Peripheral nerve growth conduit

Abstract

The present invention provides a peripheral nerve growth conduit for peripheral nerve repair, in particular conduits through which peripheral nerves can grow. The conduit includes poly--caprolactone (PCL). Preferably, the inner (luminal) surface of the conduit comprises pits having a depth of 1-4m. Suitably, the conduit may also include poly-lactic acid (PLA). The inner surface of the conduit may have been treated with an alkaline composition. The present invention also provides a method for treating a peripheral nerve damage using a peripheral nerve growth conduit including poly--caprolactone (PCL). The present invention also provides a kit for treating a peripheral nerve damage having a peripheral nerve growth conduit including poly--caprolactone (PCL).

Claims

1. A peripheral nerve growth conduit including poly--caprolactone (PCL) and poly-lactic acid (PLA), wherein the weight ratio of PCL:PLA is in the range 20:1 to 2:1, wherein the conduit comprises a conduit wall having a thickness, and wherein the conduit wall does not include any pores extending through the thickness of the conduit wall, and wherein the conduit wall defines a luminal space and provides an inner surface of PCL and PLA for attachment of peripheral nerve cells.

2. The peripheral nerve growth conduit according to claim 1, wherein the inner surface of the conduit comprises pits and wherein the pits have an average diameter in the range 1-10m.

3. The peripheral nerve growth conduit according to claim 2, wherein the pits have an average depth in the range 1-4m.

4. The peripheral nerve growth conduit according to claim 2, wherein the pits cover the inner surface at a % surface coverage of 45% to 55%.

5. The peripheral nerve growth conduit according to claim 1 wherein the conduit comprises at least 75 wt % PCL based on the total weight of the conduit.

6. The peripheral nerve growth conduit according to claim 1, wherein the PCL has a number average molecular weight in the range 60,000 to 100,000 g/mol.

7. The peripheral nerve growth conduit according to claim 1, wherein the conduit is a tubular conduit and the thickness of the conduit walls is in the range 20m to 80m.

8. The peripheral nerve growth conduit according to claim 1, wherein the conduit has a length in the range 5 mm to 20 mm.

9. The peripheral nerve growth conduit according to claim 1, wherein the conduit has a diameter in the range 1 to 5 mm.

10. The peripheral nerve growth conduit according to claim 1, wherein the conduit is made from a film comprising PCL, by solvent evaporation and optionally opposite edges of the film are joined together by heat sealing to form the conduit.

11. The peripheral nerve growth conduit according to claim 1, wherein the inner surface of the conduit has been treated with an alkaline composition, wherein the duration of the treatment with alkaline composition is in the range 30 minutes to 3 hours, the alkaline composition is aqueous NaOH, and the concentration of the aqueous NaOH is in the range of 8N to 12N.

12. The peripheral nerve growth conduit according to claim 1, wherein the inner surface of the conduit includes COOH and/or OH terminated PCL chains, and the conduit has nanopits on the inner luminal surface of the conduit.

13. The peripheral nerve growth conduit according to claim 1, wherein the inner surface of the conduit has an average surface roughness (Ra) of at least 1 m.

14. The peripheral nerve growth conduit according to claim 1, wherein a surface roughness of an outer surface of the conduit has an average surface roughness (Ra) of less than 1 m.

15. The peripheral nerve growth conduit according to claim 1, wherein the difference in average surface roughness between the inner surface and an outer surface is at least 1 m.

16. A kit for treating a peripheral nerve in a human or animal, the kit including a peripheral nerve growth conduit according to claim 1.

17. A method of treating a damaged peripheral nerve utilizing.

18. A method of making a peripheral nerve growth conduit according to claim 1, the method comprising the step of: i) forming a film by solvent-casting a solution including PCL, PLA and a solvent, wherein the weight ratio of PCL: PLA is in a range 20:1 to 2:1, and allowing the solvent to evaporate; and ii) joining opposite edges of the film together by heat sealing to form the conduit.

19. The peripheral nerve growth conduit according to claim 1, wherein the weight ratio of PCL: PLA is in a range of 10:1 to 2:1.

