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Towards functional 3D-stacked electrospun composite scaffolds of PHBV, silk fibroin and nanohydroxyapatite: Mechanical properties and surface osteogenic differentiation

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Bone tissue engineering scaffolds have two challenging functional tasks to fulfil: to encourage cell proliferation, differentiation and matrix synthesis and to provide suitable mechanical stability upon implantation. Composites of biopolymers and bioceramics combine the advantages of both types of materials, resulting in better processability and enhanced mechanical and biological properties through matrix reinforcement. In the present study, novel thick bone composite scaffolds were successfully fabricated using electrospun flat sheets of polyhydroxybutyrate–polyhydroxyvalerate/nanohydroxyapatite/silk fibroin essence (2% nanohydroxyapatite – 2% silk fibroin essence and 5% nanohydroxyapatite – 5% silk fibroin essence, respectively). Their potential as in vitro bone regeneration scaffolds was evaluated using mouse calvarian osteoblast cells (MC3T3-E1), in terms of morphology (scanning electron microscope), cell attachment, cell proliferation, Col type I, osteopontin and bone alkaline phosphatase activity (Quantitative Real Time Polymerase Chain Reaction [qRT-PCR], enzyme-linked immunosorbent assay, immunocytochemistry). Electrospun polyhydroxybutyrate–polyhydroxyvalerate scaffolds were used as reference constructs. The results showed that the compressive and tensile mechanical properties of the scaffolds are dependent on the change in their composition, and the treatment these underwent. Furthermore, methanol-treated and autoclaved (MA) P2 (2% nanohydroxyapatite, 2% silk fibroin essence) samples appeared to exhibit more promising tensile properties. Additionally, the compressive tests results confirmed that the methanol pre-treatment and the autoclaving step lead to an increase in the P2 secant modulus when compared to the non-methanol-treated ones, P2 and P5 (5% nanohydroxyapatite, 5% silk fibroin essence), respectively. Both formulations of polyhydroxybutyrate–polyhydroxyvalerate/nanohydroxyapatite/silk fibroin essence composite promoted greater cell adhesion and proliferation than the corresponding polyhydroxybutyrate–polyhydroxyvalerate control ones. Cells seeded on the composite fibrous scaffolds were extensively expanded and elongated on the fibre surface after one day in culture, whereas those seeded on the polyhydroxybutyrate–polyhydroxyvalerate scaffolds were not completely elongated. In addition, cells grown on P2 and P5 scaffolds had higher alkaline phosphatase activity when compared to those containing no nanohydroxyapatite/silk fibroin essence.