A bioinspired mineralized collagen scaffold promotes enthesis healing and activates <i>Gli1</i> expression in preclinical models

The enthesis, a fibrocartilaginous tissue connecting tendon or ligament to bone, is critical for joint movement but lacks regenerative capacity after injury. Current clinical treatments for enthesis healing remain limited. Here, with a resolution of 2 to 3 nanometers, we found that mineral particles form a continuous cross-fibrillar phase with a discontinuous distribution in the fibrocartilage layer. Building on this finding, we developed a series of bioinspired mineralized collagen matrices, characterized by both intra- and extrafibrillar localization of crystallites, with a tunable mass percentage of inorganic content as scaffolds for enthesis repair. Our results revealed that mineralized collagen with controlled inorganic content (33% mineral content) facilitated fibrocartilage healing across multiple animal enthesis injury models, including mice, rats, rabbits, and goats. In direct comparisons with other biomaterials in a rabbit model, the bioinspired mineralized collagen resulted in 82% fibrocartilage width recovery, more than two times the healing observed with other materials. Treatment with the bioinspired mineralized collagen scaffold produced joint healing with an ability to sustain a higher maximum load in both rat and rabbit models, with the animals able to walk normally. The goat model exhibited an improvement in jumping ability. Mechanistically, we found that the bioinspired mineralized collagen modulated Hedgehog signaling intensity in a mineralization-dependent manner, which in turn up-regulated Gli1 expression. This modulation regulated the differentiation of mesenchymal progenitor cells and promoted fibrocartilage healing. Overall, we demonstrate that a bioinspired mineralized collagen scaffold effectively promotes enthesis injury repair, demonstrating potential for clinical translation.