Purpose: Over the past decade, there has been a concerted effort to develop new artificial wound coverings that effectively treat major skin wounds. Hydrogels have emerged as novel drug delivery vehicles and wound dressing adjuncts, with a clinical dressing change frequency of 3 days given their unique moisture-conferring properties at the wound site. The topical application of fibronectin (FN) in a murine irradiated skin model has demonstrated improved wound healing by >20%. FN additionally has reported wound healing efficacy at the nanogram to microgram level. However, there is no reported application of hydrogel-facilitated FN delivery to promote wound healing. In this study, the in vitro and in vivo dynamics of 3D-transglutaminase-hydrogel (3DTgH) FN release were evaluated to characterize optimal parameters for the development of a novel wound dressing. Methods: 4.5A and 7.5A stiffness 3DTgH with 1 ug FN were constructed and plated in either PBS or collagenase type 2 (Col2) to allow gel digestion. Digested samples were assayed with FN ELISA. Additionally, full-thickness 8 mm punch biopsy dorsal skin wounds were created in 8-week-old FVB/NJ mice (n=22) weighing between 20 and 25 grams. Experimental mice (n=10) were treated with 4.5A stiffness 3DTgH incorporating 31.25 ug human FN (31.25 ul volume), and control mice (n=12) were treated with 4.5A stiffness 3DTgH incorporating 31.25 ul PBS. The total volume of each hydrogel treatment construct was 50 ul, and this suspension was applied topically onto the wound surface. For wound harvest on POD1, POD2, and POD3, residual hydrogel was carefully excised from the wound surface, and wounds were then harvested with 1 mm margins. Mouse tissue was homogenized and 100 ul samples were analyzed with human FN ELISA for pharmacokinetic (PK) analysis of FN release via hydrogel enzymatic digestion in vivo. Results: Unlike 3DTgH plated in PBS, 3DTgH plated in Col2 gradually dissolved. 4.5A 3DTgH maximally released FN on day 4 and 7.5A 3DTgH maximally released FN on day 13 with an overall slower rate of FN release. When compared with controls, mice treated with FN 3DTgH demonstrated sustained and increasing FN release via hydrogel after wound creation, with an average release of 125.31 ug FN on POD1 (p=0.0039), 141.91 ug on POD2 (p=0.0145), and 167.78 ug on POD3 (p=0.0001). Mice treated with control 3DTgH treatment did not display detectable levels of human FN via ELISA. Conclusion: 3DTgH is a suitable construct for gradual FN release in vitro and in vivo. Hydrogel constructs of differing stiffnesses demonstrate differential rates of FN release and thus have the potential to be tailored to wound healing environments for incremental FN delivery. As a result, hydrogel-based dressings incorporating FN have promise for clinical translation and sustained FN delivery in the treatment of skin wounds.