Nerve conduits

Abstract

The invention provides bioresorbable nerve guidance conduits made from polymer blends which include polyhydroxyalkanoates (PHAs). In particular, the invention provides nerve guidance conduits having a body which comprises a polymer blend comprising: (a) from 60 to 98 wt. % of a first component which is a PHA copolymer comprising two or more different medium chain length hydroxyalkanoate monomer units; and (b) from 2 to 40 wt. % of a second component which is either a PHA homopolymer containing a short chain length hydroxyalkanoate monomer unit, or a polylactide (PLA). The invention further relates to polymer blends comprising (a) and (b).

Claims

1. A nerve guidance conduit having a body which comprises a polymer blend comprising: (a) from 60 to 98 wt. % of a first component which is a PHA copolymer comprising two or more different medium chain length hydroxyalkanoate monomer units; and (b) from 2 to 40 wt. % of a second component which is either a PHA homopolymer containing a short chain length hydroxyalkanoate monomer unit, or a polylactide (PLA).

2. A nerve guidance conduit as claimed in claim 1, wherein the first component is a binary or ternary PHA copolymer.

3. A nerve guidance conduit as claimed in claim 1, wherein the PHA copolymer comprises hydroxyalkanoate monomer units which, independently of one another, contain 6 or more carbon atoms.

4. A nerve guidance conduit as claimed in claim 3, wherein the PHA copolymer comprises hydroxyalkanoate monomer units which, independently of one another, contain 8, 10 or 12 carbon atoms.

5. A nerve guidance conduit as claimed in claim 1, wherein the PHA copolymer comprises hydroxyalkanoate units which are independently selected from 3-hydroxy and 4-hydroxyalkanoates.

6. A nerve guidance conduit as claimed in claim 5, wherein each hydroxyalkanoate unit is a medium chain length 3-hydroxyalkanoate.

7. A nerve guidance conduit as claimed in claim 6, wherein each hydroxyalkanoate unit is independently selected from the group consisting of 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD) and 3-hydroxydodecanoate (3HDD).

8. A nerve guidance conduit as claimed in claim 1, wherein the PHA copolymer is poly(3-hydroxyoctanoate-co-3-hydroxydecanoate) or poly(3-hydroxyoctanoate-co-3-hydroxydecanoate-co-3-hydroxydodecanoate).

9. A nerve guidance conduit as claimed in claim 1, wherein the chirality of the hydroxy-substituted carbon atom in each hydroxyalkanoate unit in the PHA copolymer is of the R-configuration.

10. A nerve guidance conduit as claimed in claim 1, wherein the first component is a binary PHA copolymer which contains 3-hydroxydecanoate (3-HD) monomer units in an amount ranging from 60 mol % to 85 mol % (based on the weight average molecular weight, Mw, of the PHA copolymer) and/or 3-hydroxyoctanoate monomer units in an amount from 20 to 35 mol % (based on the weight average molecular weight, Mw, of the PHA copolymer).

11. A nerve guidance conduit as claimed in claim 1, wherein the first component is a ternary PHA copolymer which contains 3-hydroxydecanoate (3-HD) monomer units in an amount ranging from 40 mol % to 60 mol % (based on the weight average molecular weight, Mw, of the PHA copolymer) and/or 3-hydroxyoctanoate monomer units in an amount from 20 to 40 mol % (based on the weight average molecular weight, Mw, of the PHA copolymer).

12. A nerve guidance conduit as claimed in claim 1, wherein the first component is a PHA copolymer which contains 3-hydroxydodecanoate (3-HDD) monomer units in an amount ranging from 10 to 30 mol % (based on the weight average molecular weight, Mw, of the PHA copolymer).

13. A nerve guidance conduit as claimed in claim 1, wherein the PHA copolymer has a molecular weight in a range from 50 to 600 kDa.

14. A nerve guidance conduit as claimed in claim 1, wherein the PHA copolymer is obtained or obtainable by culturing of a microorganism selected from Pseudomonas putida, Pseudomonas oleovorans, Pseudomonas mendocina CH50, Pseudomonas fluorescence, Pseudomonas aeruginosa, Pseudomonas raguenesii, Pseudomonas guezennei, Pseudomonas stutzeri, Pseudomonas cepacia, and Comamonas testosteronii.

15. A nerve guidance conduit as claimed in claim 14, wherein said microorganism is Pseudomonas mendocina CH50.

16. A nerve guidance conduit as claimed in claim 14, wherein said microorganism is grown in a culture medium which comprises glucose or coconut oil as a carbon source.

17. A nerve guidance conduit as claimed in claim 1, wherein the second component of the polymer blend is a PHA homopolymer containing a short chain length hydroxyalkanoate monomer unit.

18. A nerve guidance conduit as claimed in claim 17, wherein the PHA homopolymer comprises hydroxyalkanoate monomer units which each contain 3, 4 or 5 carbon atoms.

