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bioRxiv · 10.64898/2026.07.02.736090

Bioorthogonal Tuning of Hydrogel Stiffness Promotes Zonal Redifferentiation of Passaged Chondrocytes

Manzoni, T. J.·Natu, A.·Caputo, J. E.·Ho, A.·Ewine, I.·Smull, L.·Fang, Y.·Fox, J. M.·Su, A. W.·Jia, X.·Parreno, J.
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Abstract

Generating bioengineered cartilage that recapitulates the depth-dependent phenotype, structure, and function of native articular cartilage remains a challenge. While cartilage is rich in aggrecan and type II collagen, proper function depends on depth-dependent protein expression. Superficial zone chondrocytes (SZCs) secrete proteoglycan-4 (PRG4) to lubricate the cartilage surface. Deep zone chondrocytes produce type X collagen (COLX) to support compressive loading and load transfer to subchondral bone. We previously demonstrated that passaged full-thickness chondrocytes (FTCs) and zonal chondrocytes can re-express cartilage and zone-specific markers following scaffold-free three-dimensional (3D) culture in redifferentiation media. However, in the absence of an instructive matrix, cells expressed low levels of zone-specific proteins and exhibited limited depth-dependent organization. We hypothesize that synthetic extracellular matrix with zone-specific microenvironmental cues will guide zonal differentiation. To this end, passaged primary bovine chondrocytes were encapsulated in a soft, hyaluronan (HA)-based, cell-adhesive, and protease-degradable hydrogel established via bioorthogonal tetrazine (Tz) ligation with norbornene (Nb). When supplemented with TGF{beta}3, FTCs deposited aggrecan and type II collagen with minimal type I collagen. Application of interfacial tetrazine ligation with trans-cyclooctene (TCO) during cell culture resulted in matrix stiffening, leading to upregulation of COLX expression. Conversely, SZCs cultured in soft hydrogels exhibited the greatest PRG4 expression. Establishment of a trilayered construct with region-specific stiffness via the diffusion-controlled reaction promoted PRG4 and COLX expression in defined zones. Together, these findings demonstrate that tunable HA-based hydrogels can enhance zone-specific chondrocyte phenotypes and promote the formation of zonally organized cartilage.

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Manzoni, T. J., Natu, A., Caputo, J. E., Ho, A., Ewine, I., Smull, L., Fang, Y., Fox, J. M., Su, A. W., Jia, X., Parreno, J.. 2026-07-03. Bioorthogonal Tuning of Hydrogel Stiffness Promotes Zonal Redifferentiation of Passaged Chondrocytes. https://doi.org/10.64898/2026.07.02.736090

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