The development of novel biocomposite formulations has significantly contributed to the biomedical field- specifically within translational and clinical applications. This study centres on the polyhydroxyalkanoates-based copolyester superfamily of materials, with a particular emphasis on the poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer as a novel biomimetic substrate. Both 3HB and 4HB monomers have been recognised as natural metabolites in mammalian systems and this copolymer can be synthesised using a microbial-based production system. Crucially, it has been accepted that the 4HB monomer molar fraction determines the physical characteristics of the final copolymer which, ultimately, defines its endpoint application. This project compares two different fermentation techniques used for the synthesis of P(3HB-co-4HB) in terms of yield, purity and composition. Importantly, the synthesised material is characterised in terms of its physical, mechanical and biological profiles before being further enhanced/functionalised with 45S5 bioactive glass and graphene.
P(3HB-co-4HB) copolymer, from Cupriavidus sp. USM1020, was extracted from a shake flask cultivation or bioreactor inoculation system before being purified to obtain crude copolymer. 45S5 bioactive glass was prepared using a sol-gel technique [ratio of 45SiO2-24.5CaO-24.5NaO-6P2O5 (wt%) precursors]. Graphene monolayers were fabricated using liquid exfoliation of graphite. The biocomposite mixture was prepared via facile blending by dissolving P(3HB-co-4HB) crude copolymer in chloroform followed by ultrasonication with the bioactive glass and graphene. Casting was achieved by either solvent or emulsion freeze drying techniques to assess the differences in their porosity. Characterisation was performed using SEM, FT-IR and DSC. Disc diffusion antibacterial assay was employed with Escherichia coli and Staphylococcus aureus. Biocompatibility was assessed using human dermal fibroblasts (HDF) and murine osteoblastic (MC3T3-E1) cells, and standard cell culture assays: cell attachment (SEM imaging, live/dead), proliferation and viability (metabolic activity/MTS, LDH-release), and differentiation activities (cytokine expression/western blotting). Preliminary in vivo studies were performed using male Sprague-Dawley rats.
The shake flask cultivation system generated a higher percentage (~69%) of 4HB compared to the bioreactor system. Miscibility of the biocomposite was further improved via facile blending. Morphological analyses showed that the emulsion freeze drying technique resulted in a more porous structure compared to solvent casting and the associated change in wettability confirmed with water contact angle measurements. The mechanical profiles of the biocomposite and antibacterial activities were enhanced following incorporation of graphene. Optimised composition of both bioactive glass and graphene within the biocomposite is vital in ensuring optimal level of cells adhesion, which resulted from the observed attachment and proliferation of both cell lines. The animal studies (i.e. skin flap and bone defect) demonstrated good biocompatibility and favour interaction between P(3HB-co-4HB)-bioactive glass-graphene biocomposite and native tissues with enhanced presence of nuclei and neo-vascularisation, and minimal immune response.
Taken together, this study illustrates the crucial optimisation parameters of the novel formulation P(3HB-co-4HB)-bioactive glass-graphene, which includes processing techniques that affects the final morphology and behaviour of the biocomposite. Owing to the individual benefits of each prominent material utilised in this study, increased potential in translational biomedical applications especially as therapeutic dressings and non-load bearing scaffold for bone regeneration can be considered."