20. The peripheral nerve growth conduit according to claim 1, wherein the PLA is provided as a blend with the PCL.

21. A peripheral nerve growth tubular conduit including poly--caprolactone (PCL), an inner surface of the conduit comprising pits, wherein the conduit comprises at least 50 wt % PCL based on the total weight of the conduit and the PCL has a number average molecular weight in the range 60,000 to 100,000 g/mol, and wherein the conduit has a length in the range 5 mm to 50 mm and a diameter in the range 1 to 5 mm, and wherein the peripheral nerve growth conduit further including poly-lactic acid (PLA), wherein the weight ratio of PCL:PLA is in the range 20:1 to 2:1, wherein the conduit comprises a conduit wall having a thickness, and wherein the conduit wall does not include any pores extending through the thickness of the conduit wall.

22. A peripheral nerve growth conduit including poly--caprolactone (PCL) and poly-lactic acid (PLA), wherein the weight ratio of PCL:PLA is in the range 20:1 to 2:1 wherein the conduit comprises at least 50 wt % PCL based on the total weight of the conduit, wherein the conduit comprises a conduit wall having a thickness, and wherein the conduit wall does not include any pores extending through the thickness of the conduit wall.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Embodiments of the invention and experiments illustrating the advantages and/or implementation of the invention are described below, by way of example only, with respect to the accompanying drawings, in which:

(2) FIG. 1 shows schematically the heat sealing method preferred for forming the conduit of the present invention;

(3) FIG. 2 shows SEM images of the air (inner) (2A) and glass (outer) (2B) surfaces of a PCL film;

(4) FIG. 3 shows 3-D AFM images of the air (inner) (3A) surfaces and glass surface (outer) (3B) of a PCL film;

(5) FIG. 4 shows SEM images of a cast PCLA film before (4A) and after (4B) NaOH treatment;

(6) FIG. 5 shows XPS spectra for dichloromethane cast PCL films before (5A) and after (5B) NaOH treatment;

(7) FIG. 6 shows a graph of wettability data for a number of different materials formed as films;

(8) FIG. 7 shows a graph of MTS cell attachment data for NG108-15 cells on different materials;

(9) FIG. 8 shows a graph of DNA cell attachment data for NG108-15 cells on different materials;

(10) FIG. 9 shows a graph of proliferation data for NG108-15 cells on untreated PCL films; NaOH treated PCL films, and PLA;

(11) FIG. 10 shows the results of Schwann cell proliferation on treated and untreated PCL films;

(12) FIG. 11 shows (A) an SEM image of differentiated NG108-15 cells on NaOH treated PCL film; (B) SEM image of (A) at higher resolution; (C) Confocal microscope image of phalloidin stained cells; (D) Confocal microscope image of anti-neurofilament antibody stained cells. Bar=100 m in (C) and (D).

(13) FIG. 12 shows (A) an SEM image of Schwann cells growing on NaOH treated PCL film; (B) Immunohistochemical-stained cells, using antibody against marker protein S100; (C) Toluidine Blue O stained Schwann cells.

(14) FIG. 13 shows images of haematoxylin stained nuclei of NG108-15 cells on different materials;

(15) FIG. 14 shows a photograph of a PCL conduit sutured in place to bridge a 10 mm nerve gap;

(16) FIG. 15 shows a photograph of the healed wound of a rat 14 days/2 weeks after surgery;

(17) FIG. 16 shows a photograph of a PCL conduit 14 days/2 weeks after implantation;

(18) FIG. 17 shows photographs of a harvested PCL nerve conduit after 14 days/2 weeks of in vivo testing (A) and regenerated nerve tissue after the removal of the PCL conduit (B);

(19) FIG. 18 shows photographs of peripheral nerve regrowth in the conduit of FIG. 17, being anti-PGP9.5 antibody stained regenerated nerve fibres (18A) and anti-S100 antibody stained Schwann cells (18B); and

(20) FIG. 19 shows an SEM image of the inner surface of a PCL conduit after 14 days/2 weeks in vivo;

DETAILED DESCRIPTION OF THE INVENTION

(21) Definitions

(22) The term scaffold as used herein is well known to the skilled reader. In particular, a scaffold in the context of the present invention is a structure adapted for peripheral nerve growth. Suitably the scaffold promotes or enhances peripheral nerve growth.

