MEDICAL DEVICE FOR USE IN THE REGENERATION OF AN INJURED NERVE

Abstract

A medical device having a tubular membrane with openings adapted to receive the ends of an injured nerve and a lumen to allow the regeneration of the nerve, such that the tubular membrane includes fibers of polyhydroxyalkanoate (PHA) containing 3-hydroxybutyrate monomer units. The fibers have an average diameter from 500 nm to 2000 nm. Preferably, the PHA is a poly-3-hydroxybutyrate homopolymer and the tubular membrane further includes silver nanowires (Ag). The medical device has the advantages of not causing inflammation of the surrounding tissues surrounding the injured nerve, thus demonstrating a high biocompatibility, and at the same time exhibiting high ability to induce the regeneration of the injured nerve, providing a valid support for the maturation of the newly formed nerve fibers.

Claims

1. A medical device comprising: a tubular membrane having openings adapted to receive the ends of an injured nerve and a lumen to allow the regeneration of said nerve, wherein said tubular membrane comprises fibers of polyhydroxyalkanoate (PHA) containing 3-hydroxybutyrate monomer units, said fibers having an average diameter comprised from 500 nm to 2000 nm.

2. The medical device according to claim 1, wherein the PHA fibers are oriented in a direction substantially parallel to the longitudinal axis of the tubular membrane.

3. The medical device according to claim 1, wherein the PHA fibers are produced by electrospinning.

4. The medical device according to claim 1, wherein the tubular membrane further comprises silver (Ag) nanowires.

5. The medical device according to claim 4, wherein the tubular membrane comprises from 0.2% to 5% by weight, with respect to the overall weight of the membrane, of Ag nanowires.

6. The medical device according to claim 4, wherein the Ag nanowires are oriented in a direction substantially parallel to the longitudinal axis of the tubular membrane.

7. The medical device according to claim 4, wherein the Ag nanowires are coated by a binding agent.

8. The medical device according to claim 7, wherein the binding agent has a functionalized hydrocarbon chain with at least one group capable of binding with silver.

9. The medical device according to claim 4, wherein the Ag nanowires coated with the binding agent comprise from 60% to 90% by weight of Ag and from 10% to 40% by weight of the binding agent.

10. The medical device according to claim 1, wherein the PHA containing 3-hydroxybutyrate monomer units is a poly-3-hydroxybutyrate homopolymer (P3HB) or a copolymer containing at least 20% moles of 3-hydroxybutyrate monomer units, the remainder being hydroxyalkanoate monomer units other than 3-hydroxybutyrate.

11. The medical device according to claim 10, wherein the hydroxyalkanoate monomer units other than 3-hydroxybutyrate derive from: 4-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxyundec-10-enoate, and 4-hydroxyvalerate.

12. A method for producing a medical device according to claim 1, the method includes the following steps: preparing a spinning solution by solubilization of polyhydroxyalkanoate (PHA) containing 3-hydroxybutyrate monomer units in an organic solvent, and subjecting the spinning solution to an electrospinning process on a rotating support so as to obtain a tubular membrane.

13. The method according to claim 12, further including the step of: dispersing Ag nanowires into the spinning solution, so as to achieve a co-deposition of PHA fibers and Ag nanowires on the rotating support.

14. The method according to claim 12, wherein the solvent is selected from the group consisting of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), chloroform, N,N-dimethylformamide (DMF), and mixtures thereof.

15. The method according to claim 12, wherein in the spinning solution, the PHA containing 3-hydroxybutyrate monomer units has a concentration comprised from 1% to 20% w/v.

16. The method according to claim 13, wherein the Ag nanowires are present in the spinning solution in a concentration preferably comprised from 0.05% to 2.0% w/v.

Description

DETAILED DESCRIPTION OF THE DISCLOSURE

[0060] The following embodiment examples are provided purely for illustrative purposes of the present disclosure and should not be interpreted as limiting the scope of protection defined by the accompanying claims.

