Scaffolds mimicking the native annulus fibrosus (AF) extracellular matrix (ECM) structure are crucial to guide the seeding cells to regenerate aligned tissue, while fabricating such a scaffold by synthetic material is challengeable. Native acellular scaffolds derived from AF tissue certainly possess the advantages of natural structure and composition. Based on previous studies, we modified decellularization procedure and especially compared two drying methods, including gradient dehydration and freeze-drying. The decellularization process can effectively remove the host cells and antigens such as α-Gal, while maintaining the original ECM including GAG and collagen I. Compared with gradient dehydration, freeze-drying not only rendered the decellularized scaffold in dry state for storage but also gave the scaffold more aligned porous structure and hydrophilicity. And, the acellular porous scaffold manifested better capacity of supporting cell ingrowth when seeded human bone marrow mesenchymal stem cells (hBMSCs) or implanted in vivo. Furthermore, this optimized freeze-dried scaffold showed similar mechanical elastic modulus as native AF and demonstrated rare inflammatory granuloma and immune rejection as observed in HE staining and immunohistochemistry staining (IHC) of CD8 and MAC387 epitopes when implanted subcutaneously in vivo. To sum up, through our decellularization and freeze-drying procedure, an aligned porous three-dimensional scaffold derived from the natural AF ECM was successfully fabricated with good retention of ECM components and benign biocompatibility. It will be a promising scaffold for AF tissue engineering.

Mesenchymal stem cells (MSCs) and scaffolds offer promising perspectives for annulus fibrosus (AF) repair. The repair effect was linked to features of the local mechanical environment related to the differentiation of MSCs. In this study, we established a Fibrinogen-Thrombin-Genipin (Fib-T-G) gel which is sticky and could transfer strain force from AF tissue to the human mesenchymal stem cells (hMSCs) embedded in the gel. After the Fib-T-G biological gel was injected into the AF fissures, the histology scores of intervertebral disc (IVD) and AF tissue showed that Fib-T-G gel could better repair the AF fissure in caudal IVD of rats, and increase the expression of AF-related proteins including Collagen 1 (COL1), Collagen 2 (COL2) as well as mechanotransduction-related proteins including RhoA and ROCK1. To clarify the mechanism that sticky Fib-T-G gel induces the healing of AF fissures and the differentiation of hMSCs, we further investigated the differentiation of hMSCs under mechanical strain in vitro. It was demonstrated that both AF-specific genes, including Mohawk and SOX-9, and ECM markers (COL1, COL2, aggrecan) of hMSCs were up-regulated in the environment of strain force. Moreover, RhoA/ROCK1 proteins were also found to be significantly up-regulated. In addition, we further -demonstrated that the fibrochondroinductive effect of the mechanical microenvironment process could be significantly blocked or up-regulated by inhibiting the RhoA/ROCK1 pathway or overexpressing RhoA in MSCs, respectively. Summarily, this study will provide a therapeutic alternative to repair AF tears and provide evidence that RhoA/ROCK1 is vital for hMSCs response to mechanical strain and AF-like differentiation.

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