(23) The term pit as used herein means a closed-end pore or blind hole. In short, a pit as used herein does not extend all of the way through the wall of the scaffold.

(24) The term nanopit as used herein means a pit having at least one dimension on the nano- or sub-m scale.

(25) Film Formation

(26) PCL pellets (Sigma-Aldrich) were dissolved in dichloromethane (3.0%, wt/v) and gentle heating at a temperature of approximately 50 C. could be used to assist dissolving. PCL solution was evenly applied onto borosilicate glass slides (7525 mm.sup.2), which had been degreased with acetone/ethanol (1:1, v/v).

(27) Complete solvent evaporation was allowed in a fume cupboard for at least 48 hours, to provide films with a thickness of 605 m.

(28) The polymer films were washed in distilled H.sub.2O and sterilized by UV irradiation for 1 hour prior to in vitro and in vivo testing.

(29) Complete solvent evaporation was confirmed by FTIR (Thermo Nicolet Nexus FTIR (Cambridge, UK) controlled by OMNIC Software Version 6.1a), which ensured that no solvent toxic effect would occur in the subsequent cell growth and in vivo testing.

(30) Using the same method, a mixture of PCL and PLA was formed as a film (the PCLA film). The weight ratio of PCL to PLA was 4:1.

(31) Alkaline (Hydroxide) Treatment

(32) PCL films were soaked in 10N NaOH for 1 hour with horizontal shaking at 150 rpm at room temperature and then rinsed thoroughly with distilled H.sub.2O to return the pH to neutral (pH 7.2-7.4). Subsequent XPS analysis (discussed below) confirmed the cleavage of the ester bond (ester hydrolysis) as follows:

(33) ##STR00001##

(34) For comparison, a film of PHB was treated with NaOH. However, the PHB film did not withstand NaOH treatment; it was too brittle and shattered into pieces.

(35) Conduit Formation

(36) FIG. 1 illustrates schematically the methodology used to form the PCL and PCLA conduits. The films 2 were wrapped around a 16G cannula 4, to form a tubular conduit. Sealing of the overlapping edges of the film was carried out by briefly (several seconds) pressing the edges on to a hot plate 6 at 60 C. A thin layer of tin foil was provided (at location 8) between the outer surface of the conduit and the hot plate. This provided a durable seal and the resultant tubular conduit was self supporting.

(37) The inner (luminal) surface of the PCL and PCLA conduits was unchanged as a result of the heating step.

(38) Surface AnalysisAFM & SEM

(39) PCL and PCLA films prepared as described above were imaged using Atomic Force Microscopy (AFM, Veeco CP II) and Philips XL30 Field Emission Gun Scanning Electron Microscopy (SEM) techniques. 3-D images were created, and dimension of individual pores measured using IP Image Analysis 2.1 software (Image Processing and Data Analysis version 2.1.15. TM Microscopes, copyright 1998-2001).

(40) FIG. 2A shows an SEM image of the PCL film, being the air surface of the film that is destined to become the inner (luminal) surface of the conduit.

(41) FIG. 2B shows an SEM image of the glass surface of the film, which when formed as the conduit will be the outer surface.

(42) It is clear from FIGS. 2A and 2B that the outer surface is considerably smoother (i.e. has a lower surface roughness) than the inner surface. In particular, FIG. 2A shows that the inner surface is pitted and that the plurality of pits have diameters in the range 1 to 10 m. FIG. 2B shows that the outer surface has smaller and shallower pits.

(43) Indeed, SEM imaging revealed that PCL films comprised pits on the air surface in the range of 1-10 m in diameter; the depth of these pits was between 1-5 m. The glass (outer) surface was also pitted, with pores in the diameter of 1-5 m. However, the depth of pits on this side of the films was down to 100 nm-800 nm.

(44) The diameter and depth of the pits for the inner surface of both PCL and PCLA films are set out in Table 1. Also included is diameter and depth data for the same surfaces after treatment with NaOH.

