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Description
Introduction
Bone tumour removal, traumas with large defects or infections, and degenerative diseases are the main catastrophic events impeding complete bone healing. Autologous and allogenic bone grafting, and biologically inert metallic devices have limitations such as non-availability of autogenous bone, risk of infectious disease transmission, subsequent surgical removal, and bacterial infections [1]. Therefore, to overcome the limitations, we fabricated composite scaffolds based on a combination of Poly(3-hydroxybutyrate) [P(3HB)], a natural biocompatible and bioresorbable polymer of bacterial origin, and a Borosilicate-based bioactive glass doped with Zinc (BS-Zn). Moreover, the Zinc-doped bioglass is used to provide antibacterial activity, as Zn2+ is known for its strong anti-inflammatory and bactericidal properties. [2]
Methodology
P(3HB) was produced by bacterial fermentation of B. sacchari in the presence of excess of carbon and nitrogen limitation. Gas chromatography-mass spectrometry (GC-MS), Fourier-transform Infrared spectroscopy (FTIR), Nuclear Magnetic Resonance spectroscopy (NMR), and Differential Scanning Calorimetry (DSC) have been conducted to characterise the resulting polymer. Solvent casting has been used to produce composite films and X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM), High-resolution X-ray CT and tensile testing were conducted to determine their properties.
MG63 human osteoblast cell line was cultured on the composite and neat polymer films to assess biocompatibility, quantified using the resazurin assay. Subsequently, Live/Dead assay was performed using calcein green and ethidium bromide. In order to evaluate the antimicrobial activity, Minimum Inhibitory (MIC) and Minimum Bactericidal Concentration (MBC) were determined using ISO20776 against E. coli 8739, S. aureus 2569 and S. aureus 6538P for P(3HB)/BS films loaded with Gentamicin and P(3HB)/BS-Zn. The HALO Test was also carried out for the composite films.
Results
Chemical analysis confirmed that the polymer produced was Poly(3-hydroxybutyrate). The thermal properties of the extracted polymer analysed by DSC were found to be very similar to previous studies and commercially available products [3]. XRD analysis resulted in profiles which indicated the amorphous nature of the bioactive glasses. The addition of nanosized bioglasses induced a nanostructured topography on the surface of the composites, as visible by SEM images. Moreover, X-ray CT confirmed the homogeneous distribution of the bioglass filler in the polymeric matrix. The resazurin assay demonstrated the biocompatibility of P(3HB) and the related composites. The MIC/MBC test and the HALO test confirmed the antimicrobial activity of the composites.
Conclusions
In this study, a short-chain length PHA was produced by bacterial fermentation and its properties were investigated. Composite films have been synthesised using Borosilicate-based bioactive glasses. These have been further characterised to confirm the properties of the composite materials. The biocompatibility of the films was confirmed using the MG63 human osteoblast cell line. Also, the antimicrobial activity of P(3HB)/BS composite films loaded with gentamicin and P(3HB)/BS-Zn were investigated. The final aim of this project is the development of a promising material with antimicrobial activity for bone regeneration.
References
1. Koons, G.L. et al., Nat. Rev. Mater., vol. 5, n. 8, 584-603, 2020
2. Schuhladen, K. et al., Journal of Non-Crystalline Solids, n. 502, 22-34, 2018
3. Misra, S.K. et al., Biomacromolecules, n. 8, 2112-2119, 2007
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