WO2009125196A1

WO2009125196A1 - Neural circuits and stereotactic apparatus

Info

Publication number: WO2009125196A1
Application number: PCT/GB2009/000945
Authority: WO
Inventors: Monte. Alan Gates; Ying Yang; Andrzej Bonawentura Jozwiak
Original assignee: Keele University
Priority date: 2008-04-10
Filing date: 2009-04-09
Publication date: 2009-10-15
Also published as: GB0806555D0

Abstract

Herein are disclosed: - solid or semi-solid implantable neural circuits conduit comprising an elongate polymer sheath and one or a plurality of neurons in the lumen of the sheath or embedded in the scaffold; - methods of manufacturing said implantable neural circuits; - a stereotactic syringe apparatus having a frame and a syringe support mounted on the frame, the syringe support having : (i) a translation stage on which a medical syringe is mounted, the syringe having a single cannula, the cannula having a tip at one end suitable for insertion into tissue or other matter, wherein the translation stage is moveable to position the cannula, the cannula having a plunger slideably disposed in the lumen of the cannula, (ii) a plunger control element configured to control the position of the plunger in the cannula, wherein the translation stage is moveable to withdraw the cannula from the tissue or other matter independently of the plunger control element; - and a corresponding method of implanting neural circuits.

Description

NEURAL CIRCUITS AND STEREOTACTIC APPARATUS

Field of the Invention

The present invention relates to formation and implantation of a neural circuit useful in establishing point-to-point neural connections. The present invention also relates to the stereotactic implantation of material into biological tissue or other media.

Background to the Invention

Transplantation of immature cells has been highlighted as a potential therapy for the damaged adult brain and spinal cord. Cell replacement strategies for central nervous system (CNS) deficits would be greatly benefited by the re-establishment of point-to-point connections for functional recovery in various CNS deficits (such as Huntington's and Parkinson's disease, stroke, or spinal cord injury) . Re-establishing point-to-point connections in the adult CNS has proven very difficult due to the fact that the adult CNS expresses molecules that inhibit axonal growth, or fails to express precise gradients of growth-promoting cues that stimulate and direct axonal growth. Transplanted cells are very limited in their growth potential, and rarely extend beyond the immediate confines of their transplant environment. This greatly limits the potential for cell transplantation in the CNS, in that the repair of the complex circuitry of the CNS requires the re-establishment of connections and not merely the replacement of cells. Currently, attempts to re-establish CNS connections in Parkinson's disease is limited to the injection of a suspension of dopamine producing neurons into the striatum and not their place of origin (the substantia nigra) .

The existing approaches to re-establishment of point-to-point neural connections are limited to attempts to regenerate peripheral nervous system (as opposed to central nervous system) connections. Neural guide tubes such as those described in US 7,135,040 have been provided for this purpose. This does not involve the transplantation of neurons, or a neural circuit, but the grafting of a synthetic guide designed to encourage regeneration and reconnection of the severed nerve in situ.

Others have considered the transplantation of cells into the CNS, but the transplanted cells are not pre-constructed to provide a neural circuit bridging, and therefore capable of providing a connection between, two sites in the CNS. In contrast, the existing techniques and constructs rely on mere transplantation of cells, often as a liquid solution of cells or constructs, to a single site in the hope that the transplanted cells will act or grow in situ in a desirable manner. Thus, effective treatment is dependent on the way in which those cells grow following transplantation.

In US6,264,693 cells for transplantation into the brain were cultured on a support matrix but maintained on the surface of the matrix and were not encapsulated by the matrix. In US 5,853,385 PC12 cells were encapsulated in an immunoisolatory conduit. The use of an immunisolatory conduit is also described in US 6,264,865.

During the stereotactic implantation of neural circuit conduits and other matter into biological tissue it is desirable to minimise the damage caused to the tissue. Known stereotactic implantation apparatus and methods include those described in SE 468270 and US 5,006,122. These disclosures describe a two step implantation method in which a first cannula is inserted into the brain, and then the stylet within the cannula is subsequently withdrawn. A second cannula containing the material to be implanted is then inserted through the lumen of the first cannula. Once the second cannula is in place, both cannulas are withdrawn to deposit the material in the tissue.

These methods suffer from a disadvantage in that when the stylet is withdrawn from the first (external) cannula brain fluid is sucked into the lumen of the first cannula and is then pushed back out when the second cannula is inserted. This creates a pumping action which can damage the brain tissue .

Summary of the Invention

In an attempt to circumvent the complex milieu of the adult CNS (which negatively affects the growth of transplanted cells) the inventors have devised methods to implant "neural wires", herein referred to as neural circuits, that have been constructed in vitro. This strategy creates a completely new concept for repair of CNS defects where neural circuits pre- constructed in vitro nullify the problems associated with the aberrant expression of (known and unknown) inhibitory and growth promoting molecules in the adult brain. This means that two points ("A" and "B") can be bridged at the moment of grafting without requiring any influence from the host tissue.

Pre-construction of circuitry has the additional benefit of: (a) providing a quality control period for assessing cell viability among the to-be transplanted cells; (b) flexibility in the time of application of the cells for surgical implantation; and (c) optional additional health screening of tissue prior to cell grafting.

