su, †,§ Benjamin D. Fairbanks, § and Justin J. Cooper-White* ,†,‡,§ † Tissue Engineering and Microfluidics Laboratory, The Australian Institute for Bioengineering and Nanotechnology (AIBN), and ‡ School of Chemical Engineering, University of Queensland, St. Lucia QLD Australia § Manufacturing Flagship, Commonwealth Scientific and Industrial Research Organization (CSIRO), Clayton, Victoria 3169, Australia * S Supporting Information ABSTRACT: Capturing cell-secreted extracellular matrix (ECM) proteins through cooperative binding with high specificity and affinity is an important function of native tissue matrices during both tissue homeostasis and repair. However, while synthetic hydrogels, such as those based on poly(ethylene glycol) (PEG), are often proposed as ideal materials to deliver human mesenchymal stem cells (hMSCs) to sites of injury to enable tissue repair, they do not have this capabilitya capability that would enable cells to actively remodel their local extracellular microenvironment and potentially provide the required feedback control for more effective tissue genesis. In this work, we detail a methodology that engenders poly- (ethylene glycol) (PEG)-based two-dimensional substrates and three-dimensional porous hydrogels with the ability to capture desired extracellular matrix (ECM) proteins with high specificity. This “encoded” ECM protein capture is achieved by decorating the PEG-based materials with protein binding peptides (PBPs) synthesized to be specific in their binding of fibronectin, laminin, and collagen I, which are not only the most omnipresent ECM proteins in human tissues but, as we confirmed, are also secreted to differing extents by hMSCs under in vitro maintenance conditions. By encapsulating hMSCs into these PBP-functionalized hydrogels, and culturing them in protein-free maintenance media, we demonstrate that these PBPs not only actively recruit targeted ECM proteins as they are secreted from hMSCs but also retain them to much higher levels compared to nonfunctionalized gels. This novel approach thus enables the fabrication of encoded surfaces and hydrogels that capture cell- secreted proteins, with high specificity and affinity, in a programmable manner, ready for applications in many bioengineering applications, including bioactive surface coatings, bioassays, stem cell culture, tissue engineering, and regenerative medicine. 1. INTRODUCTION Extracellular matrix (ECM) proteins are an essential component of any stem cell niche within tissues, as they directly (through integrin-mediated interactions) and indirectly (through co-operative binding interactions with other ECM components) modulate the maintenance, proliferation, self- renewal, and differentiation of stem cells. 1 This dependence on ECM composition is paralleled in vitro, where it is clear that mesenchymal stem cells (MSCs) produce a range of different ECM proteins as they differentiate (in response to inductive media) into defined tissue cell types. 2 Furthermore, it has also been shown that the type and amount of ECM presented to them during in vitro culture can actively drive these stem cells to bias fate choice, including toward specific differentiated cell types. 3 In an effort to take advantage of this “feed-forward” control loop, biomaterial scientists have for some time incorporated full length ECM proteins, or fragments/peptides thereof, into synthetic surfaces or hydrogels to create both 2D and 3D biosynthetic microenvironments representative (in terms of ECM composition and architecture) of different tissues or stem cell niches. 4,5 In this endeavor, poly(ethylene glycol) (PEG)- based biomaterials are one of the more commonly utilized systems due to their favorable property slate, including their bioinert and nonfouling properties 6 as well as the ease with which end-group functionalization can be achieved. Functionalization of PEG chain ends can be utilized to introduce cross-linking moieties to enable facile formation of hydrogels from linear or branched PEG macromers. A plethora of cross-linking schemes have been developed 7 in order to generate hydrogels that mimic biochemical and/or biophysical matrix properties of the ECM. Of these, photoinitiated cross- linking offers temporal and spatial control over gelation, allowing mechanical and/or chemical patterning of PEG Received: October 14, 2017 Revised: January 18, 2018 Published: February 13, 2018 Article pubs.acs.org/Biomac Cite This: Biomacromolecules 2018, 19, 721-730 © 2018 American Chemical Society 721 DOI: 10.1021/acs.biomac.7b01482 Biomacromolecules 2018, 19, 721-730