Novel bilayered silk fibroin-based scaffolds incorporating Sr- and Zn-ions for osteochondral tissue engineering

Osteochondral tissue engineering (OCTE) has been proposing monolithic bilayered scaffolds consisting of a cartilaginous layer and an interconnected underlying osseous layer. These scaffolds hold unique composition, organization, structural strength and specific biological properties according to the target bone and cartilage tissues [1]. Among others natural-based polymers used for scaffolds production, silk fibroin (SF) exhibits high chemical versatility, biocompatibility and tunable mechanical properties [2]. Moreover, our recent approach of combining enzymatically cross-linked SF hydrogels (HRP-SF) with pore-inducing technologies (i.e., salt-leaching and freeze-drying) allowed to create novel scaffolds with high porosity and viscoelastic properties. On the other side, bioresorbable inorganic materials, such as β-tricalcium phosphate (β-TCP) have outstanding osteoconductivity [3]. The incorporation of ionic dopants into β-TCP with trace elements existing in bone, enhance osteogenesis and the neovascularization of scaffolds [3].

In this study, we aim to produce novel monolithic bilayered scaffolds composed by HRP-SF for the cartilage layer, and 80/20 (w/w) HRP-SF/undoped and ZnSr-doped β-TCP for the underlying osseous layer, using salt-leaching followed by freeze-drying techniques.
Physicochemical characterization was assessed through FTIR, XRD, SEM, and micro-CT. The structural integrity of the scaffolds was evaluated by degradation profile studies and the mechanical properties were determined after immersion in PBS. The bioactivity of the scaffolds was assessed by immersion in SBF up to 30 days. The in vitro cell adhesion, migration and proliferation were evaluated by co-culturing human chondrocytes (hCh) and human osteoblasts (hOB) in the scaffolds up to 14 days. Monocultured hOB and hCh on the individual scaffold layers were used as controls. Biochemical characterization of ALP activity and GAGs production were also performed.

The results showed porosity index of 50-60% and highly interconnected pores of 130-140 μm. An homogeneous distribution of the β-TCP into the HRP-SF on the osseous layer was also observed. The mechanical properties of ZnSr-doped bilayered scaffolds were superior then the undoped

scaffolds. Co-cultured cells were able to adhere and proliferate on the bilayered scaffolds and higher ALP activity levels were detected on the monocultured HRP-SF/undoped and ZnSr-doped β-TCP constructs. A positive effect was induced by the co-culture system for GAGs production and deposition. The preliminary in vitro results of the SrZn-incorporating bilayered scaffolds together with their physicochemical and mechanical features endorse these monolithic and gradient structures for OCTE applications.

References

[1]L.-P.Yan,et al.,Acta biomaterialia12(2015):227-241. 

[2]C.Vepari,D.L.Kaplan,Progress in polymer science32(8)(2007):991-1007. 

[3]S.Pina,et al.,Cells Tissues Organs204(2017).