(45) TABLE-US-00001 TABLE 3 Inner surface pit size of PCL and PCLA films. Samples Pore diameter (m) Pore depth (m) PCL 1-10 1-5 PCL (NaOH treated) 1-10 1-5 PCLA 1-8 1-3 PCLA (NaOH treated) 1-8 1-3

(46) The results in Table 1 show that NaOH treatment didn't affect the overall morphology of the materials but that some reduction in the surface roughness was observed. In addition, the results show that PCLA films have smaller pit size than PCL films.

(47) The % coverage of pits on the inner surface is 51%, measured using SEM image and data and Image J software [2].

(48) The 3-D image generated from AFM data of the PCL film inner (air) and outer (glass) surfaces are shown in FIG. 3. The scanned area of 3A is 3030 m.sup.2; for 3B it is 1010 m.sup.2. The pits (closed end holes) can be seen clearly.

(49) The average surface roughness (Ra) of the untreated inner surface is 3.883 m, and of the NaOH treated surface is 3.041 m.

(50) The average surface roughness (Ra) of the outer surface is 0.569 m and 0.576 m respectively before and after NaOH treatment.

(51) The average surface roughness (Ra) and pit size were measured using AFM images and IP Image Analysis 2.1 software.

(52) FIG. 4, being SEM images of a PCLA film before (4A) and after (4B) treatment with NaOH, shows that the pitted morphology is maintained after treatment. Nanoscale structure (nanopits) can also be seen in 4B indicating that NaOH treatment causes formation of nanopits.

(53) Measurement of % coverage of pits using Image J software [2] and SEM image data showed that the % coverage of pits for the PCL film is 51%, and for the PCLA film it is 35.8%. In addition, the size of the pits on the PCLA film is smaller than that for the PCL film.

(54) Surface AnalysisXPS

(55) X-ray Photoelectron Spectroscopy (XPS, AXIS Ultra) was used to analyse the chemical and electronic state of the carbon and oxygen elements existing in the PCL film before and after treatment with NaOH.

(56) FIG. 5 shows XPS spectra for a PCL film before (5A) and after (5B) NaOH treatment. The reduced peak of CO group confirms that alkaline hydrolysis has cleaved the ester bond.

(57) Wettability

(58) The hydrophilicity of the PCL and PCLA films before and after NaOH treatment was compared by measuring the static contact angles using Krss DSA 100 Drop Size Analyser. Ten treated or untreated films were tested and five randomly selected areas were measured on each film. A glass coverslip was tested for comparison. The results are reported in Table 2 below, where OH denotes NaOH treatment.

(59) TABLE-US-00002 TABLE 2 Water contact angle for PCL and PCLA films (S designates smooth outer surface; P pitted inner (luminal) surface) Samples Water Content Angle (%) Standard Deviation (%) PCL-OH-S 36.7 4.65 PCL-OH-P 52.79 10.8 PCL-S 43.81 6.3 PCL-P 64.58 2.8 PCLA-OH-S 61.49 4.6 PCLA-OH-P 74.45 9.2 PCLA-S 69.36 4.2 PCLA-P 76.63 6.5 PLA-P 71.33 3.3 Glass Coverslips 31.25 9.87

(60) The results are graphed in FIG. 6.

(61) The results show that the smooth outer surface is more hydrophilic than the porous inner surface and that NaOH treated materials are more hydrophilic than the untreated counterparts. Also that PCL is more hydrophilic than the PCLA composite either before or after the NaOH treatment.

(62) For comparison, the wettability of Poly(3-hydroxybutyrate) (PHB) was tested. PHB (Astra Tech, Sweden) was dissolved into chloroform at 70 C. and then applied evenly onto the surface of glass slides. PHB (1% wt/v) film had a contact angle of 80.03.

(63) Mechanical Testing

(64) The tensile strength, Young's modulus and maximum strain of PCL and PCLA films were measured, before and after NaOH treatment.

(65) Tensile strength is defined as the maximum amount of tensile stress that a material can be subjected to before failure. Young's modulus is a measurement of stiffness. Maximum strain is measured as the total elongation per unit length of material subject to same applied stress.

