Eumelanin from the Black Soldier Fly as Sustainable Biomaterial: Characterisation and Functional Benefits in Tissue-Engineered Composite Scaffolds

Abstract

An optimized extraction protocol for eumelanins from black soldier flies (BSF-Eumel) allows an in-depth study of natural eumelanin pigments, which are a valuable tool for the design and fabrication of sustainable scaffolds. Here, water-soluble BSF-Eumel sub-micrometer colloidal particles were used as bioactive signals for developing a composite biomaterial ink for scaffold preparation. For this purpose, BSF-Eumel was characterized both chemically and morphologically; moreover, biological studies were carried out to investigate the dose-dependent cell viability and its influence on human mesenchymal stem cells (hMSCs), with the aim of validating suitable protocols and to find an optimal working concentration for eumelanin-based scaffold preparation. As proof of concept, 3D printed scaffolds based on methacrylated hyaluronic acid (MEHA) and BSF-Eumel were successfully produced. The scaffolds with and without BSF-Eumel were characterized in terms of their physico-chemical, mechanical and biological behaviours. The results showed that MEHA/BSF-Eumel scaffolds had similar storage modulus values to MEHA scaffolds. In terms of swelling ratio and stability, these scaffolds were able to retain their structure without significant changes over 21 days. Biological investigations demonstrated the ability of the bioactivated scaffolds to support the adhesion, proliferation and osteogenic differentiation of human mesenchymal stem cells.

Keywords: 3D printing; bone tissue engineering; eumelanins; hyaluronic acid; methacrylated hyaluronic acid; scaffolds.

Figure 1

Biosynthetic pathway of eumelanins.

Figure 2

Fabrication steps from biomaterial ink synthesis to the 3D printed MEHA and MEHA/BSF-Eumel scaffolds.

Figure 3

(a) UV-vis absorption spectra of BSF-Eumel in PBS, pH 7.4. (b) EPR analysis of BSF-Eumel at the solid state (S) and as PBS solution (L).

Figure 4

TEM micrographs of BSF-Eumel: (a) scale bar: 1 µm, (b) scale bar: 200 nm. (c) Zeta (ξ) potential of BSF-Eumel as function of different conductivities and ionic strengths of medium (diH₂O, PBS, DMEM and DMEM + 10% (v/v) FBS) measured by DLS. (* p < 0.05).

Figure 5

(a) Alamar blue cell viability assay (%, normalized to control) performed on L929 cells grown in the presence of BSF-Eumel serial dilution (2.5, 1.25, 0.625, 0.3125, 0.125, 0.05 mg/mL) after 24 h of treatment. Data are presented as mean ± SD. Statistical analysis of variance on mean values is assessed by one-way ANOVA followed by Tukey’s post hoc test with multiple comparisons. (* p < 0.05; ° p < 0.001). (b) Optical micrographs and (c) fluorescent images of L929 with and without BSF-Eumel treatment at 24 h of in vitro cell culture (Scale bar: 500 μm).

Figure 6

(a) ATR-FTIR spectra of HMwHAs and MEHA, highlighting the success of chemical modification. (b) Dynamic mechanical analysis performed on MEHA and MEHA/BSF-Eumel scaffolds. * p < 0.05. (c) Swelling behaviour and (d) degradation profile of MEHA and MEHA/BSF-Eumel scaffolds up to 21 days. (e) Release profile of BSF-Eumel from 3D printed scaffolds up to 15 days in diH₂O. Data reported as mean value ± S.D., n = 3.

Figure 7

Cell proliferation and differentiation of hMSCs in contact with 0.3125 mg/mL of BSF-Eumel solution at 7 and 14 days of cell culture (a,b). Cell proliferation and differentiation of hMSCs seeded on MEHA- and MEHA/BSF-Eumel-based scaffolds after 1, 3 and 7 days of cell culture (c,d). * p < 0.05; ° p < 0.001; # p < 0.0001 vs control group.