19. A nerve guidance conduit as claimed in claim 18, wherein the PHA homopolymer is poly(3-hydroxybutyrate).

20. A nerve guidance conduit as claimed in claim 1, wherein the PHA homopolymer has a molecular weight in a range from 200 kDa to 2 MDa.

21. A nerve guidance conduit as claimed in claim 1, wherein the PHA homopolymer is obtained or obtainable by culturing of a microorganism selected from Cupriavidus necator, Alcaligenes latus, Bacillus cereus, Aeromonas caviae, Rhodospirillum rubrum, Methylobacterium extorquens, Halomonas boliviensis LC1, Bacillus subtilis, and Bacillus megaterium.

22. A nerve guidance conduit as claimed in claim 21, wherein said microorganism is Bacillus subtilis OK2.

23. A nerve guidance conduit as claimed in claim 21, wherein said microorganism is grown in the presence of a culture medium which comprises glucose as a carbon source.

24. A nerve guidance conduit as claimed in claim 1, wherein the second component of the polymer blend is a polylactide (PLA).

25. A nerve guidance conduit as claimed in claim 24, wherein the second component of the polymer blend is poly(L-lactic acid).

26. A nerve guidance conduit as claimed in claim 1, wherein the polymer blend is selected from one of the following: P(3HO-3HD)/P(3HB) P(3HO-3HD-3HDD)/P(3HB) P(3HO-3HD-3HDD)/PLA P(3HO-3HD)/PLA P(3HO-3HD-3HDD)/PLLA P(3HO-3HD)/PLLA P(3HO-3HD-3HDD)/PDLA P(3HO-3HD)/PDLA.

27. A nerve guidance conduit as claimed in claim 1, wherein the first component is present in an amount in a range from 80 to 85 wt. % (based on the total weight of the blend).

28. A nerve guidance conduit as claimed in claim 1, wherein the second component is a PHA homopolymer which is present in an amount in a range from 15 to 25 wt. % (based on the total weight of the blend).

29. A nerve guidance conduit as claimed in claim 1, wherein the second component is a polylactide which is present in an amount in a range from 3 to 10 wt. % (based on the total weight of the blend).

Description

EXAMPLES

(1) The invention will now be described in more detail by way of the following non-limiting examples and with reference to the accompanying figures, in which:

(2) FIG. 1 shows the .sup.1H NMR (upper panel) and .sup.13C NMR (lower panel) of the P(3HO-3HD) polymer produced by P. mendocina CH50 with glucose as the carbon source in Example 2.

(3) FIG. 2 shows the .sup.1H NMR (upper panel) and .sup.13C NMR of the P(3HO-3HD-3HDD) polymer produced by P. mendocina CH50 with coconut oil as the carbon source in Example 3.

(4) FIG. 3 shows the .sup.1H NMR (upper panel) and .sup.13C NMR (lower panel) of the P(3HB) polymer produced by Cupriavidus necator with walnut oil as carbon source in Example 8.

(5) FIG. 4 shows the .sup.1H NMR (upper panel) and .sup.13C NMR (lower panel) of the P(3HO-3HD) polymer produced by Pseudomonas mendocina

(6) CH50 with sugarcane molasses as carbon source in Example 9.

Measurement of Mechanical Properties

(7) In the following examples, tensile strength, Young's modulus and elongation at break are determined using a 5942 Testing Systems (Instron) equipped with 500 N load cell at room temperature. The test is conducted using films which are 5 mm in width and 3.5 to 5.0 cm in length. Before measurement, the thickness and width of the specimen are measured in several places and an average value used to calculate the cross-sectional area. The gauge length of the sample holder is set at 23 mm and a deformation rate of 5 mm per minute for SCL-PHA based materials and 10 mm per minute for MCL-PHA based materials is employed. Young's modulus, tensile strength and elongation at break are calculated from the stress-strain curve and average values calculated for 3-6 specimens. Data analysis is carried out using BlueHill 3 software.

Measurement of Molecular Weight

(8) Unless otherwise specified, the molecular weight of the polymer materials is determined using a PLgel 5 μm MIXED-C (300×7.5 mm) column which is calibrated using narrow molecular weight polystyrene standards from 162 Da to 15,000 kDa. The eluent used was chloroform. 5 mg/mL of the polymer is introduced into the GPC system at a flow rate of 1 mL/min. The eluted polymer is detected with a refractive index detector. Data is collected and analysed using “Agilent GPC/SEC” software.