Example 1: Production of the P3HB Tubular Membrane

[0061] The purified P3HB was dissolved in HFIP (1,1,1,3,3,3-hexafluoro-2-propanol) so as to obtain a spinning solution with a concentration of 4% w/v. The solution was stirred on a plate at 800 rpm at a temperature of 80° C. for about 2 hours.

[0062] Thereafter, the solution was cooled, poured into a 10 mL plastic syringe and placed inside the electrospinning system to be processed. The deposition of the fibers was carried out on a steel cylindrical support (spindle) placed in rotation by an electric motor.

[0063] The following parameters were used for the electrospinning process:

[0064] Spinning solution: purified P3HB dissolved in HFIP at 4% w/v;

[0065] solution volume processed: 1 mL;

[0066] solution rate: 1 mL/hour;

[0067] translational speed of the spinneret along the spindle: 3 mm/s;

[0068] distance between the tip of the spinneret and the spindle (gap): 15 cm;

[0069] voltage applied to the electrodes: 20 kV;

[0070] spindle rotation speed: 200 rpm;

[0071] diameter of the spinneret outlet hole: 2.15 mm; spindle diameter: 1.5 cm;

[0072] auxiliary electrode (16 cm×16 cm) placed about 1 cm from the circular ground electrode.

[0073] FIG. 1 shows the images of the tubular membrane, obtained by electronic scanning microscopy (SEM).

Example 2: Ag Nanowires Production

[0074] In a 100 mL flask, 0.485 g of polyvinyl pyrrolidone (PVP) were dissolved in ethylene glycol (40 mL) while the solution was stirred to complete dissolution. Thereafter, 0.6 mL of an anhydrous ferric chloride solution in glycerol (11.2 mM) and 0.725 g of silver nitrate were added to the solution, stirring to complete dissolution.

[0075] The solution was placed in a pre-heated oven at 140° C. and kept at this temperature for 4 and a half hours without stirring and protected from light. Then, the solution was allowed to cool to room temperature, then acetone (50 mL) was added to induce the precipitation of Ag nanowires.

[0076] Ag nanowires were separated from the suspension by centrifugation, with cycles of 15 minutes each at 4500 rpm. The nanowires thus obtained were purified from possible impurities by washing them in a centrifuge with ethanol (2×50 mL) and, finally, with purified and deionized water (2×50 mL times). The resulting precipitate was dispersed in water and filtered with woven or nylon filters characterized by pores with a diameter of 30-50 μm, in order to eliminate the too small nanowires, having an unsuitable length. The final suspension was diluted up to 100 mL with water.

[0077] The concentration of Ag in the suspension of nanowires thus obtained was determined by atomic absorption spectroscopy (AAS), obtaining a value equal to 6.8 g/L of Ag which corresponds to a yield of 94%. The concentration of PVP remaining in the final suspension, after purification, was determined by thermogravimetric analysis (TGA) and was found to be equal to 52% of the total mass.

[0078] Ag nanowires thus obtained had an average length of 60 μm and an average diameter of 100 nm.

[0079] FIG. 2 shows the images of Ag nanowires, obtained by electronic scanning microscopy (SEM).

Example 3: Coating of Ag Nanowires with a Binding Agent

[0080] To the suspension of Ag nanowires (PVP-coated) in water (10 g/L), obtained according to Example 2, an equal volume of a solution of the binding agent of formula (II) (11-(4-mercaptobenzamide)ethyl undecanoate) in ethanol (10 g/L) was added dropwise. The resulting mixture was left under slow stirring by means of a Vortex mixer for 16 hours at room temperature. At the end, the nanowires were isolated by centrifugation (3000 rpm for 10 minutes) or alternatively they are filtered with woven or nylon filters characterized by pores with a diameter of 30-50 μm.

[0081] Thereafter, the Ag nanowires thus treated were purified by ethanol washes to remove the excess binder, and finally they were suspended in chloroform.

[0082] The concentration of Ag in the suspension thus obtained was determined by atomic absorption spectroscopy (AAS), obtaining a value equal to 4.9 g/L. The sample was also analyzed by thermogravimetric analysis (TGA) obtaining a composition equal to 70% by weight of Ag and 30% by weight of binder of formula (II).