(66) Tensile strength, Young's modulus and maximum strain were measured on a mechanical tensile tester (Instron 1122) at 231 C., 50%2% relative humidity. The cross sectional area was (3.80.06) mm.sup.2; grip distance was 35 mm; strain rate was set at 50 mm/min and the full scale load 0.005 KN.

(67) Young's modulus was measured from the initial slopes in the elastic region and the tensile strength was the average of ultimate stress at the breaking point of the films.

(68) The results are set out in Table 3.

(69) TABLE-US-00003 TABLE 3 Mechanical strength of PCL and PCL + PLA (=PCLA) films, before and after NaOH treatment. Young's Samples (3% Thickness Max. STR. Max. STN. Modulus weight/volume) (m) (MPa) (mm/mm) (MPa) PCL 0.057 16.3 7.67 115.48 PCL (NaOH 0.054 14.98 7.14 118.89 treated) PCL + PLA 0.053 11.59 2.86 175.52 PCl + PLA (NaOH 0.053 10.73 2.44 156.48 treated)

(70) The results show that mechanical strength of the PCLA film is lower than that of the PCL film. It is expected that the PCLA film will have a faster degradation rate than the PCL film. Thus, the inclusion of 20 wt % PLA has modified the mechanical properties of PCL and provides a favourable balance in terms of handling ex vivo (e.g. by a surgeon) and performance in vivo.

(71) The results also show that PCL films (with or without a PLA component) can be fabricated at micro-thickness and at the same time retain mechanical strength and flexibility.

(72) Cell Compatibility Analysis

(73) Cell Source

(74) The NG108-15 cell line was purchased from ECACC (Porton Down, UK). Schwann cells were isolated from neonate rats as previously described [3] and maintained with 63 ng/ml glial growth factor (GGF) and 10 M forskolin mitogen supplemented media.

(75) Cell Culture

(76) NG108-15 cells were maintained in DMEM (Dulbecco's Modified Eagle's Medium), containing 4.5 g/L glucose; 5% foetal bovine serum; 1% antibiotics, and supplemented with 1HAT (a liquid mixture of sodium hypoxanthine, aminopterin and thymidine) solution, at 37 C. in a 5% CO.sub.2 humidified atmosphere.

(77) Schwann cells were cultured in DMEM containing 10% serum and antibiotics (penicillin 100 IU/ml and streptomycin 100 g/ml).

(78) Cell Attachment Analysis

(79) 1 ml of NG108-15 cells (10.sup.5/ml) were seeded onto PCL and/or PCLA films (3.14 cm.sup.2) and cultured for 3 hours at 37 C. in a 5% CO.sub.2 humidified atmosphere.

(80) For the MTS assay, films were transferred into fresh cell culture plates and washed gently twice in 37 C. cell culture medium to ensure that only attached cells were tested. The CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS) (Promega UK) is a colorimetric method for determining the number of viable cells. The active component is a tetrazolium compound called MTS which is reduced by cells to a colored formazan product. The amount of formazan product is directly proportional to the number of living cells; therefore, cell proliferation or death can be quantified by reading the plate at 490 nm.

(81) DNA assay for the attachment of NG108-15 cells was conducted using the Hoechst stain reagent (Hoechst 33258 from Sigma-Aldrich), which specifically binds onto DNA and as such can be used to detect the contents of a sample DNA by plotting a standard emission-to-content curve. After 3 hours of culturing films were washed twice in PBS followed by three freeze and thaw cycles in dH.sub.2O to release the DNA from cells. FLUOstar OPTIMA fluorescence microplate reader was used to measure the fluorescence.

(82) The results of the MTS analysis are shown in FIG. 7. FIG. 7A shows measured absorbance for the inner surfaces of PCL and PCLA films, with and without NaOH treatment. FIG. 7B shows cell number for the inner surfaces of PCL and PCLA films, with and without NaOH treatment.

(83) The results show that NaOH treated materials are more compatible with NG108-15 cells than untreated materials. This is quantified in Table 4, which provides the ratio (as a %) of the cell attachment achieved with untreated material compared to treated material.