Example 1—Production and Characterisation of poly(3-hydroxybutyrate) “P(3HB)” from Glucose by Bacillus subtilis OK2

(9) Producer Organism: Bacillus subtilis OK2 (Obtained from the National Institute of Genetics)

(10) Media Composition:

(11) Production Media

(12) Ammonium sulphate: 5 g/L Potassium chloride: 3 g/L Yeast extract: 2.5 g/L (autoclaved at 121° C. for 15 minutes)
Carbon Source Glucose: 35 g/L (autoclaved at 110° C. for 11 minutes) pH of all the media components was adjusted to 6.8

(13) Production: A single colony of Bacillus subtilis OK2 was used to inoculate the autoclaved nutrient broth. This nutrient broth was incubated for 16 hours at 30° C. at 150 rpm. Glucose was used as the sole carbon source. Autoclaved production media (modified Kannan and Rehacek media) was inoculated using the nutrient broth as the seed culture. Inoculated production media was then incubated for 48 hours at 30° C. at 200 rpm.

(14) Harvesting: The cells were harvested at 48 hours by centrifugation at 4600 rpm for 30 minutes. They were washed thrice, first with distilled water followed by 10% ethanol and then again with distilled water. The cells were homogenized using a homogenizer for approximately 15 minutes. The cells were then kept at −20° C. overnight after which they were placed in a freeze dryer for lyophilisation.

(15) Extraction: Polymer was extracted from the cells using the soxhlet extraction method. The cells were treated in a soxhlet apparatus with methanol for 24 hours, under reflux conditions, as a washing step to remove the impurities. After this, the methanol was replaced with chloroform which was used to extract the polymer from the cells. The cells were treated with chloroform for 4 hours under reflux conditions. This chloroform solution was concentrated using a rotary vacuum evaporator and the polymer was precipitated using ice-cold methanol solution. Extraction was continued using the same batch of cells by incubating them twice with chloroform for 24 hours under reflux conditions. This chloroform solution was again concentrated using the rotary vacuum evaporator and the polymer was precipitated using ice-cold methanol solution.

(16) Mechanical properties: Film samples were prepared by casting 10 ml of 10 w/v % polymer solution in chloroform into a glass petri dish (6 cm diameter). The samples were left covered at room temperature until solvent evaporation was complete (monitored by weight change). Mechanical properties were determined using tensile testing with samples cut from the solvent-cast film into strips of 5 mm width and 40 mm length. The thickness of the sample was around 300 μm. The crosshead speed was 10 mm/min.

(17) Elongation at break (ε.sub.U) was determined as 2-4%. Young's modulus (E) of P(3HB) was determined as 1.3 GPa. Ultimate tensile strength (σ.sub.U) reached 26 MPa.

Example 2—Production and Characterisation of poly(3-hydroxyoctanoate-co-3-hydroxydecanoate) “P(3HO-3HD)” from Glucose by Pseudomonas mendocina CH50

(18) Producer organism: Pseudomonas mendocina CH50 obtained from the National Collection of Industrial and Marine Bacteria, NCIMB (deposit No. 10542).

(19) Media composition:

(20) Second stage (Mineral salt medium)

(21) Ammonium sulphate: 0.45 g/L Sodium hydrogen phosphate: 3.42 g/L Potassium dihydrogen phosphate: 2.38 g/L
Production media (Mineral salt medium) Ammonium sulphate: 0.50 g/L Sodium hydrogen phosphate: 3.80 g/L Potassium dihydrogen phosphate: 2.65 g/L
Carbon source glucose: 20 g/L Trace element solution: 1 ml/L Magnesium sulphate heptahydrate: 0.8 g/L

(22) Production: A single colony of Pseudomonas mendocina CH50 was used to inoculate the autoclaved nutrient broth. This nutrient broth was incubated for 16 hours at 30° C. at 150 rpm. Autoclaved second stage media was inoculated using the nutrient broth culture as the seed culture. Inoculated second stage media was then incubated at 30° C. at 150 rpm until the optical density reached 1.6 without dilution. This was used as the inoculum to inoculate the production media (10% culture volume). Inoculated production media was then incubated for 48 hours at 30° C. at 200 rpm.

(23) Harvesting: The cells were harvested at 48 hours by centrifugation at 4600 rpm for 30 minutes. They were washed thrice, first with distilled water followed by 10% ethanol and then again with distilled water. The cells were homogenized using a homogenizer for approximately 15 minutes. The cells were then kept at −20° C. overnight after which they were placed in a freeze dryer for lyophilisation.

(24) Extraction: Polymer was extracted from the cells using the soxhlet extraction method. The cells were incubated in the soxhlet with methanol for 24 hours under reflux conditions as a washing step to remove the impurities. After this, the methanol was replaced with chloroform which was used to extract the polymer from the cells. Extraction was continued using the same batch of cells by incubating them with chloroform for 24 hours under reflux conditions. This chloroform solution was concentrated using a rotary vacuum evaporator and the polymer was precipitated using ice-cold methanol solution. Yield: 43%, dry cell weight 0.57 g/l.

(25) Characterisation: The resulting polymer was characterised by GC-MS and NMR. The GC-MS results are presented in Table 1:

(26) TABLE-US-00001 TABLE 1 HO content HD content (mol %) (mol %) Mean 26.2 73.8 SD 1.0 1.7

(27) .sup.1H NMR and .sup.13C NMR are presented in FIG. 1.

