Production of Polyhydroxyalkanoates and their application for coronary stent development

Development of coronary artery stents have revolutionised the treatment of coronary artery disease. However, the currently available stents have several limitations and side effects, which still need to be addressed. Within this group of biomaterials Polyhydroxyalkanoates (PHAs), which are biopolyesters produced by bacteria, have a great potential in coronary stent development. They are biodegradable, non-toxic and biocompatible. PHAs have a broad range of properties, which can be tailored based on the requirements of the application by an appropriate bacterial strain, carbon source or fermentation conditions.

The main aim of this study was the production of PHAs and their application as a platform material for coronary stent. Poly(3-hydroxybutyrate), P(3HB), short chain length PHA (scl-PHA), was obtained from B.subtilis OK2 using glucose as the sole carbon source. P. mendocina was selected for the economical production of medium chain length PHA (mcl-PHAs) using waste frying oil as the carbon source. Produced mcl-PHA copolymer was hydrolysed in order to obtain lower molecular weight PHA (oligo-PHA), which was used as the plasticising agent for P(3HB) in order to alter material properties of the scl-PHA polymer. Novel P(3HB)/oligo-PHA blends, using different ratios of P(3HB) and oligo-PHA, which were 100:0 (P(3HB), 95:5 (P(3HB): oligo-PHA), 90:10 (P(3HB): oligo-PHA) and 80:20 (P(3HB): oligo-PHA) have been developed. Addition of oligo-PHA to P(3HB) resulted not only in the enhancement of mechanical properties of the material, but also improved surface topography and increased the biocompatibility of the scaffolds in comparison to neat P(3HB).

The P(3HB)/oligo-PHA 90/10 blend was selected for the fabrication of radiopaque composites using addition of 1%, 3% and 5% wt barium sulphate. BaSO4 is well-known contrast agent, commonly used in biomedical applications. The composites were fully characterised with respect to material properties, surface topography and biocompatibility. Preliminary studies, performed by MicroCT confirmed the radiopacity of the composite material. The composites exhibited an increased surface roughness, lower contact angle values and higher protein adsorption in comparison to the neat blend without barium sulphate. The observed cell viability was significantly better in comparison to standard Tissue Culture Plastic (TCP).

The P(3HB)/oligo-PHA 90:10 blend was selected as a base material for tube manufacturing by the dip moulding technique. Mechanical properties of the blend tubes were similar to those obtained for flat films, except for tensile strength, which was slightly higher in tubes. An incubation of tubes in DMEM media at 37°C for 7 weeks resulted in the decrease values of tensile strength, Young’s Modulus values and elongation at break in comparison to the tubes stored at room temperature without any media. Additionally, In vitro degradation study was carried out on the tubes. After 6 months of incubation in PBS,The P(3HB)/oligo-PHA 90/10 tubes underwent a 30% weight loss. pH of the media has dropped insignificantly, due to release of slightly acidic degradation products. Scanning Electron Microscope (SEM) images confirmed an enlarged porosity in the tube wall with an increased time of incubation.

Furthermore, an investigation of the P(3HB)/oligo-PHA 90/10 tubes coated with PCL-PEG550 with incorporated drugs: rapamycin or tacrolimus was carried out for the development of a drug eluting biodegradable stent. The percentage viability of the HMEC-1 cells was significantly higher on the tubes with tacrolimus compared to the tubes with rapamycin. Controlled release of both drugs was observed within 90 days, which is an encouraging result for the development of biodegradable drug eluting stents.There was a clear cytotoxic effect of rapamycin on the HMEC-1 cells, whereas, presence of tacrolimus did not exhibit similar effect on cell attachment and viability.

Haemocompatiblity studies were performed in order to investigate the reaction of blood cells after direct contact with PHAs polymer samples. Obtained results confirmed the non-haemolytic effect of PHAs on erythrocytes and low risk of thrombogenicity. Although all tested materials demonstrated slightly elevated levels of monocytes and neutrophil activation in comparison to fresh whole blood, obtained values were much lower in comparison to poly(L-lactic acid) (PL38), a well-established medical polymer currently used for coronary stent application.

In conclusion, the results obtained in this work confirmed that PHAs have an excellent potential as suitable platform matrices for coronary stent application.