Co-cultivation of Komagataeibacter sp. and Lacticaseibacillus sp. strains to produce bacterial nanocellulose-hyaluronic acid nanocomposite membranes for skin wound healing applications
Marcello Brugnoli, João P. F. Carvalho, Mattia Pia Arena, Helena Oliveira, Carla Vilela, Carmen S. R. Freire, Maria Gullo
Abstract
Inspired by natural microbial cooperation, a co-culture approach was used to synthesize bacterial nanocellulose (BNC)-based nanocomposites for potential wound healing applications. By co-culturing either Komagataeibacter xylinus (K1G4) or the never tested strain K. rhaeticus (K2G46) with the hyaluronic acid (HA)-producer Lacticaseibacillus casei UMCC 2535, two BNC-HA nanocomposites were obtained (C1-K1 and C2-K2). The membranes showed a HA content of 0.49 ± 0.05 mg (C1-K1) and 1.40 ± 0.07 mg (C2-K2), and both revealing a nearly complete release of HA after 1 h in PBS. Compared to pure BNC membranes, the nanocomposites showed enhanced properties, including higher crystallinity (K1G4 = 84.6 %; K2G46 = 76.5 %; C1-K1 = 89.1 %; C2-K2 = 88.1 %), and Young's modulus (K1G4 = 3.38 ± 0.56 GPa; K2G46 = 2.22 ± 0.65 GPa; C1-K1 = 10.00 ± 1.32 GPa; C2-K2 = 7.90 ± 1.54 GPa). Additionally, both BNC-HA membranes exhibited increased moisture uptake (K1G4 = 9.06 ± 0.47 %; K2G46 = 9.27 ± 1.33 %; C1-K1 = 13.65 ± 0.53 %; C2-K2 = 16.26 ± 1.05 %) and water absorption (K1G4 = 82.18 ± 5.25 %; K2G46 = 86.54 ± 7.86 %; C1-K1 = 160.04 ± 9.33 %; C2-K2 = 144.42 ± 13.86 %) capacity. Moreover, they were non-cytotoxic towards human keratinocyte (HaCaT) cells, with >90 % cell viability for up to 72 h. The in vitro scratch assays showed a complete wound closure within 48 h for cells exposed to BNC-HA membranes. These findings underscore the potential of co-culturing system to develop BNC-HA nanocomposites for wound healing applications. • A novel co-culture system with K. rhaeticus for BNC-HA nanocomposites is described. • BNC-HA nanocomposite membranes were successfully produced by co-culture. • BNC-HA nanocomposites exhibited enhanced mechanical properties compared to pure BNC. • Higher water absorption and moisture uptake were observed for the BNC-HA nanocomposites. • BNC-HA nanocomposites were non-cytotoxic and promoted wound closure in vitro .