Development of hierarchical scaffolds for bone tissue engineering applications

The bone tissue engineering field has been pursuing new strategies to recreate bone matrix structure, composition, and hierarchy at different length scales. In this reasoning, the present study's main aim was to develop an advanced hierarchical scaffold capable of mimicking the structure, mechanical properties, and composition of bone tissue to some extent. For that, silk fibroin was combined with decellularized cell-derived extracellular matrix and reinforced with carbon nanotubes. The scaffolds were prepared using enzymatic cross-linking, freeze modeling, and decellularization methods. First, their structure and mechanical properties were assessed, showing that the developed scaffolds were elastic and with pore sizes (≈ 112 ± 22 µm), total porosity (75 ± 3%), and stiffness’s (≈ 5 kPa) within the range described for the stimulation of cell’s differentiation along the osteogenic lineage. Then, the bioactivity in vitro of the developed scaffolds was investigated, showing the formation of mineralization.

The cellular in vitro studies using human adipose-derived stem cells (hASCs) demonstrated that scaffolds supported cell adhesion, spreading, proliferation, and ultimately, osteogenic differentiation. The positive influence on osteogenic differentiation was confirmed by collagen secretion, increased ALP activity, and expression of osteogenic-related genes (e.g., ALP, Runx-2, Col Iα, and OPN). Furthermore,  the histological stainings showed cells infiltration into the scaffolds, and the hemolytic assay established the hemocompatibility of the hierarchical scaffolds.

The promising results showed that the developed carbon nanotubes-reinforced cell-derived matrix-silk fibroin hierarchical scaffolds could be used for bone tissue engineering scaffolding applications.