Crosslinking stabilization strategy: A novel approach to cartilage-like repair of annulus fibrosus (AF) defects.

Lumbar disc degeneration due to annulus fibrosus (AF) defects poses a significant challenge in clinical treatment Current treatments exhibit limited repair efficacy and a high recurrence rate. To address this, we devised a novel approach of crosslinking stabilization strategy. We integrated fibrinogen, thrombin, genipin, and human bone marrow-derived mesenchymal stem cells (hBMSCs) hydrogel (FTGB) with acellular scaffold and fascia (FTGB@S@F) to remediate AF defects. FTIR analysis confirmed stable chemical crosslinking within the FTGB hydrogel. FTGB hydrogel demonstrated superior biocompatibility compared to the FB hydrogel, with significantly higher cell viability (97.60 ± 2.02 % vs 81.43 ± 4.50 %, P < 0.01) and enhanced proliferation and migration, as shown in DAPI, Edu and phalloidin staining. Atomic force microscopy (AFM) revealed that FTGB@S has a dense reticular structure, enhancing material performance with higher elastic modulus than FB@S. MTS testing showed that FTGB@S@F outperformed other groups in resisting cyclic axial load (25.53 ± 1.17 MPa) and maintaining disc height (0.57 ± 0.12 mm), with stable axial compression resistance and minimal deformation. It also exhibited the lowest rupture ROM (1.45 ± 0.17 mm) and a rupture modulus close to the Intact control, demonstrating its potential to restore AF mechanical function. MRI imaging revealed that the FTGB@S@F group preserved an intact AF structure with high signal intensity, a significantly larger NP area (223.64 ± 73.32 mm(2) vs 137.30 ± 75.31 mm(2), P < 0.05), and higher disc height (102.5 ± 73.32 % vs 88.50 ± 12.86 %, P < 0.05). Histology confirmed superior AF repair and reduced NP degeneration in the FTGB@S@F group compared to the Un-repair and FB@S@F groups. Transcriptomic analysis identified upregulation of PIGR and downregulation of COL4A3, linked to the PI3K-Akt pathway. Immunohistochemical and qPCR analyses showed enhanced expression of COL1, Aggrecan, and RhoA, indicating effective regeneration.