(84) TABLE-US-00004 TABLE 4 Comparison of NG108-15 cell attachment on NaOH treated and untreated PCL and PCLA films (inner surface). Samples 2 hour 3 hour 4 hour PCL/NaOH treated PCL 39.6% 45.7% 47.5% PCLA/NaOH treated PCLA 66.9% 77.5% 84.9%

(85) The data obtained is in keeping with the results from DNA attachment analysis, discussed below.

(86) The results of the DNA (Hoechst) analysis are presented in FIG. 8. The results show that NaOH treated materials are more compatible with NG108-15 cells than untreated ones and the NaOH treated pitted surface of PCLA showed the best result. These results confirmed those of the MTS assay.

(87) Cell Proliferation Analysis

(88) The proliferation rate of NG108-15 cells on PCL and PCLA films (both NaOH treated and untreated) was also analyzed using the MTS method. NG108-15 cells (5000/cm.sup.2) were seeded onto films in each well of the 12-well plate and cultured as described above. Another resorbable biomaterial, poly(D,L-lactic acid) (PLA) was included as a comparison.

(89) The results are provided in FIG. 9. The results show that in six days cell number increased approximately 9 fold on PLA films and NaOH treated PCL films. The effect of NaOH treatment on the PCL film is remarkable and demonstrates that NaOH treatment of PCL provides a surface having a significantly enhanced compatibility for peripheral nerve cells and provides an active environment that encourages peripheral nerve cell proliferation.

(90) Schwann cell proliferation was also studied using the MTS method. Schwann cells were grown on NaOH treated and untreated PCL films cast from DCM. 6000/cm.sup.2 cells were seeded onto the surface of PCL and NaOH-treated PCL films. Cells were cultured in DMEM containing 10% serum and antibiotics (penicillin 100 IU/ml and streptomycin 100 g/ml). Cell culturing was conducted for 8 days; readings were taken on every second day (antibody staining was carried out after 7 days of culturing; see below). The results were graphed in FIG. 10. The results show that Schwann cells proliferate on PCL regardless of whether or not there has been hydroxide treatment.

(91) In vitro testing showed that the PCL and PCLA films, with and without hydroxide treatment, supported the attachment and proliferation of both NG108-15 cells and Schwann cells, which are involved in maintenance of axons and are crucial for neuronal survival and regeneration. Importantly, NG108-15 cells could also be induced into differentiated phenotype with long branched neurites extending across the surface of the material. FIGS. 11A and 11B show the differentiated NG108-15 cells branching and extending over the pitted PCL surface. FIG. 11C shows phalloidin stained cells and FIG. 11D shows anti-neurofilament antibody stained cells, which confirms proper differentiation. The excellent neurite elongation and branching indicates good cell-material compatibility.

(92) FIGS. 12A to C show Schwann cell growth on the NaOH treated PCL film. FIG. 12A shows a typical bipolar spindle-shaped phenotype. The immunohistochemical-stained cells shown in FIG. 12B confirms expression of marker protein and this together with the Toluidine Blue O stained cells of FIG. 12C indicates excellent cell-material compatibility.

(93) Haematoxylin Staining

(94) Images of haematoxylin stained nuclei of NG108-15 cells on PCL films, NaOH treated PCL films and PLA films (reference) were obtained after 5 days in culture. The images are shown in FIG. 13. For each material, experiments were carried out in triplicates and repeated three times.

(95) As can be seen from FIG. 13, there is excellent reproducibility between each of the 3 films for each material. Furthermore, good levels of cell proliferation are seen for PCL. Most impressive is the result provided by NaOH treated PCL where surprisingly high levels of cell proliferation were observed. Furthermore, the cells are distributed evenly on the surface.

(96) Nerve Re-Growthin vivo

(97) NaOH treated PCL films were cut into rectangular sheets and rolled around a 16G intravenous cannula (16G Abbocath-T, Abbott Ireland, Sligo, Republic of Ireland). The standardised internal diameter of the conduits is 1.6 mm, more than 1.5 times the diameter of rat sciatic nerve, thus allowing space for post-injury swelling. Conduits were sealed by controlled heating at 60 C. while still mounted on the cannula. Prior to surgical implantation, the conduits were sterilised using UV radiation.

