Combinatorial approach for developing innovative silk fibroin-based porous scaffolds for cartilage tissue engineering applications

The combination of several processing technologies can open the possibility for producing scaffolds with superior performance for tissue engineering (TE) applications. Hydrogels are structurally similar to the natural extracellular matrix (ECM) microenvironment presenting high elasticity and resistance to compression forces. They have been extensively used in biomedical devices fabrication, as drug delivery systems and for TE applications, including for cartilage defects repair [1]. Recently, thefast-formation of hydrogels through enzyme-mediated cross-linking has attracted much attention. Some authors reported that polymers/proteins with tyrosine groups can be used to prepare hydrogels under physiological temperature conditions, by using horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂) [2]. Silk fibroin (SF) has been particularly studied as a biomaterial, possessing high versatility, processability and tailored mechanical properties [3, 4]. Furthermore, it contains 5 mol% tyrosine groups, which enables theproduction of hydrogels via peroxidase-mediated cross-linking[5]. In this context, the present study proposes the development and characterization of SF-based scaffolds derived from a high-concentrated SF solution (16wt%), enzymatically cross-linked by a HRP/H₂O₂ complex after 37ºC incubation. The macro-/micro-porous scaffolds were prepared by combining salt-leaching and freeze-drying methodologies, which allowed for obtaining highly porous and interconnected structures with specific features regarding biodegradation and mechanical properties. Compression testing (Instron) showed a dramatic decrease of compressive modulus (MPa) for samples in hydrated state. Quantitative chemical analysis (FTIR) indicated that the β-sheet conformation was present in the SF scaffolds. Swelling ratio data demonstrated a large swelling capacity, higher for samples immersed in ultrapure water. Based on these results, an innovative approach was used to produce fast-formed porous SF scaffolds by using an enzymatically cross-linked SF solution structured by the combination of salt-leaching and freeze-drying methodologies, revealing that SF can be a very tunable and versatile biomaterial with great potential for finding applications in cartilage TE scaffolding.