(28) Thermal and morphological properties: The resulting P(3HO-3HD) polymer is a semi-crystalline polymer characterized by slow crystallization. Melting of the crystalline phase was observed only for aged samples. The crystalline phase was not detected by differential scanning calorimetry after melting of the polymer in a first heating cycle and cooling the sample at the rate of 20 K/min. The glass transition temperature determined by differential scanning calorimetry was in the range between −41° C. to −46° C. The crystalline phase of P(3HO-3HD) melted between 26 to 66° C. The highest melting rate (endothermic peak maximum) was 57±2° C. Enthalpy of fusion (crystallinity degree) changed with polymer storage and for polymer aged at room temperature (for a period of 5 weeks) reached 27±2 J/g.

(29) TABLE-US-00002 TABLE 2 PHA Tg (° C.) Tm (° C.) ΔH (J/g) P(3HD-co-3HO) −45.1 54.3 19.0

(30) Mechanical properties: Film samples were prepared by casting 10 ml of 10 w/v % polymer solution in chloroform into a glass petri dish (6 cm diameter). The samples were left covered at room temperature until solvent evaporation was complete (monitored by weight change). Mechanical properties were determined by tensile testing using samples cut from the solvent-cast film into strips of 5 mm width and 40 mm length. The thickness of the sample was around 300 μm. The crosshead speed was 10 mm/min.

(31) P(3HO-3HD) is a soft ductile polymer with elongation at break (ε.sub.U) 580±30%. Young's modulus (E) of P(3HO-3HD) was determined as 8.7±1.1 MPa. Ultimate tensile strength (σ.sub.U) reached 10.4±1.0 MPa.

(32) Molecular weight (Mw) and polydispersity index (PDI) were determined by GPC. Mw was determined as 340 kDa and PDI as 2.7.

Example 3—Production and Characterisation of poly (3-hydroxyoctanoate-co-3-hydroxydecanoate-co-3-hydroxydodecanoate) “P(3HO-3HD-3HDD)” from coconut oil by Pseudomonas mendocina CH50

(33) Producer organism: Pseudomonas mendocina CH50 obtained from NCIMB.

(34) Media composition:

(35) Second stage (Mineral salt medium)

(36) Ammonium sulphate: 0.45 g/L Sodium hydrogen phosphate: 3.42 g/L Potassium dihydrogen phosphate: 2.38 g/L
Production media (Mineral salt media) Ammonium sulphate: 0.50 g/L Sodium hydrogen phosphate: 3.80 g/L Potassium dihydrogen phosphate: 2.65 g/L
Carbon source Coconut oil: 20 g/L (obtained from Sigma Aldrich) Trace element solution: 1 ml/L Magnesium sulphate heptahydrate: 0.8 g/L

(37) Production: A single colony of Pseudomonas mendocina CH50 was used to inoculate the autoclaved nutrient broth. This nutrient broth was incubated for 16 hours at 30° C. at 150 rpm. Coconut oil was used as the sole carbon source. Autoclaved second stage media (MSM media) was inoculated using the nutrient broth culture as the seed culture. Inoculated second stage media was then incubated at 30° C. at 150 rpm until the optical density reached 1.6 without dilution. This was used as the inoculum to inoculate the production media (MSM media) (10% of the culture volume). The inoculated production media was then incubated for 48 hours at 30° C. at 200 rpm.

(38) Harvesting: The cells were harvested at 48 hours by centrifugation at 4600 rpm for 30 minutes. They were washed thrice, first with distilled water followed by 10% ethanol and then again with distilled water. The cells were homogenized using a homogenizer for approximately 15 minutes. The cells were then kept at −20° C. overnight after which they were placed in a freeze dryer for lyophilisation.

(39) Extraction: Polymer was extracted from the cells using the soxhlet extraction method. The cells are incubated in the soxhlet with methanol for 24 hours at 90° C. under methanol refluxing conditions to remove the impurities. After this, the methanol solution was replaced with the chloroform which was used to extract the polymer from the cells. Extraction was continued using the same batch of cells by incubating them with chloroform for 24 hours under refluxing conditions. This chloroform solution was concentrated using the rotary vacuum evaporator and the polymer was precipitated using ice-cold methanol solution. Yield: 54% dry cell weight, 1.43 g/l.

(40) Characterisation: The resulting polymer was characterised by GC-MS and NMR. The GC-MS results are presented in Table 3:

(41) TABLE-US-00003 TABLE 3 HO content HD content HDD content (mol %) (mol %) (mol %) Mean 30.4 48.4 21.2 SD 2.1 0.8 2.0

(42) .sup.1H NMR and .sup.13C NMR are presented in FIG. 2.

