ARTICLE Please do not adjust margins Please do not adjust margins Received 00th January 20xx, Accepted 00th January 20xx DOI: 10.1039/x0xx00000x Carbon nanotubes-reinforced cell- derived matrix-silk fibroin hierarchical scaffolds for bone tissue engineering applications Rafael Lemos a, b, c , F. Raquel Maia a, b,† , Viviana P. Ribeiro a, b , João B. Costa a, b , Paulo J. G. Coutinho c , Rui L. Reis a, b , and Joaquim M. Oliveira a, b In 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. Introduction The increasing rate of musculoskeletal pathologies affects the world population due to ever-higher life expectancy and aging. Since bone-related diseases dramatically affect functional and patient quality of life, it is important to develop improved treatment strategies that promote bone tissue regeneration. (1) Bone tissue engineering solutions based on advanced scaffolds are a promising strategy and alternative to a. 3B’s Research Group, I3B’s – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra, 4805-017 Barco, Guimarães, Portugal; b. ICVS/3B's - PT Government Associated Laboratory, Braga/Guimarães, Portugal; c. Centre of Physics (CFUM), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal. † Corresponding author: F. Raquel Maia (raquel.maia@i3bs.uminho.pt). Page 1 of 14 Journal of Materials Chemistry B Journal of Materials Chemistry B Accepted Manuscript Published on 25 October 2021. Downloaded by Universidade do Minho (UMinho) on 10/27/2021 11:51:45 AM. View Article Online DOI: 10.1039/D1TB01972D