No abstract

Cartilage tissue engineering strategies often rely on hydrogels with fixed covalent crosslinks for chondrocyte encapsulation; yet the resulting material properties are largely elastic and can impede matrix deposition. To address this limitation, hydrazone crosslinked poly(ethylene glycol) hydrogels were formulated to achieve tunable viscoelastic properties and to study how chondrocyte proliferation and matrix deposition vary with the time-dependent material properties of covalent adaptable networks. Hydrazone equilibrium differences were leveraged to produce average stress relaxation times from hours (4.01×103s) to months (2.78×106s) by varying the percentage of alkyl-hydrazone (aHz) to benzyl-hydrazone (bHz) crosslinks. Swelling behavior and degradation associated with adaptability was characterized to quantify temporal network changes that can influence the behavior of encapsulated chondrocytes. After four weeks, mass swelling ratios varied from 36±3 to 17±0.4 and polymer retention ranged from 46±4% to 92±5%, with higher aHz content leading to loss of network connectivity with time. Hydrogels were formulated near the Flory-Stockmayer bHz percolation threshold (17% bHz) to investigate chondrocyte response to distinct levels of covalent architecture adaptability. Four weeks post-encapsulation, formulations with average relaxation times of 3 days (2.6×105s) revealed increased cellularity and an interconnected articular cartilage-specific matrix. Chondrocytes embedded in this adaptable formulation (22% bHz) deposited 190±30% more collagen and 140±20% more sulfated glycosaminoglycans compared to the 100% bHz control, which constrained matrix deposition to pericellular space. Collectively, these findings indicate that incorporating highly adaptable aHz crosslinks enhanced regenerative outcomes. However, connected networks containing more stable bHz bonds were required to achieve the highest quality neocartilaginous tissue.

Mechanobiological cues influence chondrocyte biosynthesis and are often used in tissue engineering applications to improve the repair of articular cartilage in load‐bearing joints. In this work, the biophysical effects of an applied dynamic compression on chondrocytes encapsulated in viscoelastic hydrazone covalent adaptable networks (CANs) is explored. Here, hydrazone CANs exhibit viscoelastic loss tangents ranging from (9.03 ± 0.01) 10–4 to (1.67 ± 0.09) 10–3 based on the molar percentages of alkyl‐hydrazone and benzyl‐hydrazone crosslinks. Notably, viscoelastic alkyl‐hydrazone crosslinks improve articular cartilage specific gene expression showing higher SOX9 expression in free swelling hydrogels and dynamic compression reduces hypertrophic chondrocyte markers (COL10A1, MMP13) in hydrazone CANs. Interestingly, dynamic compression also improves matrix biosynthesis in elastic benzyl‐hydrazone controls but reduces biosynthesis in viscoelastic alkyl‐hydrazone CANs. Additionally, intermediate levels of viscoelastic adaptability demonstrate the highest levels of matrix biosynthesis in hydrazone CANs, demonstrating on average 70 ± 4 µg of sulfated glycosaminoglycans per day and 31 ± 3 µg of collagen per day over one month in dynamic compression bioreactors. Collectively, the results herein demonstrate the role of matrix adaptability and viscoelasticity on chondrocytes in hydrazone CANs during dynamic compression, which may prove useful for tissue engineering applications in load‐bearing joints.

Adaptable dynamic covalent crosslinks temporally modulate the biophysical transmission of physiologically relevant compressive strains to encapsulated chondrocytes for cartilage tissue engineering.