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Ligament/tendon disorders and injuries are among the most common health problem influencing the adult and sport population. Anterior cruciate ligament (ACL), is one of the ligaments most injured in the knee, and the soft-to-hard tissue integration (enthesis) shows high rupture rates. To overcome problems related to therapies currently used in medical practice, tissue engineering (TE) has recently emerged as an alternative strategy. TE is a field that applies principles of biology, engineering and medicine toward the development of biological tissue substitutes to restore, repair or improve damaged tissues. Ligament/tendon TE focuses on the combination of specific cell types with a biodegradable scaffold. Once implanted, the scaffold has gradually to degrade, while the tissue is regenerating. The scaffold should be able to promote cell adhesion, differentiation, proliferation and extracellular matrix (ECM) production. Different scaffold materials have been successfully studied for TE application, and among them, silk fibroin has emerged as a promising one. In this work, silk fibroin has been used to fabricate a ligament scaffold comprising the ligamentous part and the enthesis. The enthesis was fabricated with fibroin sponges with a region of anisotropic porosity (ligament site) and a region of isotropic porosity (bone site). The enthesis construct was functionalized with heparin molecules to increase the growth factors affinity. Specifically, the effects of TGF-β2 and GDF5 combined with the structural features of the scaffold led to differentiation of AdMSCs. The alignment of the pores and the biological cues synergistically influenced the gene expression and the protein expression. In fact, the tendon/ligament markers were promoted in the anisotropic part of the construct, as well as the cartilage and the enthesis markers in the isotropic and transition part, respectively. The central part of the ligament was realized with a composite of silk fibroin yarns in a spongeous matrix of silk fibroin, where the yarns have the role to sustain the applied load and the fibroin sponge the role of allowing cell penetration, organization and ECM production. The amount of yarns was decided on the basis of the reported loading of the ligament scaffold after a surgery and during rehabilitation. A homemade perfusion bioreactor was used to evaluate cell penetration and activity in the scaffold. It was proven that cell culture perfusion stimulation induced cells to populate the model scaffold and to produce ECM components, especially with the presence of TGF-β2 growth factor, indicating the starting point of the regeneration process. Additionally, animal studies have been performed and the inflammatory response was evaluated. Even if not final, the results indicate that the above strategy could be explored for the fabrication of a full ligament TE scaffold able to bear the physiological loads until the ligament regenerates. |
