Nanocomposite Methacrylated Silk Fibroin-Based Scaffolds for Bone Tissue Engineering

Abstract

The treatment of bone defects is a clinical challenge. Bone tissue engineering is gaining interest as an alternative to current treatments, with the development of 3D porous structures (scaffolds) helpful in promoting bone regeneration by ensuring temporary functional support. In this work, methacrylated silk fibroin (SilMA) sponges were investigated as scaffolds for bone tissue engineering by exploiting the combination of physical (induced by NaCl salt during particulate leaching) and chemical crosslinking (induced by UV-light exposure) techniques. A biomimetic approach was adopted to better simulate the extracellular matrix of the bone by introducing either natural (mussel shell-derived) or synthetic-origin hydroxyapatite nanoparticles into the SilMA sponges. The obtained materials were characterized in terms of pore size, water absorption capability and mechanical properties to understand both the effect of the inclusion of the two different types of nanoparticles and the effect of the photocrosslinking. Moreover, the SilMA sponges were tested for their bioactivity and suitability for bone tissue engineering purposes by using osteosarcoma cells, studying their metabolism by an AlamarBlue assay and their morphology by scanning electron microscopy. Results indicate that photocrosslinking helps in obtaining more regular structures with bimodal pore size distributions and in enhancing the stability of the constructs in water. Moreover, the addition of naturally derived hydroxyapatite was observed to be more effective at activating osteosarcoma cell metabolism than synthetic hydroxyapatite, showing a statistically significant difference in the AlamarBlue measurement on day 7 after seeding. The methacrylated silk fibroin/hydroxyapatite nanocomposite sponges developed in this work were found to be promising tools for targeting bone regeneration with a sustainable approach.

Keywords: bone tissue engineering; hydroxyapatite; methacrylated silk fibroin; scaffold.

Figure 1

Schematic of the nanocomposite SilMA sponge manufacturing process. Created with BioRender.

Figure 2

FE-SEM images of the (a) as-synthesised mussel shell-derived hydroxyapatite and (b) the commercial hydroxyapatite used in the sponge production; scale bar: 200 nm. (c) XRD pattern of the mussel shell-derived hydroxyapatite.

Figure 3

S-TEM images of the HAPm nanoparticles dispersed in SilMA solution at three different concentrations (0.5–1.0–2.0 wt%); scale bar: 2 µm.

Figure 4

FE-SEM images of the internal porous structure of the dry SilMA sponges and the pore size bimodal distributions measured from FE-SEM images in both non-UV treated and UV-treated conditions; scale bar: 100 µm.

Figure 5

Water uptake curves of the sponges, both non-UV treated (a) and UV-treated (b), incubated in PBS at 37 °C. Data are represented as means with standard deviations (n = 3).

Figure 6

(a) Representative stress–strain curves for the tested sponges with and without the UV treatment. (b) Compressive elastic moduli (in kPa) of the tested sponges measured after 24 h of incubation in PBS (pH = 7.4) at 37 °C presented as means with standard deviations (n = 4).

Figure 7

AlamarBlue assay measured over 7 days of MG63 culture on the UV-treated SilMA sponges (SilMA + UV), either with mussel-shells derived hydroxyapatite (SilMA_HAPm + UV), or with synthetic hydroxyapatite (SilMA_HAPs + UV) sponges and the non-UV treated sponge (SilMA). **** p < 0.0001.

Figure 8

Scanning electron microscope images at day 1, day 3 and day 7, representative of the cells seeded on the UV-treated sponges: SilMA + UV (SilMA only), SilMA_HAPm + UV (with mussel-shells derived hydroxyapatite) and SilMA_HAPs + UV (with synthetic hydroxyapatite). Scale bar: 50 µm. Red arrows indicate cells and cellular morphology details.