Hydrazone covalent adaptable networks modulate extracellular matrix deposition for cartilage tissue engineering

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.

Thiol-ene cross-linked alginate hydrogel encapsulation modulates the extracellular matrix of kidney organoids by reducing abnormal type 1a1 collagen deposition

“…Besides hydrogel stiffness, the relative relaxation time has been observed to affect type 1 collagen deposition. For example, the encapsulation of chondrocytes in hydrazone covalently adapatible network PEG hydrogels resulted in lower levels of type 1 collagen in hydrogels with faster relaxation times, with no effect on type 2 collagen [43,46]. Interestingly, tuning the hydrogel's mechanical properties (specifically stiffness or relaxation time) did not affect the expression and modulation of type 2 [46] or type 3 collagen [43] in these studies, which is similar to our observations for type 6 collagen.…”

Toward Stimuli‐Responsive Dynamic Thermosets through Continuous Development and Improvements in Covalent Adaptable Networks (CANs)

“…Further, this material system was applied to biophysical studies measuring cellular forces, wherein fundamental properties of viscoelastic materials were used to calculate the energy required by neuronal processes to travel through the stress‐relaxing network 78. Underscoring the importance of scaffold integrity in adaptable systems for cell culture, more recent work using hydrogels containing dynamic hydrazone crosslinks of varied stability indicates that moderate incorporation of slowly relaxing bonds is necessary for optimal tissue engineering,79 although rapidly degradable DCCs like thiyl–thiol chain transfer provide excellent opportunities for tissue culture and subsequent cell release 80. In efforts to prepare biochemically accurate mimics of native ECM, imine‐linked natural products from the cellular microenvironment were incorporated in adaptable hydrogels to facilitate cellular adhesion and enzyme‐responsiveness of the polymer network 38b,81.…”

The Plot Thickens: The Emerging Role of Matrix Viscosity in Cell Mechanotransduction

“…Moreover, fast relaxing gels allow the formation of an interconnected cartilage matrix by dissipating elastic stresses and undergoing plastic deformation . This role of stress relaxation in non‐adhesive hydrogels was recently confirmed by Richardson et al Using dynamic hydrazone crosslinking to modulate stress relaxation times in a range from hours to months, they found that an intermediate relaxation time of about 3 days prompted enhanced cellularity and cartilage matrix deposition within their gels.…”