With the advances on tissue engineering (TE) field, several processing technologies have been combined to produce scaffolds with superior performance in several applications. Cartilage TE is a good example of that where the natural extracellular matrix microenvironment presents high elasticity and resistance to compression forces. Different biodegradable materials have been proposed as matrices for cartilage scaffolding. Among them, silk fibroin (SF) presents a high versatility and tailored mechanical properties which make this natural polymer attractive for the development of innovative matrices for cartilage TE applications. In our previous study SF protein has been used to produce fast forming hydrogels with controlled gelation properties, through horseradish peroxidase (HRP) crosslinking reaction, taking advantage of the presence of tyrosine groups.
This work aims at creating robust SF-based scaffolds derived from these HRP-mediated hydrogels in combination with salt-leaching and freeze-drying methodologies to generate interconnected macro-/micro-porous structures with tailored biodegradation and mechanical properties. The in vitro ability to support the adhesion, proliferation and differentiation into the chondrogenic lineage was tested using human adipose-derived stem cells (hASCs). These cells were cultured over 28 days in basal and chondrogenic conditions. Cell behaviour in the presence of the macro-/micro-porous structures was evaluated through different quantitative (DNA and GAGs) and qualitative (live/dead, SEM, histology and immunocytochemistry) assays. The results showed that the high porosity and interconnectivity of the SF-based scaffolds allowed for cell adhesion over the scaffolds surface as well as a deeply cell penetration and colonization into the scaffolds interior. Cell viability and proliferation were also observed over the 28 days of culturing in basal conditions and a significant increase of GAGs content was detected on constructs cultured in the presence of chondrogenic differentiation medium. The obtained results demonstrated that the innovative approach of combining enzymatically cross-linked SF hydrogels with the salt-leaching and freeze-drying methodologies allowed to produce more versatile scaffold architectures with positive influence over in vitro biological performance, making this a valuable system not only for cartilage regeneration but also in other musculoskeletal TE strategies.
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