(43) Thermal and morphological properties: P(3HO-3HD-3HDD) is a semi-crystalline polymer which is characterized by slow crystallisation. Melting of the crystalline phase was observed only for aged samples. The crystalline phase was not detected by differential scanning calorimetry after melting the polymer in a first heating cycle and cooling the sample at the rate of 20 K/min. The glass transition temperature determined by differential scanning calorimetry was in the range between −42° C. to −45° C. The crystalline phase of P(3HO-3HD-3HDD) melted between 25 to 56° C. The highest melting rate (endothermic peak maximum) was 48±2° C. Enthalpy of fusion (crystallinity degree) changed with polymer storage and for polymer aged at room temperature (for a period of 5 weeks) reached 16±1 J/g.

(44) TABLE-US-00004 TABLE 4 PHA Tg (° C.) Tm (° C.) ΔH (J/g) P(3HO-co-3HD-co-3HDD) −42.8 48.6 11.4

(45) Mechanical properties: Film samples were prepared by casting 10 ml of 10 w/v % polymer solution in chloroform into a glass petri dish (6 cm diameter). The samples were left covered at room temperature until solvent evaporation was complete (monitored by weight change). Mechanical properties were determined by tensile testing using samples cut from the solvent-cast film into strips of 5 mm width and 40 mm length. The thickness of the sample was around 300 μm. The crosshead speed was 10 mm/min.

(46) P(3HO-3HD-3HDD) is a soft ductile polymer with elongation at break (ε.sub.U) 580±50%. Young's modulus (E) of P(3HO-3HD-3HDD) was determined as 2.1±0.1 MPa. Ultimate tensile strength (σ.sub.U) reached 6.0±1.0 MPa.

(47) Molecular weight (Mw) and polydispersity index (PDI) were determined by GPC. Mw was determined as 333 kDa and PDI as 2.37.

Example 4—Production of Blends

(48) All PHAs used in the procedure were purified by dissolving in chloroform followed by precipitation with methanol solution. This was repeated several times. The PLLA was a commercially available product (PL38 PURASORB) which was used without further purification.

(49) Blends were produced by dissolving the blend components in a common solvent, chloroform, which is a good solvent for all PHAs and PLLA. Film samples were prepared by solvent casting of polymer solutions.

(50) Polymer solutions were prepared by completely dissolving specified amounts of the first and second blend components in chloroform to achieve a final total polymer concentration of 5 w/v %. The blend solution was kept on the magnetic stirrer to allow mixing for 24 hours at room temperature. 10 ml of the resulting polymer solution was poured into a glass petri dish (6 cm diameter) and left covered at room temperature until solvent evaporation was complete (monitored by weight change).

Example 5—Testing of Blends

(51) The blends prepared according to Example 4 were subjected to various tests to determine their mechanical properties. As a comparison, the same tests were carried out in respect of the known P(3HB)/P(3HO) blends (Lizarraga-Valderrama et al., Engineering in Life Sciences 15(6): 612-621, 2015).

(52) Tensile Strength Testing: The following test method was used to determine tensile properties of the blend materials in the form of films (less than 1.0 mm in thickness).

(53) Apparatus: Device for Measurements of Specimen Geometry: LUJII 150-mm Electronic Digital Caliper or equivalent device accurate and precise to 0.01 mm. Tensile Tester: Instron Model 5940 Single Column Tabletop Testing System with a 0.5 kN load cell or equivalent. Gripping Devices: Instron 2710-102 Advanced Screw Side-Action Grips: capacity—500 N. Grip Faces: Rubber coated flat faces (Instron, Cat.: 2702-002)

(54) Materials: Thin films (thinner than 1 mm) of PHA-based materials were prepared by solvent casting. After the films were dried to constant weights, they were packed into non-sealed polyethylene bags and stored at room temperature for 6 weeks.

(55) Method: Preparation of Test Specimens: Cut strips with width of approximately 5 mm from a test film. A circular film disk prepared in a 60 mm Petri dish can be cut into 5 strips with the shortest strips around 36 mm. Cut at least 4 specimens. No specimen shall vary by more than 2% in width along its entire length. Utmost care must be exercised in cutting specimens to prevent nicks and tears along the edges of the specimen that are likely to cause premature failure.

(56) Testing: Measure and record the thickness of the test specimen to an accuracy of 0.01 mm, at least, in five different places within the gauge length area. Set the initial gauge length (grip separation) at 23.0 mm and the rate of grip separation at 10.0 mm/min. Place the specimen in the grips of the testing machine, taking care to align the long axis of the specimen with an imaginary line joining the points of attachment of the grips to the machine. The specimen should be aligned as perfectly as possible with the direction of pull so that no rotary motion that may induce slippage will occur in the grips. Tighten the grips evenly and firmly to the degree necessary to minimize slipping of the specimen during testing. Start the test and record the load versus extension. Repeat the testing for the series of specimens prepared.

