Crosslinking stabilization strategy: A novel approach to cartilage-like repair of annulus fibrosus (AF) defects
Zihan Wang, Lei Wang, Shaoshuo Li, Xin Chen, Bo Chen, Zhichao Lou, Zheng Li, Rongrong Deng, Lin Xie, Jianwei Wang, Xin Liu, Ran Kang
Abstract
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. Figure. Schematic diagram of the crosslinking stabilization strategy and mechanism diagram of the crosslinking stabilization strategy promoting the differentiation of MSCs into fibrocycle-like cells.