(98) All work was conducted in keeping with the terms of the Animals (Scientific Procedures) Act 1986, and the experimental design recognised the need to optimise animal welfare.

(99) Eight-week-old female adult Sprague-Dawley rats (Harlan, Inc. USA) (weighing between 180-220 g) were anesthetised with isofluorane (Abbott Laboratories Ltd.). The site for implantation was shaved and sterilised with surgical alcohol. The left sciatic nerve of the rat was exposed through a gluteal muscle-splitting incision at the mid-thigh level after a dorsolateral skin incision and splitting of the fascia between the gluteus and biceps femoris muscle. The surrounding tissues were separated and a piece of 8 mm in length was removed from the sciatic nerve, leaving a 10-mm nerve gap after retraction of both ends.

(100) Under an operating microscope (Zeiss, Germany), the proximal and distal nerve stumps of the transected nerve were secured epineurially within the 14 mm long guidance conduit using a 9-0 ETHILON suture. Both the nerve ends were positioned 2 mm from the conduit ends to ensure the proximal and distal nerve stumps were separated by a 10 mm gap (20, FIG. 14). A single 4-0 coated VICRYL was used to suture the muscle and skin. After the operation, 4 g of buprenorphine (20 g/kg) was injected into the rats as an analgesic intramuscularly. The depth of anaesthesia, heart rate and breathing were checked periodically to ensure the rat was in a good surgical condition. A total of 9 animals were implanted in the same manner. The animals were caged in a temperature- and humidity-controlled room with a 12-hour light/dark cycle. Food and water was provided immediately.

(101) 14 days/2 weeks post-operation, the site was well-healed without any sign of swelling and inflammation (22, FIG. 15). The animals were killed using Schedule I method. FIG. 16 shows that the conduit 20 was integrated with both proximal 24 and distal 26 stumps of the natural nerve. No severe inflammatory response was found in all nine animals. The conduits didn't open or collapse in all samples (n=9). (FIG. 16, Bar=10 mm).

(102) FIG. 17A shows the harvested PCL peripheral nerve conduit after 14 days of in vivo testing. FIG. 17B shows the regenerated nerve tissue after the removal of PCL conduit.

(103) For immunohistochemical studies, the entire implants with a 2 mm length of proximal and distal nerve were harvested en bloc, pinned onto a plastic card to avoid shrinkage and marked at the proximal end. Fixation was carried out in 4% (wt/v) paraformaldehyde solution for 24 h at 4 C. and then washed three times with phosphate buffered saline (PBS) containing 15% sucrose and 0.1% sodium azide.

(104) Blocks for cryostat sectioning were prepared by rapid freezing of samples into OCT mounting medium in liquid nitrogen. Systematic longitudinal 15 m transversal sections were cut using Bright (Model OTE) cryostat instrument at 23 C. and collected onto glass slides coated with Vectabond (Vector Laboratories). Samples were dried overnight in 37 C. oven. Immunostaining was performed by using polyclonal rabbit antibodies directed against protein gene product (PGP9.5) (Dako, dilution 1:200) in order to identify neurites. Schwann cells were identified using polyclonal rabbit anti-protein S100 (Dako, dilution 1:500). Secondary antibody used in the staining was FITC conjugated anti-rabbit IgG (Vector labs, F1-1000; 1:100).

(105) Schwann Cell Detection (Immunostaining)

(106) FIG. 18 shows the results of immuno-staining of neurofilament and Schwann cells in the PCL conduit used in the in vivo testing discussed above.

(107) FIG. 18A shows anti-PGP9.5 antibody stained neurofilaments and FIG. 18B shows anti-S100 antibody stained Schwann cells.

(108) The results of the preclinical testing show that the regenerating neurites have grown through the whole length (i.e. 10 mm) of the conduit together with the infiltrated Schwann cells.

