Reengineering 3D cellular-scale stiffness gradient at the enthesis with biomimetic hydrogels to promote in situ chondrogenesis.

Authors

Yukuan Wu, Lang Bai, Jin Tian, Qiaonan Liu, Yixiang Ai, Xiangyang Li, Feng Xu, Guoyou Huang, Zhanhai Yin

Published in

Biomaterials. Volume 338. Issue Pt A. Pages 124612. Sep 03, 2026. Epub Sep 03, 2026.

Abstract

The enthesis links tendon/ligament to bone, forming a soft-hard interface characterized by unique cellular-scale compositional and biomechanical gradients. Enthesis injuries are common and increase with age, yet the lack of biomaterials that faithfully mimic its cellular-scale gradient mechanical environment hinders understanding of the mechanobiological basis of this vulnerability. We showed that the enthesis cellular-scale stiffness gradient progressively flattens with aging. This age-dependent mechanical remodeling diminishes chondrogenesis and may predispose the attachment site to degeneration. To investigate this, we developed a simple, tunable, and cytocompatible three-dimensional hydrogel capable of generating a sharp stiffness gradient (∼1 kPa μm^-1) within a physiologically relevant range (2-119 kPa) confined to the cellular scale. Using this platform, we found that the cellular-scale stiffness gradient directs enthesis resident stem cell chondrogenesis. Mechanistically, the adhesion sensor integrin β1/FAK and the mechanosensitive calcium channel TRPV4 converge on the PI3K/AKT pathway to activate the SOX9-driven chondrogenesis. FAK promotes TRPV4 channel opening, while TRPV4-mediated calcium influx amplifies FAK signaling. Furthermore, in an aged rat model of enthesis injury, restoration of young-like stiffness gradient with the cell-laden hydrogel enhanced chondrogenic responses and improved histological, imaging-based, and functional healing outcomes. These findings identify the age-dependent cellular-scale stiffness gradient as an important mechanical feature regulating cellular differentiation and highlight potential strategies for enthesis regeneration.

PMID:
42702118
Bibliographic data and abstract were imported from PubMed on 07 Sep 2026.

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