{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,5,23]],"date-time":"2026-05-23T08:05:33Z","timestamp":1779523533185,"version":"3.53.1"},"posted":{"date-parts":[[2026,5,20]]},"group-title":"Bioengineering","reference-count":34,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,5,20]],"date-time":"2026-05-20T00:00:00Z","timestamp":1779235200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"name":"UW Endowment"},{"name":"NIH NIGMS","award":["R35GM143081"],"award-info":[{"award-number":["R35GM143081"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,5,20]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Hydrogels are widely used as three-dimensional cell culture systems to understand the impact of cellular mechanotransduction for tissue engineering applications. Photoinitiated thiol-ene click chemistry is a commonly utilized hydrogel crosslinking mechanism that provides spatial and temporal control over hydrogel network formation and resulting mesh size and compressive properties. Despite historically documented efficiency as step-growth reactions, these reactions do not always proceed as predicted. To understand the impact of cell confinement and microenvironmental mechanics on cellular function, thiol-ene network formation must be thoroughly characterized. To this end, the objective of this work was to investigate the crosslinking dynamics to determine hydrogel network formation as assessed via mesh size and mechanical properties using a pentenoate-functionalized hyaluronic acid thiol-ene reaction. Hydrogel parameters including polymer concentration and thiol:-ene crosslinker molar ratio were modulated (4, 6, or 8 polymer weight percent and 0.15:1, 0.5:1, or 1:1 molar ratio of thiol groups to reactive -ene groups) to tune network properties including shear storage modulus and relative mesh size. Molecular Dynamics (MD) simulations were used to simulate the thiol-ene crosslinking reaction and establish a method for predicting thiol-ene reaction efficiency. Lastly, the feasibility of this hydrogel system for\n                  <jats:italic>in vitro<\/jats:italic>\n                  modeling was confirmed via assessment of metabolic activity of encapsulated primary human meniscal cells.\n                <\/jats:p>","DOI":"10.64898\/2026.05.17.725744","type":"posted-content","created":{"date-parts":[[2026,5,20]],"date-time":"2026-05-20T18:26:37Z","timestamp":1779301597000},"source":"Crossref","is-referenced-by-count":0,"title":["Network Formation Dynamics in Thiol-ene Crosslinked Hyaluronic Acid Hydrogels: Design Principles for In Vitro Tissue Models"],"prefix":"10.64898","author":[{"given":"Kyley","family":"Burkey","sequence":"first","affiliation":[{"name":"Bioengineering Graduate Program, University of Kansas, Lawrence, KS, United States"},{"name":"Department of Orthopaedic Surgery & Sports Medicine, University of Washington, Seattle, WA, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yiwen","family":"Zheng","sequence":"additional","affiliation":[{"name":"Department of Mechanical Engineering, University of Washington, Seattle, WA, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kinsey","family":"Drake","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Washington, Seattle, WA, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryan","family":"Brady","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering, University of Washington, Seattle, WA, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0337-3577","authenticated-orcid":false,"given":"Cole A.","family":"DeForest","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Washington, Seattle, WA, United States"},{"name":"Department of Chemical Engineering, University of Washington, Seattle, WA, United States"},{"name":"Department of Bioengineering, University of Washington, Seattle, WA, United States"},{"name":"Institute for Protein Design, University of Washington, Seattle, WA, United States"},{"name":"Institute of Stem Cell & Regenerative Medicine, University of Washington, Seattle, WA, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alshakim","family":"Nelson","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Washington, Seattle, WA, United States"},{"name":"Molecular Engineering & Sciences Institute, University of Washington, Seattle, WA, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aniruddh","family":"Vashisth","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering, University of Washington, Seattle, WA, United States"},{"name":"Department Mechanical Engineering, University of Washington, Seattle, WA, United States"},{"name":"Department of Materials Science & Engineering, University of Washington, Seattle, WA, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jennifer","family":"Robinson","sequence":"additional","affiliation":[{"name":"Department of Orthopaedic Surgery & Sports Medicine, University of Washington, Seattle, WA, United States"},{"name":"Department of Mechanical Engineering, University of Washington, Seattle, WA, United States"},{"name":"Department of Bioengineering, University of Washington, Seattle, WA, United States"},{"name":"Institute of Stem Cell & Regenerative Medicine, University of Washington, Seattle, WA, United States"},{"name":"Molecular Engineering & Sciences Institute, University of Washington, Seattle, WA, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026052300250931000_2026.05.17.725744v1.1","doi-asserted-by":"crossref","first-page":"114","DOI":"10.3390\/gels10020114","article-title":"Advances in Hydrogels for Meniscus Tissue Engineering: A Focus on Biomaterials, Crosslinking, Therapeutic Additives","volume":"10","year":"2024","journal-title":"Gels"},{"key":"2026052300250931000_2026.05.17.725744v1.2","doi-asserted-by":"crossref","first-page":"4551","DOI":"10.1039\/D4SM01458H","article-title":"Designing hydrogel dimensionality to investigate mechanobiology","volume":"21","year":"2025","journal-title":"Soft Matter"},{"key":"2026052300250931000_2026.05.17.725744v1.3","doi-asserted-by":"publisher","DOI":"10.1002\/anie.200903924"},{"key":"2026052300250931000_2026.05.17.725744v1.4","doi-asserted-by":"crossref","first-page":"1900752","DOI":"10.1002\/adhm.201900752","article-title":"Thiol\u2013Gelatin\u2013Norbornene Bioink for Laser-Based High-Definition Bioprinting","volume":"9","year":"2020","journal-title":"Adv. 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