[0083] The Ag nanowires were finally dried out and stored at room temperature protected from light and moisture.

[0084] FIG. 3 shows the images of Ag nanowires coated with the binding agent, obtained by electronic scanning microscopy (SEM).

Example 4: Coating of Ag Nanowires with Animal Gelatin

[0085] A solution of pork gelatin (240 Bloom degrees) was prepared in water at 40° C., to which an equal volume of suspension in water (20 g/L) of Ag nanowires obtained according to Example 2 was added dropwise, so as to have, in the final mixture, a gelatin concentration equal to 35 g/L and an Ag concentration equal to 10 g/L. The mixture was stirred for 16 hours at 40° C. At the end of stirring, the Ag nanowires were isolated from PVP and from excess gelatin by centrifugation and repeated washes with warm water (40° C.). The final product was then stored in a suspension at a concentration of 40 g/L of Ag (concentration obtained following the removal of excess gelatin and removal of the initial PVP). Following thermogravimetric analysis (TGA), it was found that Ag constituted 90% of the dry mass of the suspension, while gelatin constituted the remaining 10%. The final product was then dried by freeze-drying and stored away from light, moisture and at room temperature.

[0086] FIG. 4 shows the images of Ag nanowires coated with animal gelatin, obtained by electronic scanning microscopy (SEM).

Example 5: Production of the P3HB and Ag Nanowires Tubular Membrane

[0087] The purified P3HB was dissolved in HFIP (1,1,1,3,3,3-hexafluoro-2-propanol) so as to obtain a spinning solution with a concentration of 4% w/v. The solution was stirred on a plate at 800 rpm at a temperature of 80° C. for about 2 hours.

[0088] Once the polymer was dissolved, 0.1 mL of a suspension of Ag nanowires in HFIP (92.7 mM, 10 g/L) were added, prepared and treated with the binding agent of formula (II) as described in Example 3. The solution thus added was stirred on a plate at 800 rpm for about 2 hours at room temperature.

[0089] The solution was then poured into a 10 mL plastic syringe and placed inside the same electrospinning system used in Example 1.

[0090] The following parameters were used for the electrospinning process:

[0091] spinning solution: purified P3HB dissolved in HFIP at 4% w/v+0.2% w/v Ag in HFIP;

[0092] solution volume processed: 5 mL;

[0093] solution rate: 3 mL/hour;

[0094] translational speed of the spinneret along the spindle: 3 mm/s;

[0095] distance between the tip of the spinneret and the spindle (gap): 15 cm;

[0096] voltage applied to the electrodes: 15 kV;

[0097] spindle rotation speed: 300 rpm;

[0098] diameter of the spinneret outlet hole: 2.15 mm; spindle diameter: 1.5 cm;

[0099] auxiliary electrode (16 cm×16 cm) placed about 1 cm from the circular ground electrode.

[0100] The following composition of the tubular membrane thus obtained was determined by atomic absorption spectroscopy (AAS): 98.5% by weight of P3HB and 1.5% by weight of Ag.

[0101] FIG. 5 shows the images of the tubular membrane, obtained by electronic scanning microscopy (SEM).

Example 6: Pre-Clinical Study of Medical Devices

[0102] (a) Animals and Surgery.

[0103] Nerve regeneration analysis conducted in vivo was performed under general anesthesia using Zolazepam (Zoletil, Virban)+Xylazine (Bayer) by intraperitoneal injection (40 mg/kg+5 mg/kg), on adult female Wistar rats, weighing about 200 g. The median nerves were repaired by medical devices comprising a tubular membrane comprising P3HB (with or without silver nanowires) or by autotransplantation and collected 2 or 4 weeks after surgery, as follows:

[0104] a) the nerves were repaired with a tubular membrane comprising P3HB (n=6), or with a tubular membrane comprising P3HB and silver nanowires (n=4) or by autotransplantation (n=3) and the regenerated nerves were collected 2 weeks after surgery;

[0105] b) the nerves were repaired with a tubular membrane comprising P3HB (n=6), or with a tubular membrane comprising P3HB and silver nanowires (n=4) or by autotransplantation (n=3) and the regenerated nerves were collected 4 weeks after the surgery.

