Carbon nanotubes-reinforced cell-derived matrix-silk fibroin hierarchical scaffolds for bone tissue engineering applications

Accepted ManuscriptIn bone tissue engineering, the development of advanced biomimetic scaffolds has led to the quest for biomotifs in scaffold design that better recreate bone matrix structure and composition and hierarchy at different length scales. In this study, an advanced hierarchical scaffold consisting of silk fibroin combined with decellularized cell-derived extracellular matrix and reinforced with carbon nanotubes was developed. The goal of the carbon nanotubes-reinforced cell-derived matrix-silk fibroin hierarchical scaffolds is to harvest the individual properties of its constituents to introduce hierarchical capacity in order to improve standard silk fibroin scaffolds. The scaffolds were fabricated using enzymatic cross-linking, freeze modeling, and decellularization methods. The developed scaffolds were assessed for pore structure and mechanical properties showing satisfying results to be used in bone regeneration. The developed carbon nanotubes-reinforced cell-derived matrix-silk fibroin hierarchical scaffolds showed to be bioactive in vitro and expressed no hemolytic effect. Furthermore, cellular in vitro studies on human adipose-derived stem cells (hASCs) showed that scaffolds supported cell proliferation. The hASCs seeded onto these scaffolds evidenced similar metabolic activity to standard silk fibroin scaffolds but increased ALP activity. The histological stainings showed cells infiltration into the scaffolds and visible collagen production. The expression of several osteogenic markers was investigated, further supporting the osteogenic potential of the developed carbon nanotubes-reinforced cell-derived matrix-silk fibroin hierarchical scaffolds. The hemolytic assay demonstrated the hemocompatibility of the hierarchical scaffolds. Overall, the carbon nanotubes-reinforced cell-derived matrix-silk fibroin hierarchical scaffolds presented the required architecture for bone tissue engineering applications.Research and Innovation Staff Exchanges (RISE) action (H2020 Marie Skłodowska-Curie actions) for funds obtained through the BAMOS project (H2020-MSCA-RISE-2016-73415) and the R&D Project KOAT PTDC/BTMMAT/29760/2017 (POCI-01-0145- FEDER-029760), financed by Fundação para a Ciência e a Tecnologia (FCT) and co-financed by FEDER and POCI. F.R.M. acknowledges FCT for her contract under the Transitional Rule DL 57/2016 (CTTI-57/18-I3BS(5)). V.P.R. acknowledges the Junior Researcher contracts (POCI-01-0145-FEDER-031367; POCI-01-0145-FEDER-029139) attributed by the FCT under the projects Fun4TE project (PTDC/EMD-EMD/31367/2017) and BLiver (PTDC/EMD-EMD/29139/2017). J.B.C. acknowledges the Junior Researcher contract (POCI-01-0145-FEDER-031367) attributed by FCT to the Fun4TE project (PTDC/EMDEMD/31367/2017)