(57) Fixing a specimen of film sample in the grips of the testing machine always results in a degree of bending of the specimen. This results in an actual length of the sample larger than the set separation between the grips. Therefore the raw load (tensile stress) vs strain curves do not start from “0” separation distance. The initial specimen length is corrected by adding the separation distance, where load starts increasing, to the set distance between the grips. This correction factor is also used for correcting the current specimen deformation by deducing it from the measured separation.

(58) Calculations:

(59) Tensile Strength: Tensile stress (σ) is calculated by dividing the load (F) at a specific time point by the original cross-sectional area (A). The result is expressed in megaPascals (MPa) and reported to three significant figures:

(60) $\sigma =\frac{F}{A_0}$
The ultimate tensile strength is defined as the maximal value of tensile stress in the stress-strain curve.

(61) Elongation: Percent elongation (ε) is calculated by dividing the corrected distance (l.sub.i) of grip separation by the corrected initial length of specimen (l.sub.0) and multiplying by 100:

(62) $\mathrm{.Math.}=\frac{l_i-l_0}{l_0}\times 100=\frac{\Delta l}{l_0}\times 100$

(63) Young's Modulus: Young's modulus is calculated as a tangent to the initial linear portion of the stress-strain curve. Obtain the stress-strain curve, select a linear region usually between 0.5 to 1.5% of elongation of the specimen. Use data from this region to calculate a tangent using appropriate software. The result is expressed in gigapascals (GPa) and reported to three significant figures.

(64) Results:

(65) TABLE-US-00005 TABLE 5 Mechanical Characterisation of PHA polymer components Mechanical P(3HB) P(3HO-3HD) P(3HO-3HD-3HDD) Properties Example 1 Example 2 Example 3 σ, MPa 26 10.4 6 E, MPa 1300 8.7 2.1 ε.sub.b, % 2-4 580 580

(66) TABLE-US-00006 TABLE 6 Mechanical Characterisation of known P(3HB)/P(3H0) BLENDS Mechanical P(3HB)/P(3HO) P(3HB)/P(3HO) P(3HB)/P(3HO) Properties 25:75 50:50 75:25 σ, MPa 0.71 2.17 17.80 E, MPa 12 21 140 ε.sub.b, % 73.8 94.1 41.30

(67) TABLE-US-00007 TABLE 7 Mechanical Characterisation of P(3HB)/P(3HO-3HD) BLENDS P(3HB)/ P(3HB)/ P(3HB)/ P(3HB)/ P(3HB)/ Mechanical P(3HO-3HD) P(3HO-3HD) P(3HO-3HD) P(3HO-3HD) P(3HO-3HD) Properties 5:95 10:90 15:85 20:80 25:75 σ, MPa 10 5.7 4.1 3.2 1.9 E, MPa 12 19 19 22 28 ε.sub.b, % 540 430 330 280 200

(68) TABLE-US-00008 TABLE 8 Mechanical Characterisation of P(3HB)/P(3HO-3HD-3HDD) BLENDS P(3HB)/P(3HO-3HD-3HDD) Mechanical Properties 5:95 σ, MPa 8.4 E, MPa 51 ε.sub.b, % 510

(69) TABLE-US-00009 TABLE 9 Mechanical Characterisation of PLLA/P(3HO-3HD) BLENDS PLLA/ PLLA/ PLLA/ PLLA/ PLLA/ Mechanical P(3HO-3HD) P(3HO-3HD) P(3HO-3HD) P(3HO-3HD) P(3HO-3HD) Properties 5:95 10:90 15:85 20:80 25:75 σ, MPa 14.3 4.9 3.6 6.5 7.2 E, MPa 84 21 240 470 500 ε.sub.b, % 640 250 10.5 5.7 4.2

(70) TABLE-US-00010 TABLE 10 Mechanical Characterisation of PLLA/P(3HO-3HD-3HDD) BLENDS PLLA/P(3HO-3HD-3HDD) Mechanical Properties 5:95 σ, MPa 5.8 E, MPa 40 ε.sub.b, % 350

Example 6—Dip Moulding Preparation of Tubes for Use as Nerve Conduits

(71) P(3HB)/P(3HO-3HD) tubes with composition 15/85 were made from polymers by dip moulding using polymer solutions in chloroform.

(72) Solutions of a polymer mixture of P(3HB)/P(3HO-3HD) were prepared by dissolving the required amounts of polymer in chloroform in order to obtain a total polymer concentration of 6 wt %. Clear solutions were used to form tubes on a cylindrical stainless steel mandrel with diameter 1.8 mm. Tubes were formed by multiple dipping of the mandrel into the polymer solutions. The tube formation was carried out at 25° C. Dipping and withdrawal rate was 200 mm/min. After complete mandrel withdrawal from the polymer solution, 30 sec drying time was used before conducting the next dip. After each series of 5 dippings, 4 min drying time was applied.

