Development of novel scl-mcl polyhydroxyalkanoates blends for soft tissue engineering

Abstract

Event Abstract Back to Event Development of novel scl-mcl polyhydroxyalkanoates blends for soft tissue engineering Pooja P. Basnett1, Barbara B. Lukasiewicz1, Rinat R. Nigmatullin1 and Ipsita Roy1 1 University Of Westminster, Faculty of science and technology, United Kingdom Introduction: Polyhydroxyalkanoates (PHAs) are group of natural biopolymers produced by various microorganisms as a reserve of carbon and energy under nutrient limiting conditions and excess of carbon source[1]. PHAs are biodegradable, non-toxic and biocompatible[2]. Furthermore PHAs as biomaterials are characterized by a wide diversity of monomeric structures. In fact depending on the number of carbon atoms present in the monomer, PHAs are classified into (1) short chain length (SCL) PHA which contains C3 to C5 carbon atoms and (2) medium chain length (MCL) PHA that contains C6 to C14 carbon atoms. This wider variance in their chemical composition enables PHA family to exhibit a broad spectrum of material properties. For instance, SCL-PHAs are brittle and semi-crystalline, whereas MCL-PHAs are highly elastomeric[3]. All these positive attributes make PHAs a potential candidate for a variety of applications, particularly in the area of medicine such as wound management, drug delivery, medical devices, regeneration of both hard and soft tissues etc. engineering[4]-[6]. In this study we have presented results obtained from blending two different types of Polyhydroxyalkanoates: poly-3-hydroxybutyrate P(3HB) an SCL PHA with hydrolysed P(3HHx-3HO-3HD-3HDD) an MCL PHA to develop matrix amenable for soft tissue engineering application. Experimental Methods: MCL-PHAs copolymer was produced using Pseudomonas sp. with waste frying oil as the sole carbon source. Similarly, P(3HB) was produced using Bacillus sp. and glucose as the carbon source. Bacterial cultures were grown on mineral salt medium under nitrogen limitation with excess of carbon. Polymer was extracted from biomass by solid phase extraction. Pure polymers were analysed by Fourier Transformed Infrared Spectroscopy (FTIR) and Gas Chromatography – Mass Spectrometry (GC-MS). Acidic hydrolysis of the mcl-PHA was performed for 20 hours. Growth profile of bacteria and polymer content has been monitored. Three different blends of P(3HB) and hydrolysed P(3HHx-3HO-3HD-3HDD) were prepared by solvent cast method (95/5, 90/10 and 80/20). Studies were then carried out to look into aspects such as thermal, mechanical, microstructural and protein adsorption of the developed blend systems. Biocompatibility assessment was performed by carrying out in vitro interaction of the films with the mouse myoblasts cell line (C2C12). Fluorescence staining by Calcein AM and Ethidium homodimer-1 were performed for confocal microscope. Results and Discussion: Polymers were produced and chemically identified by FTIR, GC-MS and NMR as P(3HB) and P(3HHx-3HO-3HD-3HDD). Additionally P(3HHx-3HO-3HD-3HDD) copolymer was hydrolysed to reduce molecular weight and thereby increase the compatibility between the two polymers within the blend matrix With the increase in the concentration of hydrolysed mcl-PHA copolymer, the glass transition temperature dropped and melting temperature decreased. The surface roughness of blend films was higher when compared to the neat P(3HB). The highest surface roughness (Rq=1.22±0.017μm) was obtained for the blend with 20 wt% addition of P(3HHx-3HO-3HD-3HDD). With the increased concentration of the mcl-PHAs, protein adsorption was higher for the blend composition 80/20 when compared with their blends and neat P(3HB). In vitro biocompatibility studies confirmed higher cell proliferation on the all blends in comparison with TCP at day 1, 3 and 7. SEM images and confocal micrographs confirm high biocompatibility of the cells for the blend films in all the compositions (Figure 1a-c). Figure 1a-c. SEM images of the C2C12 cells seeded on the blend films P(3HB)/P(3HHx-3HO-3HD-3HDD): 95/5, 90/10 and 80/20 at day 7. Conclusion: Both SCL and MCL PHAs were successfully produced and chemically characterised as P(3HB) and P(3HHx-3HO-3HD-3HDD). Three different blend compositions were fabricated and further studied. The most favourable blend for cell proliferation and adhesion was with the maximum amount of mcl-PHAs, 20%wt. In fact blend material demonstrated greater adhesion of the C2C12 cells on its surface when compared to the TCP control. Taken together, these results suggest the potential of the developed SCL-MCL blend system as promising new matrix for soft tissue engineering application. The authors would like to thank technical staff from University College London and ReBiostent EU FP7 project funding. ReBioStent is supported by grant agreement No 604251 from the European Union’s Seventh Programme for research, technological development and demonstration (FP7).