Leveraging the nanotopography of filamentous fungal chitin-glucan nano/microfibrous spheres (FNS) coated with collagen (type I) for scaffolded fibroblast spheroids in regenerative medicine.

Numerous naturally occurring biological structures have inspired the development of innovative biomaterials for a wide range of applications. Notably, the nanotopographical architectures found in natural materials have been leveraged in biomaterial design to enhance cell adhesion and proliferation and improve tissue regeneration for biomedical applications. In this study, we fabricated three-dimensional (3D) chitin-glucan micro/nanofibrous fungal-based spheres coated with collagen (type I) to mimic the native extracellular matrix (ECM) microenvironment. These collagen-coated fungal nano/microfibrous spheres (C-FNS) were utilized to construct 3D scaffolded spheroids of human fibroblasts through suspension culture for tissue engineering and regenerative medicine. The particle sizes of C-FNS ranged from 1.4 to 3.25 µm (average: 2.27 ± 0.38 µm), with a porosity of 81.17 %. Field emission-scanning electron microscopy (FE-SEM) revealed that C-FNS comprised continuous chitin-glucan fibers with an average diameter of 363 ± 61 nm (range: 203-512 nm), exhibiting a highly interconnected structure. The reduced arithmetic average roughness (Ra) and root mean square roughness (Rq) values of C-FNS compared to uncoated FNS suggested that collagen coating reduced surface roughness, resulting in a smoother surface that enhanced hydrophilicity, crucial for mammalian cell adhesion and spheroid formation. Moreover, the in vitro cytocompatibility of C-FNS with fibroblasts was evaluated using a resazurin-based PrestoBlue assay, which demonstrated a time-dependent increase in the metabolic activity of C-FNS/fibroblast spheroids during suspension culture for up to 14 days. FE-SEM images of C-FNS/fibroblast spheroids further revealed enhanced adhesion and proliferation of fibroblasts on the nano/microfibrous mycelial architecture, accompanied by the secretion of ECM components and formation of multilayered cell sheets over the 14-day culture period. Similarly, an assessment of the hemocompatibility of C-FNS with erythrocytes revealed the non-hemolytic properties of the biomaterial. Overall, the interaction between collagen-coated fungal chitin-glucan nano/microfibrous structures and mammalian cells holds significant potential for the development of novel, sustainable biomaterials with tailored properties for a myriad of biomedical applications, including tissue engineering, regenerative medicine, drug screening, and wound healing.