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.
Keywords: annulus fibrosus; decellularization; freeze-drying; gradient dehydration; porous structure; tissue engineering.