Nerve tissue engineering using blends of Polyhydroxyalkanoates

PHAs are a family of linear polyesters consisting of 3, 4, 5 and 6-hydroxyacids, synthesized by a variety of bacterial species. They can be produced from renewable carbon sources, they are biodegradable, biocompatible and exhibit thermoplastic and elastomeric properties. Therefore, PHAs are a potential substitute for petroleum-derived plastics and have a wide range of biomedical applications, particularly in tissue engineering and drug delivery. The properties of PHA devices can be successfully modified through the fabrication of composites and blends. Such modifications allow for further tailoring of properties such as biocompatibility, mechanical properties and degradation times required for specific physiological conditions. PHAs have been used for the manufacture of a wide variety of surgical materials and implants in the areas of skin, nerve, dental, cardiac and bone tissue engineering. The latter two fields being where the PHAs have been the most extensively explored. These unique polymers possess significant advantages compared to their chemically-synthetized counterparts. Their structural diversity, adaptable properties, controllable surface degradation and biocompatibility with a wide range of cells, place PHAs as a biomaterial with immense potential for biomedical applications.

The main aim of this project was to explore the use of Polyhydroxyalkanoates in nerve tissue engineering ultimately leading to the development of novel PHA-based nerve guidance conduits (NGCs). Production of the scl-PHA, P(3HB) and the mcl-PHA, P(3HO) through bacterial fermentation was performed to obtain appropriate amounts of polymer for chemical characterisation and further manufacturing of scaffolds for nerve tissue engineering applications. The average polymer yields achieved using a 20 L fermenter were 42.27 % dcw of P(3HO) using Pseudomonas mendocina CH50 and 49.22 % dcw of P(3HO) using Bacillus cereus SPV respectively.

Novel PHA blends as resorbable biomaterials for use in the manufacture of NGCs were fabricated. PHA blend films with varying ratios of poly(3-hydroxyoctanoate)/poly(3-hydroxybutyrate), (P(3HO)/P(3HB), were produced using the solvent-casting method. Neat films of P(3HO) and P(3HB) along with 25:75, 50:50 and 75:25 blend films of P(3HO)/P(3HB) were characterised with respect to their chemical, material and biological properties in order to evaluate them as potential base materials for nerve tissue engineering. In the surface analysis the blends exhibited higher values of roughness compared with the neat films. The DSC characterisation of the blends confirmed that P(3HO) and P(3HB) formed immiscible blends. FTIR and XRD analysis of the blends showed a decrease in the crystallinity with increase in the proportion of P(3HO). An increase in the stiffness of the blends was observed when the proportion of P(3HB) increased. Although all of the blends were biocompatible with NG108-15 neuronal cells, the 25:75 P(3HO)/P(3HB) blend showed significantly better support for the growth and differentiation of these cells. Mechanical properties of PHA blends corresponded to the reported properties of peripheral nerves providing potential materials for their use as base material for the manufacture of NGCs.

The 25:75 P(3HO)/P(3HB) blend was used for the manufacturing of electrospun fibres as resorbable scaffolds for their use in the manufacture of NGCs as lumen structure. The biocompatibility of these fibres with NG108-15 neuronal cells as well as the influence of RN22 Schwann cells on their growth and differentiation was studied. Highly aligned and uniform fibres with varying diameters were successfully fabricated by controlling electrospinning parameters. The resulting fibre diameters were 2.42 ± 0.34 μm, 3.68 ± 0.26 μm and 13.50 ± 2.33 μm for small, medium and large fibres respectively. The effect of the fibers on the growth of neuronal cells NG108-15 was investigated by live/dead cell test. Cell migration observed on the electrospun fibres showed directional alignment in accordance with the direction of the fibres. The correlation between 25:75 P(3HO)/P(3HB) micro-fibre diameter and neuronal growth under two conditions; individually and in co-culture with RN22 Schwann cells were evaluated. This was investigated using two types of cell staining; live/dead cell test and anti-beta tubulin immunolabelling. Results displayed from both assays revealed that all 25:75 P(3HO)/P(3HB) blend fibre groups were able to support growth and guide aligned distribution of neuronal cells when grown individually and in the presence of RN22 Schwann cells. Results also revealed a direct correlation between fiber diameter and neuronal growth and differentiation. Although neuronal cell viability was similar for all the substrates (approximately 99%) except on glass, large fibres supported the highest number of live neuronal cells grown individually compared to the rest of substrates.

Biocompatibility and neuron regenerating properties of various Bioactive glasse (BG)/PHA blend composites were assessed in order to study their suitability for peripheral nerve tissue applications. BG/PHA blend composites were fabricated using Bioglass® 45S5 (BG1) and BG 1393 (BG2) along with 25:75 P(3HO)/P(3HB) blend. Different proportions of each BG (0.5, 1.0 and 2.5 % w/v) were used to determine the BG concentration that resulted in superior neuronal growth and differentiation of NG108-15 in single culture and in co-culture system with Schwannoma RN22 cells. NG108-15 cells displayed good growth and differentiation performance on all the PHA blend composites showing that both BGs (BG1 and BG2) have good biocompatibility at 2.5, 1.0 and 0.5 % w/v in the PHA blend solution. The Young’s modulus values displayed by all the PHA blend/BG composites ranged from 385.6 MPa to 1792.6 MPa, which are much higher than that of peripheral nerves. However, the tensile strength obtained in PHA blend/BG1 (1% w/v) (10.0 ± 0.6 MPa) was found to be similar to that of rabbit peroneal nerve measured in another study. Therefore, although PHA
blend/BG1 (1% w/v) does not provide the adequate elasticity, it has the appropriate strength that NGCs require. PHA blend/BG1 (1% w/v) showed the best performance in supporting growth and neuronal differentiation of NG108-15 amongst all the substrates in all the cell culture experiments (Live/dead cell test, neurite outgrowth assessment on NG108-15 neuronal cell and on NG108-15/Schwann cell co-cultures). Moreover, neurite extension found on the PHA blend/BG1 (1% w/v) was remarkable as neurites formed a complex connection network. No correlation was found between the surface characteristics (roughness, hydrophilicity and pore size) of PHA blend/BGs at different concentrations of BG and cell growth and differentiation.

Two prototypes of NGCs were fabricated using blends of PHAs as base materials. Since 75:25 P(3HO)/P(3HB) blend films have shown to possess the required flexibility to be implanted in peripheral nerves, this polymer blend was chosen for manufacturing of NGCs. The NGC prototype 1 consisted of a nerve guidance conduit made from 75:25 P(3HO)/P(3HB) blend with luminal electrospun aligned fibres fabricated with 25:75 P(3HO)/P(3HB) blend. The prototype 2 comprised of a hollow tube made from 75:25 P(3HO)/P(3HB) blend manufactured by dip-moulding using various dipping conditions. The NGC prototype 2 was implanted in rats for in vivo work, which was carried out in Neuroscience Institute Cavalieri Ottolenghi, Italy. The regenerated nerves were removed and processed for high-resolution light microscopy, transmission electron microscopy and immunohistochemistry analysis. Myelinated and unmyelinated fibres, Schwann cells, connective tissue and vessels were observed in the middle of NGCs through transmission electron microscopy. Although, quantitative estimation of myelinated and unmyelinated nerve fibres was not carried out, these preliminary in vivo results revealed the potential of 75:25 P(3HO)/P(3HB) NGCs to support regeneration.

To summarize, the biocompatibility and mechanical properties of theP(3HO)/P(3HB) blends made these polymers excellent materials for nerve tissue engineering and for the manufacture of novel nerve guidance conduits.