(73) The total number of dips was 20 and produced polymeric tubes with a wall thickness of 150 μm. After the last dipping the tubes were left on a mandrel for complete solvent evaporation at room temperature for 5 days.

Example 7—Extrusion of Tubes for Use as Nerve Conduits

(74) Porous P(3HB)/P(3HO-3HD) tubes with composition 15/85 were made by extrusion of a pre-mix of the polymers with 70 wt % of a powder of sodium chloride. Ground and sieved NaCl with a particle fraction smaller than 50 μm was used as porogen. For the preparation of the pre-mix the required amount of polymers were dissolved in chloroform in order to obtain polymer concentration of 8 wt %. The required amount of NaCl particles was added to the polymer solution under mechanical stirring. After dispersion of the NaCl particles in the polymer solution, the solution was poured into glass trays and the solvent was allowed to evaporate for 3 days at room temperature.

(75) The pre-mix of polymer blend with NaCl was cut into small pieces using a kitchen blender. The pre-mix was used for tube extrusion at barrel temperature of 130° C. to produce tubes with a wall thickness around 200 μm. The extruded tubes were further processed to achieve porous tubes by leaching NaCl in water. The tubes were kept in a portion of water under gentle stirring for 7 days. The water was replaced on a daily basis. After washing out NaCl, the tubes were dried at room temperature resulting in porous tubes.

Example 8—Production and Characterisation of poly(3-hydroxybutyrate) “P(3HB)” from Walnut Oil by Cupriavidus necator

(76) Producer Organism: Cupriavidus necator (Formerly Known as Ralstonia eutropha)

(77) Production Media

(78) Ammonium chloride: 4 g/L Disodium hydrogen phosphate. 12 H.sub.2O: 11 g/L Potassium dihydrogen phosphate: 1.2 g/L (autoclaved at 121° C. for 15 minutes)
Carbon Source Walnut oil: 20 g/L (autoclaved at 121° C. for 15 minutes) (obtained from Waitrose Ltd.)

(79) Trace Element Solution: 1 ml/L (Filter Sterilized)

(80) Magnesium Sulphate Heptahydrate: 1.4 g/L (Autoclaved at 121° C. for 15 Minutes)

(81) Production: A single colony of Cupriavidus necator was used to inoculate the autoclaved nutrient broth. This nutrient broth was incubated for 24 hours at 30° C. at 150 rpm. Autoclaved production media was inoculated using the nutrient broth as the seed culture. Inoculated production media was then incubated for 48 hours at 30° C. at 200 rpm.

(82) Harvesting: The cells were harvested at 48 hours by centrifugation at 4600 rpm for 30 minutes. They were washed thrice, first with distilled water followed by 10% ethanol and then again with distilled water. The cells were homogenized using a homogenizer for approximately 15 minutes. The cells were then kept at −20° C. overnight after which they are placed in the freeze dryer for lyophilisation.

(83) Extraction: Polymer was extracted from the cells using soxhlet extraction method. The cells were incubated in the soxhlet with methanol for 24 hours under methanol refluxing conditions as a washing step to remove the impurities. After this, the methanol was replaced with chloroform which was used to extract the polymer from the cells. The cells are incubated with the chloroform for 4 hours under reflux conditions. This chloroform solution was concentrated using the rotary vacuum evaporator and the polymer was precipitated using ice-cold methanol solution. Extraction was continued using the same batch of cells by incubating them twice with chloroform solution at 70° C. for 24 hours. This chloroform solution was again concentrated using the rotary vacuum evaporator and the polymer was precipitated using ice-cold methanol solution. Yield: 50% dry cell weight, 1.97 g/l.

(84) Characterisation: The resulting polymer was characterised by GC-MS and NMR. .sup.1H NMR and .sup.13C NMR are presented in FIG. 3.

(85) Thermal and morphological properties: P(3HB) is a semi-crystalline polymer. Glass transition temperature determined by differential scanning calorimetry was in the range between 0° C. to 3° C. The crystalline phase of P(3HB) melted in the wide temperature range between 120 to 175° C. The highest melting rate (endothermic peak maximum) was 171±2° C. Enthalpy of fusion (crystallinity degree) changed with polymer storage and for polymer aged at room temperature reached 88±2 J/g.

(86) TABLE-US-00011 TABLE 11 PHA Tg (° C.) Tm (° C.) ΔH (J/g) P(3HB) 2.9 168.4 71.4

(87) Mechanical properties: Film samples were prepared by casting 10 ml of 10 w/v % polymer solution in chloroform into a glass petri dish (6 cm diameter). The samples were left covered at room temperature until solvent evaporation was complete (monitored by weight change). Mechanical properties were determined by tensile testing using samples cut from the solvent-cast film into the strips of 5-mm width and 40-mm long. The thickness of the sample was around 200 μm. The crosshead speed was 5 mm/min.

