Dose and Timing of N-Cadherin Mimetic Peptides Regulate MSC Chondrogenesis within Hydrogels

The transmembrane glycoprotein N-cadherin (NCad) mediates cell-cell interactions found during mesenchymal condensation and chondrogenesis. Here, NCad-derived peptides (i.e., HAV) are incorporated into hyaluronic acid (HA) hydrogels with encapsulated mesenchymal stem cells (MSCs). Since the dose and timing of NCad signaling are dynamic, HAV peptide presentation is tuned via alterations in peptide concentration and incorporation of an ADAM10-cleavable domain between the hydrogel and the HAV motif, respectively. HA hydrogels functionalized with HAV result in dose-dependent increases in early chondrogenesis of encapsulated MSCs and resultant cartilage matrix production. For example, type II collagen and glycosaminoglycan production increase ≈9- and 2-fold with the highest dose of HAV (i.e., 2 × 10^-3 m), respectively, when compared to unmodified hydrogels, while incorporation of an efficient ADAM10-cleavable domain between the HAV peptide and hydrogel abolishes increases in chondrogenesis and matrix production. Treatment with a small-molecule ADAM10 inhibitor restores the functional effect of the HAV peptide, indicating that timing and duration of HAV peptide presentation is crucial for robust chondrogenesis. This study demonstrates a nuanced approach to the biofunctionalization of hydrogels to better emulate the complex cell microenvironment during embryogenesis toward stem-cell-based cartilage production.