(109) In contrast, the results reported in [1] (using the same preclinical testing method) show that only a much smaller extent of nerve re-growth was achieved when a PHB conduit is used. The effect of fibrin matrix (Tisseel) and Schwann cells (SC)/differentiated mesenchymal stem cells (dMSC) on the regeneration of peripheral nerves in PHB conduits is shown in Table 5. PHB conduits were used to bridge a 10 mm gap in the left sciatic nerve of adult Sprague-Dawley rats (Harlan Inc. USA). Regeneration was analysed by immunohistochemical staining to identify PGP9.5 for neurofilament and S100 for Schwann cells two weeks post-implantation.

(110) The results from [1] are set out in Table 5 below.

(111) TABLE-US-00005 TABLE 5 PHB conduits filled with fibrin gel matrix and/or cells were used to bridge a 10 mm gap in the left sciatic nerve of adult Sprague-Dawley rats. PHB with Antibodies used for Empty PHB with fibrin PHB with immunohistochemical PHB fibrin matrix- fibrin staining conduit matrix dMSC matrix-SC PGP9.5 1.91 mm* 2.28 mm* 3.16 mm* 3.17 mm* S100 Proximal 2.2 mm 2.4 mm 3.30 mm* 3.40 mm* Distal 1.7 mm 2.1 mm 2.80 mm* 2.91 mm* *Numbers in bold were accurate data from the original work in [1]. The other four measurements were extracted from the figure in [1].

(112) It is clear from the above results that the PCL scaffold of the present invention is an active scaffold in that it encourages and promotes peripheral nerve growth.

(113) FIG. 19 shows an SEM image obtained for the inner (luminal) surface of a PCL conduit after 14 days/2 weeks in vivo. The arrows are pointing at the regenerated nerve fibres. The SEM image also serves to show that the pitted surface morphology was not affected by the heat sealing method used to the form the conduit.

(114) 18 Week in vivo Study

(115) A 1 cm sciatic nerve gap in adult Sprague-Dawley rats was created and repaired with either NaOH treated PCL conduits or a nerve autograft (9 subjects in each group).

(116) In both groups, 3 rats were prematurely culled due to autotomy, a commonly reported phenomenon occurring as a result of the surgical procedure.

(117) The remaining 6 rats in each group adopted a normal living style without any visible difference in behaviour. Before sacrifice, the rats treated with PCL conduits were observed to support themselves on both hind-limbs, indicative of significant distal regeneration. This was supported by electrophysiological measurements.

(118) Briefly, after induction of anaesthesia (week 18), the sciatic nerves were exposed from the sciatic notch to the distal branches emanating from the popliteal fossa. A stimulating electrode was placed in the proximal nerve segment and a recording electrode distal to the repair site. In response to the electrical stimulation, we were able to record action potentials (nerve conduction) in the sural, medial gastrocnemius and tibial nerve branches indicating significant regeneration across the nerve conduit and distal towards the end organs.

(119) Reinnervation of hind-limb muscles was indicated by recovery of gastrocnemius muscle weight. In previous studies of nerve repair, we have shown that peak muscle atrophy (loss of weight) occurs at 7 weeks post-injury. At this time point, muscle weight on the operated side was 27.873.04% of the contra-lateral side.

(120) However, 18 weeks after repair with the PCL conduits, the muscle weight was significantly (P<0.05) increased to 44.644.67% and to 61.372.37% (P<0.01) with autografts.

(121) These results indicate the capacity of the PCL nerve conduit to support nerve regeneration and reinnervation comparable to the gold standard nerve autograft.

(122) It is to be understood that variants of the above described examples of the invention in its various aspects, such as would be readily apparent to the skilled person, may be made without departing from the scope of the invention in any of its aspects.

REFERENCES

(123) A number of publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference. [1] Kalbermatten, D. F. et al., Fibrin matrix for suspension regenerative cells in an artificial nerve conduit, Journal of Plastic, Reconstructive & Aesthetic Surgery (2008), Volume 61, Issue 6, Pages 669-675. [2] Rasband, W. S., Image J, U. S. National Institutes of Health, Bethesda, Md., USA, http://rsb.info.nih.gov/ij/, 1997-2008. [3] Caddick, J. et al., Phenotypic and functional characteristics of mesenchymal stem cells differentiated along a Schwann cell lineage, Glia 54 (2006), pp. 840-849.