Combinatory approach for developing silk fibroin-based scaffolds to support human adipose-derived stem cells chondrogenic differentiation

INTRODUCTION

With the advances on tissue engineering (TE) field, several processing technologies have been combined to produce scaffolds with superior performance in several applications. Hydrogels have been extensively used in cartilage TE field, presenting structural similarities to the natural extracellular matrix microenvironment of cartilage tissue^1,2. Silk fibroin (SF) is an especially attractive protein for this purpose, since it contains tyrosine groups that can be used to produce fast-formed hydrogels with controlled gelation properties³. In this work, SF-based scaffolds derived from high-concentrated SF (16wt%) enzymatically cross-linked by a HRP/H₂O₂ complex and combined with salt-leaching and freeze-drying methodologies were produced. The in vitro ability of the scaffolds towards supporting human adipose-derived stem cells (hASCs) chondrogenic differentiation was evaluated. The in vivo biocompatibility test was also carried out in a mice subcutaneous model.

EXPERIMENTAL METHODS

SF-based scaffolds were assessed in terms of morphology and mechanical properties by mean of SEM, micro-CT, Instron, FTIR and XRD analysis. In order to evaluate scaffolds structural integrity, swelling ratio and degradation profile studies were performed. Human adipose-derived stem cells (hASCs) were cultured over 28 days in basal and chondrogenic conditions. In vitro chondrogenesis in the presence of the macro-/micro-porous structures was analyzed through different quantitative (DNA, GAGs and RT-PCR) and qualitative (live/dead, SEM, histology and immunocytochemistry) assays. In vivo biocompatibility was analyzed by subcutaneous implantation in mice (2 and 4 weeks), by hematoxylin & eosin (H&E) staining and immunohistochemical analysis of the angiogenic marker CD31.

RESULTS AND DISCUSSION

The results showed highly porous and interconnected macro-/micro-porous SF scaffolds (Fig. 1a) with specific features regarding biodegradation and mechanical properties. The compressive modulus decreased for samples in hydrated state and the chemical characterization of SF scaffolds showed the typical peaks for β-sheet conformation. Scaffolds maintained the structural integrity over 30 days of soaking in PBS, however, in the presence of protease XIV the degradation profile increased. HASCs adhered to the macro-/micro-porous SF scaffolds surface and deeply penetrate and colonize the scaffolds interior. Cell viability and proliferation were observed over 28 days of basal culture and a significant increase of GAGs content was detected on constructs cultured in chondrogenic differentiation conditions (Fig. 1b). SF scaffolds allowed tissue ingrowth’s after subcutaneous implantation in mice (Fig. 1c).

CONCLUSION

This work demonstrated that SF scaffolds presented good mechanical properties and a macro-/micro-porous structure suitable for cartilage TE purposes. HASCs were able to differentiate in chondrocytes when cultured onto the SF scaffolds. New tissue formation within the porous scaffolds was also observed, in vivo. Thus, the combination of different processing techniques allowed producing innovative fast-formed SF scaffolds as a valuable system for cartilage TE applications.