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In vivo, cells interact with the extracellular matrix (ECM), which provides a multitude of biophysical and biochemical signals that modulate cellular behavior. Inspired by this, we explored a new methodology to develop a more physiomimetic polysaccharide-based matrix for 3D cell culture. Maleimide-modified alginate (AlgM) derivatives were successfully synthesized using DMTMM to activate carboxylic groups. Thiol-terminated cell-adhesion peptides were tethered to the hydrogel network to promote integrin binding. Rapid and efficient in situ hydrogel formation was promoted by thiol-Michael addition "click" chemistry via maleimide reaction with thiol-flanked protease-sensitive peptides. Alginate derivatives were further ionically crosslinked by divalent ions present in the medium, which led to greater stability and allowed longer cell culture periods. By tailoring alginate's biofunctionality we improved cell-cell and cell-matrix interactions, providing an ECM-like 3D microenvironment. We were able to systematically and independently vary biochemical and biophysical parameters to elicit specific cell responses, creating custom-made 3D matrices. DMTMM-mediated maleimide incorporation is a promising approach to synthesizing AlgM derivatives that can be leveraged to produce ECM-like matrices for a broad range of applications, from in vitro tissue modeling to tissue regeneration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Alginate (ALG) is a widely used biomaterial to create artificial extracellular matrices (ECM) for tissue engineering. Since it does not degrade in the human body, imparting proteolytic sensitivity to ALG hydrogels leverages their properties as ECM-mimics. Herein, we explored the strain-promoted azide-alkyne cycloaddition (SPAAC) as a biocompatible and bio-orthogonal click-chemistry to graft cyclooctyne-modified alginate (ALG-K) with bi-azide-functionalized PVGLIG peptides. These are sensitive to matrix metalloproteinase (MMP) and may act as crosslinkers. The ALG-K-PVGLIG conjugates (50, 125, and 250 μM PVGLIG) were characterized for peptide incorporation, crosslinking ability (double-end grafting), and enzymatic liability. For producing cell-permissive multifunctional 3D matrices for dermal fibroblast culture, oxidized ALG-K was grafted with PVGLIG and with RGD peptides for cell-adhesion. SPAAC reactions were performed immediately before cell-laden hydrogel formation by secondary ionic-crosslinking, considerably reducing the steps and time of preparation. Hydrogels with intermediate PVGLIG concentration (125 μM) presented slightly higher stiffness while promoting extensive cell spreading and higher degree of cell-cell interconnections, likely favored by cell-driven proteolytic remodeling of the network. The hydrogel-embedded cells were able to produce their own pericellular ECM, expressed MMP-2 and 14, and secreted PVGLIG-degrading enzymes. By recapitulating key ECM-like features, these hydrogels provide biologically relevant 3D matrices for soft tissue regeneration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)