(88) P(3HB) is a rigid polymer with elongation at break (ε.sub.U) 2-4%. Young's modulus (E) of P(3HB) was 1.3±0.2 GPa. Ultimate tensile strength (σ.sub.U) reached 26±2 MPa.

(89) Molecular weight (Mw) and polydispersity index (PDI) were determined by GPC. Mw was determined as 606 kDa and PDI as 2.5.

(90) The P(3HB) polymer may be used as the second component in a blend according to the invention.

Example 9—Production and Characterisation of poly (3-hydroxyoctanoate-co-3-hydroxydecanoate) “P(3HO-3HD)”—23 mol % HO, 77 mol % HD

(91) Producer Organism: Pseudomonas mendocina CH50

(92) Media Composition:

(93) Second Stage (Mineral Salt Medium)

(94) Ammonium sulphate: 0.45 g/L Sodium hydrogen phosphate: 3.42 g/L Potassium dihydrogen phosphate: 2.38 g/L (autoclaved at 121° C. for 15 minutes)
Production Media (Mineral Salt Media) Ammonium sulphate: 0.50 g/L Sodium hydrogen phosphate: 3.80 g/L Potassium dihydrogen phosphate: 2.65 g/L (autoclaved at 121° C. for 15 minutes)
Carbon Source Sugarcane molasses: 20 g/L (bought from Holland and Barret) (autoclaved at 110° C. for 10 minutes)
Trace Element Solution: 1 ml/L (Filter Sterilized) Magnesium sulphate heptahydrate: 0.8 g/L (autoclaved at 121° C. for 15 minutes)

(95) Production: A single colony of Pseudomonas mendocina CH50 was used to inoculate the autoclaved nutrient broth. This nutrient broth was incubated for 16 hours at 30° C. at 150 rpm. Autoclaved second stage media was inoculated using the nutrient broth culture as the seed culture. Inoculated second stage media was then incubated at 30° C. at 150 rpm until the optical density reached 1.6 without dilution. This was used as the inoculum to inoculate the production media (10% culture volume). Inoculated production media was then incubated for 48 hours at 30° C. at 200 rpm.

(96) Harvesting: The cells were harvested at 48 hours by centrifugation at 4600 rpm for 30 minutes. They were washed thrice, first with distilled water followed by 10% ethanol and then again with distilled water. The cells were homogenized using a homogenizer for approximately 15 minutes. The cells were then kept at −20° C. overnight after which they are placed in the freeze dryer for lyophilisation.

(97) Extraction: Polymer was extracted from the cells using soxhlet extraction method. The cells were incubated in the soxhlet with methanol for 24 hours under reflux conditions as a washing step to remove the impurities. After this, the methanol was replaced with chloroform which was used to extract the polymer from the cells. Extraction was carried out by incubating the cells with chloroform solution for 24 hours under reflux conditions. This chloroform solution was concentrated using the rotary vacuum evaporator and the polymer was precipitated using ice-cold methanol solution. Yield: 37.5% dry cell weight, 0.46 g/l.

(98) Characterisation: The resulting polymer was characterised by GC-MS and NMR. .sup.1H NMR and .sup.13C NMR are presented in FIG. 4.

(99) Thermal and morphological properties: P(3HO-3HD) is a semi-crystalline polymer which is characterized by slow crystallization. Melting of the crystalline phase was observed only for aged samples. The crystalline phase was not detected by differential scanning calorimetry after melting the polymer in first heating cycle and cooling the sample at the rate of 20 K/min. Glass transition temperature determined by differential scanning calorimetry was in the range between −41° C. to −46° C. The crystalline phase of P(3HO-3HD) melted between 28 to 70° C. The highest melting rate (endothermic peak maximum) was 53±2° C. Enthalpy of fusion (crystallinity degree) changed with polymer storage and for polymer aged at room temperature reached 21±2 J/g.

(100) TABLE-US-00012 TABLE 12 PHA Tg (° C.) Tm (° C.) ΔH (J/g) P(3HO-co-3HD) −41.5 53.0 21.0

(101) Mechanical properties: Film samples were prepared by casting 10 ml of 10 w/v % polymer solution in chloroform into a glass petri dish (6 cm diameter). The samples were left covered at room temperature until solvent evaporation was complete (monitored by weight change). Mechanical properties were determined by tensile testing using samples cut from the solvent-cast film into the strips of 5-mm width and 40-mm long. The thickness of the sample was around 300 μm. The crosshead speed was 10 mm/min.

(102) P(3HO-3HD) is a soft ductile polymer with elongation at break (ε.sub.U) 635±25%. Young's modulus (E) of P(3HO-3HD) was 11.4±0.3 MPa. Ultimate tensile strength (σ.sub.U) reached 15.1±0.3 MPa.

(103) Molecular weight (Mw) and polydispersity index (PDI) were determined by GPC. Mw was determined as 449.3 kDa and PDI as 1.5.

(104) The P(3HO-3HD) polymer may be used as the first component in a blend according to the invention.