[0106] The median nerve was injured and a 10 mm long tubular membrane comprising P3HB (with or without silver nanowires) was used to bridge the nerve injury by inserting 1 mm of each of the two nerve ends within the membrane. The nerve guide was sutured with a 9/0 epineurial point at each end.

[0107] In the experimental autotransplant group, a 10 mm nerve segment was cut, inverted (distal−proximal) and sutured at the ends of the injured nerve with three 9/0 epineural points.

[0108] On days 0, 1, 2 and 3 from surgery, analgesic therapy with Rymadil (4 mg/kg, Zoetis Italy) was administered subcutaneously, while on days 2 and 5 after surgery, antibiotic treatment was administered by intramuscular injection (Rubrocylline 0.05 ml/500 g, MSD animal health).

[0109] 2 and 4 weeks after the surgery, the rats were sacrificed by anesthesia overdose with Zoletil+Xylazine (>60 mg/kg and >10 mg/kg) by intraperitoneal injection and the regenerated nerves were collected and analyzed.

[0110] (b) Fixation Protocol for Immunohistochemistry and Confocal Microscopy.

[0111] Regenerated nerve samples, together with the medical device comprising a tubular membrane comprising P3HB, were fixed by immediate immersion in 4% paraformaldehyde in PBS (saline phosphate buffer) (Sigma) for 2-3 hours at 4° C. The nerve samples were stored until the inclusion procedure in PBS at 4° C.

[0112] (c) Cryo-Embedding Protocol.

[0113] The samples were rehydrated with PBS (Sigma) and cryo-protected by three passages in increasing solutions of sucrose (Sigma) (7.5% for 1 hour, 15% for 1 hour, 30% overnight) in PBS 0.1 M. Thereafter, the samples were kept in a 1:1 solution of 30% sucrose and optimal cutting temperature medium (OCT, ELECTRON MICROSCOPY SCIENCES) for 30 minutes and then embedded in 100% OCT. The samples were stored at −80° C. 10 μm thick longitudinal nerve sections were cut and stored at −20° C. For staining, the sections were brought to room temperature and, as soon as they were acclimatized, they were processed.

[0114] (d) Masson's Trichrome Stain.

[0115] A Masson trichrome kit with aniline blue (Bio-Optica) was used for Masson's trichrome stain. Six drops of Weigert's iron hematoxylin (solution A) and six drops of Weigert's iron hematoxylin (solution B) were combined together and used to stain the slides for 10 minutes. Without washing, the slides were then drained and incubated with ten drops of alcoholic picric acid solution for 4 minutes. After washing with distilled water, the sections were stained with ten drops of Ponceau red acid for 4 minutes and washed again in distilled water. Thereafter, ten drops of phosphomolybdic acid solution were added to the section for 10 minutes. Without washing, the slides were dripped and 10 drops of quick dehydrating agent in ethanol were added and cleaned in xylene/Bioclear (Bio-Optica), the slides were mounted in DPX (Fluka).

[0116] (e) Immunohistochemistry and Confocal Microscopy.

[0117] For immunofluorescence, the sections were rinsed in PBS, blocked with normal serum (normal 1% serum in PBS containing 0.1% Triton) (it is recommended to use a normal serum obtained from the same species as the secondary antibody) for 1 hour and then incubated overnight with the primary antibody. For double marking, two different primary antibodies can be used at the same time, provided they are produced in different animal species. After incubation of the primary antibody (or primary antibodies), the sections were washed three times in PBS and incubated for 1 hour in a solution containing the secondary antibody (or secondary antibodies) Alexa 488 a-mouse, Cy3 a-rabbit (Life Technologies) conjugated with fluorophore and selected to recognize the primary antibody/antibodies species. After three washes in PBS, the sections were finally prepared with a Dako fluorescent mounting medium and stored at 4° C. before being analyzed.

