Poster N5 - Presenting ED-A fibronectin in a bioorthogonally crosslinked hydrogel to control myofibroblast differentiation

Introduction: Vocal fold scarring presents a significant public health challenge. Scarred vocal folds exhibit altered matrix composition and compromised vibratory capability.[1] There is a critical need for the development of an engineered model of vocal fold scarring that is physiologically relevant and can be used to test new treatment options. Fibronectin (FN) is a major structural protein in the extracellular matrix (ECM) of the vocal fold lamina propria. It is involved in cell adhesion, spreading, migration and proliferation. One of the isoforms of fibronectin includes the type III domain called Extra Domain A (ED-A) in its structure. The ED-A domain plays a critical in wound healing and tissue fibrosis.[2] The profibrotic effects of the ED-A domain have been correlated with involvement of EDA-FN in myofibroblast differentiation. It has been shown that the ED-A segment by itself is sufficient to induce myofibroblast differentiation by interacting with integrins α4β7 and α4β1[3,4]. Here, we synthesized hyaluronic acid (HA)-based hydrogels with covalently conjugated ED-A FN. We examined how the ED-A FN epitope effects transforming growth factor beta 1 (TGFβ1)-induced myofibroblast differentiation using primary porcine vocal fold fibroblasts (VFFs).

Materials and

Methods: The hydrogels were established via tetrazine ligation with slow (norbornene, Nb) and fast dienophiles (trans-cyclooctene, TCO). Hydrogel precursors, including tetrazine modified HA (HA-Tz), a norbornene (Nb) modified matrix metalloproteinase (MMP)-degradable peptide crosslinker (SMR-bisNb) and TCO modified cell adhesive peptide (RGD-TCO) was synthesized following established protocols.[5] Cysteine-tagged ED-A-FN mimetic peptide sequence (EDG) was synthesized via solid phase peptide synthesis. Cysteine-tagged recombinant ED-A FN (Type III Domain 11-FN Domain EDA) was produced by transforming the plasmid into E. coli strain BL21 (DE3). Purified EDG peptide and ED-A protein were then reacted with Sulfo-TCO Maleimide to conjugate TCO reactive sites. The purity of the TCO-conjugated peptide and protein was assessed using ESI-MS. Primary porcine VFFs, isolated from freshly harvested porcine larynxes, were cultured in HA gels in the presence of TGFβ1 (10 ng/mL) as previously reported.[5] Cultures were characterized by qPCR and immunofluorescence to assess myofibroblast differentiation.

Results, Conclusions, and Discussions: Results and discussion: TCO modified ED-A-FN mimetic peptide and protein were successfully synthesized. The UPLC trace and the ESI-MS spectrum (Fig. 1) confirmed the purity of the TCO conjugated peptide and protein. Immunoblot assay showed successful inclusion of the ED-A domain in the recombinantly produced protein construct. UV-vis analysis confirmed the high reactivity of the TCO group towards tetrazines dangling from HA backbone. MMP-degradable hydrogels with 1 mM RGD and varying amounts (0-1 mM) of EDG or ED-A protein were prepared via diffusion controlled interfacial bioorthogonal conjugation. Porcine VFFs encapsulated in the gel constructs showed greater than 85% viability at Day 14. Preliminary data suggest that ED-A FN signals synergize with TGFβ1 to promote myofibroblast differentiation.

Conclusion: EDA-FN mimetic peptide and recombinant protein were synthesized and conjugated to HA-based hydrogels via tetrazine ligation. VFFs maintained high viability in the 3D environment. Work is underway to study signalling pathways involved in myofibroblast differentiation.

Acknowledgements (Optional): This work was supported by National Institutes of Health (R01DC014461).

References: [1] Friedrich+ Eur Arch Otorhinolaryngol 270, 2491–2507 (2013). [2] Muro+ Am J Respir Crit Care Med 2008, 177 (6), 638–645. [3] Kohan+ The FASEB Journal 2010, 24 (11), 4503–4512. [4] Shinde+ Matrix Biology 2015, 41, 26–35. [5] Song+ ACS Appl. Mater. Interfaces 2022, 14, 46, 51669–51682