Bio-inspired mineralization collagen induce fibrocartilage regeneration after tendon-bone injury by activating Gli1+Dkk3+ progenitor cells

A fibrocartilaginous connection between the tendon and bone, plays a critical role in transferring force from muscle to bone to enable joint movement. However, due to the high mechanical stress it experiences, the enthesis is vulnerable to injury and incapable of regenerating. The spatial relationship and functional basis of the principal components of the fibrocartilage - mineral and collagen - have not been clearly elucidated, which is a significant remaining gap in reconstructing complex architectures for promoting interface tissue regeneration. Here, using three-dimensional electron tomography imaging and high-resolution two-dimensional electron microscopy, we discover that mineral particles form a continuous cross-fibrillar phase within the fibrocartilage region. By developing a "floating mineralization" system, we fabricate a three-layer hydrogel that mimics the hierarchical nano- to micro-scale structure of tendon-bone interface (TBI). The middle layer is noteworthy for its resemblance to the nanostructure of fibrocartilage and its superior ability to induce mineralized fibrochondrogenesis in vitro. Based on motor function analysis, imaging diagnosis, histological staining, immunofluorescence staining, and biomechanics performance, we demonstrate that in situ transplantation of the gradient hydrogel achieved tendon-fibrocartilage-bone synchronous regeneration and result in 68% maximum mechanical recovery at 8-week postoperation. Single-cell RNA sequencing analysis reveals that a unique atlas of in situ stem/progenitor cells is generated during the TBI healing in vivo. Notably, the bio-inspired hydrogel microenvironment drived endogenous Gli1+Dkk3+ progenitor cells, playing a key role in TBI regeneration. Therefore, we have successfully decoded and reconstructed the nanostructure of fibrocartilage, which has great potential in TBI regeneration.