[0118] Both the axon and the glia can be identified by immunohistochemistry using specific antibodies.

[0119] The sections were incubated in a solution containing the following primary antibodies, used alone or in pairs: [0120] anti-NF 200 kDa (monoclonal, mouse, Sigma Aldrich) [0121] S-100β (polyclonal, rabbit, Sigma Aldrich)

[0122] (f) Resin Embedding and Optical Microscope Analysis.

[0123] The regenerated nerve samples, together with the medical device comprising a tubular membrane comprising P3HB, were fixed by immediate immersion in 2.5% glutaraldehyde (SIC Società Italiana Chimici) in 0.1 M phosphate buffer (pH 7.4) for 5-6 hours at 4° C. The samples were then post-fixed in 2% osmium tetroxide (SIC Società Italiana Chimici) for 2 hours and dehydrated by passages in ethanol (Sigma Aldrich) from 30% to 100% (5 minutes for each passage). After two 7-minute passages of propylene oxide and overnight in a 1:1 mixture of propylene oxide (Sigma Aldrich) and mixture of Glauerts resins, the samples were embedded in the Glauerts resin mixture (made of equal parts of Araldite M and Araldite Harter, HY 964, Sigma Aldrich). A 0.5% dibutyl phthalate plasticizer (Sigma Aldrich) was added to the resin mixture. For the final phase, the 2% accelerator 964 was added to the resin to facilitate the polymerization of the embedding mixture, at 60° C.

[0124] Semi-thin sections (2.5 μm thick) were cut using a Ultracut UCT ultramicrotome (Leica Microsystems, Wetzlar, Germany) and stained with 1% toluidine blue for high-resolution optical microscopy examination and design-based stereology. A DM4000B microscope equipped with a DFC320 digital camera and an IM50 image management system (Leica Microsystems, Wetzlar, Germany) was used for section analysis.

[0125] (g) In Vivo Experiments on Nerve Regeneration.

[0126] The animals inclusion criteria for in vivo experiments were based on their weight, age and health condition.

[0127] Before collecting the regenerated nerve, each implant site was carefully examined to detect any signs of local tissue alterations.

[0128] Thus, to observe the qualitative morphology of the nerve in the early stages of regeneration, both sections were analyzed: longitudinal and transverse. In particular, the longitudinal sections were subjected to Masson's trichrome staining and to immunofluorescence staining to have a macroscopic overview of the initial regeneration phase; the semi-thin cross-sections were stained with toluidine blue and were analyzed by high-resolution optical microscope to study the microscopic behavior of the nerves in regeneration. The images of the cross section were collected inside the graft, both proximally and distally, to follow the regeneration of the nerve along the medical device.

[0129] All analyzes were conducted in the short term after the surgery (2 and 4 weeks).

[0130] The nerves repaired with the medical device comprising a tubular membrane comprising P3HB and silver nanowires were analyzed by high-resolution optical microscopy analyzing semi-thin transversal sections stained with toluidine blue.

[0131] (h) Qualitative Evaluation of Scar Formation.

[0132] On the day of the sacrifice, each implant site was examined for the presence of local tissue alterations with a high magnification surgical microscope. Special attention was paid to identify any signs of inflammation and/or development of scar tissue that could be attributed to the degradation of the implant. All tubular membranes comprising P3HB (with or without silver nanowires) were found at the graft site, covered with a thin layer of connective tissue. A careful peri-implant examination showed no signs of inflammation or scar tissue formation around the membranes.

[0133] (i) Two Weeks after the Surgery.

[0134] The trichrome staining of the longitudinal sections showed the presence of a thin layer of connective tissue surrounding the outer side of the tubular membrane.

[0135] After 2 weeks of surgery, several types of cells colonized the lumen of the medical device comprising P3HB (Schwann cells, fibroblasts, immune system cells) and several vessels inside the lumen of the medical device were also identified in cross sections stained with toluidine blue.

