Statement of Purpose: With the advances on tissue engineering (TE) field, several processing technologies have been combined to produce scaffolds with superior performance in several applications. Hydrogels are a good example of that and it have been extensively used for cartilage TE applications, presenting structural similarities to the natural extracellular matrix microenvironment of cartilage tissue. From the different biodegradable materials proposed as matrices for cartilage scaffolding, silk fibroin (SF) is an especially attractive protein for its high versatility, processability and tailored mechanical properties. Moreover, it presents tyrosine groups that can be used to prepare fast-formed hydrogels with controlled gelation properties, via an enzyme-mediated cross-linking reaction using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2).
Methods: In this work, SF-based scaffolds derived from high-concentrated SF (16wt%) enzymatically cross-linked by a HRP/H2O2 complex were produced. The combination of these HRP/H2O2-mediated hydrogels with salt-leaching and freeze-drying methodologies allowed to generate interconnected macro-/micro-porous structures herein proposed (Fig. 1a). The scaffolds morphology and mechanical properties were assessed by mean of different characterization techniques, including SEM, micro-CT, Instron, FTIR and XRD. In order to evaluate the scaffolds structural integrity, swelling ratio and degradation profile studies were performed for a period of 30 days. The in vitro chondrogenesis was evaluated by culturing human adipose-derived stem cells (hASCs) over 28 days in basal and chondrogenic culturing conditions. Cell behaviour in the presence of the macro-/micro-porous structures was analysed through different quantitative (DNA, GAGs and RT-PCR) and qualitative (live/dead, SEM, histology and immunocytochemistry) assays. The in vivo biocompatibility of the SF-based scaffolds was assessed by subcutaneous implantation in mice for 2 and 4 weeks. Inflammatory response of the collected explants was analyzed by means of hematoxylin & eosin (H&E) staining. Immunohistochemical analysis of the angiogenic marker CD31 was also performed.
Results and Discussion: The results showed highly porous and interconnected SF-based scaffolds with trabecular structures evenly distributed (Fig. 1b). The compressive modulus decreased for samples in hydrated state and the chemical characterization of SF scaffolds showed the characteristic peaks for β-sheet conformation. Scaffolds maintained the structural integrity over 30 days of soaking in PBS and distilled water, however, as expected in the presence of protease XIV the degradation profile increased. HASCs cultured on the macro-/micro-porous SF scaffolds were alive over the 28 days of culturing, presenting good cell adhesion over the scaffolds surface and were able to deeply penetrate and colonize the scaffolds interior. Cell proliferation was also observed over the 28 days of culturing in basal conditions and a significant increase of GAGs content was investigated on constructs cultured in the presence of chondrogenic differentiation culture medium. In vivo results showed that the implanted scaffolds allow tissue ingrowth’s, without inducing any acute inflammatory response. In addition blood vessels formation was also observed (Fig.1c).
Conclusions: The obtained results demonstrated that the innovative approach of combining enzymatically cross-linked SF hydrogels with the salt-leaching and freeze-drying methodologies allowed producing fast-formed porous scaffolds with a more versatile architecture and appropriate mechanical properties. These scaffolds were able to support cell adhesion, proliferation and chondrogenic differentiation. New tissue formation and angiogenesis within the porous scaffolds was also observed, in vivo. Thus, the proposed macro-/micro-porous SF scaffolds can be a valuable system for cartilage TE applications but also in other musculoskeletal TE strategies.