Osteochondral (OC) tissue engineeringhas been proposingbilayeredscaffolds composed of a cartilage-like layer and an underlying subchondral bone-like layer. These scaffolds hold unique composition, mechanical strength and specific biological properties according to the target tissue layer [1].Silk fibroin (SF) exhibits high chemical versatility, biocompatibility and tunable mechanical properties [2].On the other side, bioresorbable inorganic materials, including β-tricalcium phosphate(β-TCP)have shown outstanding osteoconductive properties, and the possibility of incorporating ionic dopants into β-TCPenhance osteogenesis and neovascularization of scaffolds [3]. In this study, we aim to produce novel monolithic bilayered scaffolds for OC tissue repair/regeneration, composed of enzymatically crosslinked SF scaffolds (HRP-SF) for the cartilage-like layer and an underlying subchondral layer incorporating β-TCP powdersdoped with Sr and Zn [4]. HRP-SF/undoped β-TCP scaffolds were used as controls. Physicochemical characterization was assessed through XRD, SEM and micro-CT. Structural integrity was evaluated by degradation profile studies and themechanical properties determined after immersion in PBS solution. Scaffolds bioactivity was assessed by immersion in SBF up to 30 days. The in vitrocell adhesion and proliferation were characterized by co-culturing human articular chondrocytes (hACs) and human osteoblasts (hOBs) in the scaffolds up to 14 days. Monocultured individual scaffolds were used as controls. The results showed an interconnected porosity and homogeneous β-TCP distribution into the subchondral bone layers. The mechanical properties of the HRP-SF/ZnSr-β-TCP bilayered scaffolds were superior than the undoped scaffolds. Co-cultured cells adhered and proliferated on the bilayered scaffolds, showing the formation of a mineralized ECM and GAGs deposition in the respective subchondral bone and cartilage-like layers.In brief, the structural adaptability and suitable mechanical properties of the proposed engineered OC scaffolds, combined with the biological performance achieved using a co-culturing system, make these scaffolds a viable strategy for OC defects regeneration.

References

[1] L.-P. Yan et al., Acta biomaterialia,12:227-241, 2015

[2] C. Vepari, D.L. Kaplan, Progress in polymer science, 32:991-1007, 2007

[3] S. Pina et al., Cells Tissues Organs, 204:150, 2017

[4] V.P. Ribeiro et al., Acta Biomaterialia, 1742-7061(18)30178-8, 2018

Acknowledgments:

Portuguese Foundation for Science and Technology (FCT) project M-ERA-NET/0001/2014 project. Investigator FCT programs IF/00423/2012, IF/00411/2013 and IF/01285/2015. Financial support from FCT/MCTES (Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia, e Ensino Superior) and Fundo Social Europeu através do Programa Operacional do Capital Humano (FSE/POCH), PD/59/2013, PD/BD/113806/2015.