[0136] The central portion of the medical device has not yet been completely colonized by cells or extracellular matrix, which means that the cells must still migrate to the central portion of the medical device.

[0137] In the semi-thin cross section stained with toluidine blue the regenerated myelinated fibers have not yet been identified within the medical device, both at a proximal and at a distal level; however, few positive neurofilament fibers were appreciated with immunohistochemical analysis.

[0138] On the contrary, the nerve repaired with the tubular membrane comprising P3HB and silver nanowires showed an early regeneration, in fact after 2 weeks from the implantation it was already possible to observe the formation of regenerated fibers in the proximal stump. Cellular colonization was observed in the distal stump.

[0139] The nerves repaired by autotransplantation showed many axons that were still degenerating. A few regenerating fibers with a thin myelin sheath were identified.

[0140] FIG. 6 shows, by comparison, images of a nerve repaired with the tubular membrane comprising P3HB (without Ag) and of a nerve repaired with the tubular membrane comprising P3HB enriched with silver nanowires, the images were obtained by optical microscopy. In particular, the images represent semi-thin transversal sections stained with toluidine blue of the proximal portion of a median nerve repaired with the medical device comprising P3HB (without Ag) (A-D) and of the proximal portion of a median nerve repaired with the medical device comprising P3HB enriched with silver nanowires (E-H), 2 weeks after implantation.

[0141] (j) Four Weeks after the Surgery.

[0142] The trichrome staining of the longitudinal sections showed the presence of a thin layer of connective tissue surrounding the outer side of the medical device comprising a tubular membrane comprising P3HB even after 4 weeks of nerve repair. Furthermore, it was possible to identify a rich cellular colonization, especially in transversal sections stained with toluidine blue. Unlike the results 2 weeks after surgery, in which the central portion of the medical device was still empty, after 4 weeks the entire tubular membrane comprising P3HB was colonized. Many blood vessels, even of large diameter, were easily identified with a toluidine blue staining.

[0143] Interestingly, after 4 weeks, many regenerated fibers are present in the proximal portion of the cell membrane. The regenerated fibers were easily identified both by immunofluorescence analysis, showing many positive longitudinal neurofilament fibers surrounded by Schwann positive S-100 cells, and by high resolution optical microscopy, showing many transverse regenerating fibers with their myelin sheath.

[0144] Many transverse regeneration fibers can also be identified, 4 weeks after implants, in the proximal nerve stump repaired the tubular membrane comprising P3HB enriched with silver nanowires.

[0145] In the distal stump of these repaired nerves, it is possible to observe the formation of endoneural tubes, which will guide the regenerated fibers towards the target organs.

[0146] The nerves regenerated by autotransplantation showed many myelinated fibers both proximally and distally. Few degeneration fibers have been identified.

[0147] (k) Conclusions of the Pre-Clinical Study.

[0148] All tubular membranes comprising P3HB (both with and without Ag nanowires) were found at the implant site, covered with a thin layer of connective tissue. The careful examination of the peri-implantation site shows the absence of signs of inflammation or scar tissue formation, demonstrating the biocompatibility of these membranes. From the macro and microscopic analysis, differences have emerged regarding the regeneration pattern. In particular, it was observed that in the tubular membranes comprising P3HB enriched with Ag nanowires, the nerve regeneration occurs near the duct wall, while in the tubular membranes without Ag nanowires the regenerating axons are located in the center of the medical device itself. Moreover, through the optical microscopy analysis of semi-thin sections, it was possible to observe that the tubular membranes comprising P3HB and Ag nanowires showed regenerating nerve fibers already two weeks after implantation, thus demonstrating an earlier regeneration compared to the membranes without Ag nanowires. From these analyzes it is therefore possible to conclude that the Ag nanowires present in the membrane are capable of attracting the regenerating nerve fibers, providing a support pathway that facilitates the correct orientation thereof: from proximal to distal. The correct orientation of the axons during the regeneration phase leads to a faster nerve regeneration which, as a result, involves an early re-innervation of the target muscle tissue and consequently an early motor recovery.