The present invention has the potential to enhance the therapeutic use of primary cells or stem cells for treating Parkinson's disease, and offers a new route for cell replacement to treat other CNS disorders which require the re- establishment of point-to-point contacts. For example, Huntington's disease is well placed to benefit from such circuitry replacement, as a primary source for donor cells which could be used to treat the disorder is well defined and some of the circuitry involved (i.e., the striato-pallidal circuit) is relatively short.

The present invention provides for the implantation/grafting of an intact neural circuit into the mammalian brain in order to restore point-to-point connections for functional recovery of various CNS deficits. In particular, the present invention provides methods and construction of terminally differentiated neuronal circuits for the treatment of neurological disabilities, such as Parkinson's disease, Huntington's disease and traumatic brain injury.

Accordingly, in some aspects the present invention relates to the formation of neural circuits which are useful in establishing point to point connections.

The circuits are preferably constructed in vitro by culturing tissue or cells capable of neuron growth in a support material to form a conduit in which the neurons are contained. By forming an elongate conduit having a main length corresponding to the distance between two anatomical locations in the nervous system of a human or animal neurons can be grown to extend generally along the length of the conduit between the conduit ends. The neurons thereby form a neural circuit which, following implantation between the two anatomical locations, can function to provide a neural connection between the two locations. This can restore chemical and/or electrical signalling between the anatomical locations and contribute to therapeutic treatment of a number of neurological disorders.

The neural circuits are preferably described as "conduits" or "constructs". In some embodiments they may form capsules having a sheath or membrane that partially encloses the neurons .

Neural circuit conduits according to the invention comprise a support material or scaffold and neurons, and optionally an extracellular matrix component. In one arrangement the support material is a tubular sheath having neurons and optional extracellular matrix components in the lumen of the sheath. In another arrangement the support material is a matrix material through which the neurons can grow and in which the neurons may become embedded.

By forming the conduit such that nutrients and factors required for neuron growth can be exchanged with tissue or cells contained in the conduit neuron growth can be supported both in vitro prior to implantation and in vivo following implantation to neural tissue.

According to one aspect of the present invention a solid or semi-solid implantable neural circuit conduit is provided comprising an elongate polymer sheath and one or a plurality of neurons in the lumen of the sheath, wherein the conduit is permeable to molecules required for neuron growth.

The sheath may comprise a thin film polymer tube and the sheath may further contain a polymer scaffold in the lumen of the sheath in which the neuron (s) can grow and in which they can become embedded. The scaffold is preferably made of a gel or hydrogel material.

The conduit is preferably a tubular structure in which the polymeric sheath forms the walls of the tube.

According to another aspect of the present invention a solid or semi-solid implantable neural circuit conduit is provided comprising an elongate polymer scaffold and one or a plurality of neurons embedded in the scaffold, wherein the scaffold is permeable to molecules required for neuron growth.

The scaffold is preferably made of a gel or hydrogel material. The conduit is designed to provide a neural circuit between ends of the conduit and therefore preferably has a tubular structure. The conduit may therefore be formed as a plug of material comprising scaffold and neurons. In preferred embodiments the plug is generally cylindrical.

The polymer sheath or scaffold may be a biodegradable polymer, which may be chosen from the group of: alginate, agarose, collagen, chitosan, polycaprolactone, poly (DL-lactide-co- caprolactone) , poly (L-lactide-co-caprolactone-co-glycolide) , polyglycolide, polylactide, polyhydroxyalcanoates, copolymers and blends thereof.

Alternatively, the polymer sheath or scaffold may be a nonbiodegradable polymer which may be chosen from the group of: cellulose acetate; cellulose butyrate, polysulfone, polyurethane, polyacrylonitrile, sulfonated polysulfone, polyamide, polyacrylonitrile, polymethylmethacrylate, copolymers and blends thereof.

The conduit preferably has a main longitudinal axis between a first end and a second end wherein the axon(s) of the neuron (s) predominantly extend between the first and second ends. The axons may be substantially parallel to the axis or may take any route between the first and second ends. In preferred embodiments a majority (i.e. greater than 50%) of the axons extend between the first and second ends. More preferably 60% or more, 70% or more, 80% or more or 90% or more of the axons extend between the first and second ends. The conduit is preferably non-immunogenic, i.e. does not provoke an immune response in the patient that would lead to rejection of the transplanted conduit.

In preferred embodiments, the conduit is provided for use in a method of medical treatment. More preferably the conduit is provided for use in the treatment of a neurological condition. The conduit may be provided as a pharmaceutical composition.

The conduit is preferably provided for use in providing a neuron connection between two positions in the brain of a human or animal. Accordingly, the conduit may be provided for use in the treatment of one of Parkinson's disease, Huntingdon's disease or Alzheimer's disease. The conduit may also be used for re-establisment of neural connections in any circumstance. For example, re-establishment of neural connections in the brain or spinal cord may be required following traumatic injury, e.g. a blow to the head or serious fall.

In a further aspect of the present invention a method of manufacturing an implantable neural circuit conduit is provided, the method comprising the steps of:

(a) forming a non-liquid polymeric elongate sheath having a lumen with at least one end of the sheath being open;

(b) positioning tissue or cells capable of neuron formation such that neuron formation can occur in the lumen of the sheath;

(c) culturing the tissue and sheath under conditions in which neuron growth can occur in the lumen of the sheath, thereby forming a conduit having one or a plurality of neurons contained in the lumen of the sheath. The tissue or cells in (b) may be capable of forming CNS or PNS neurons, but are preferably capable of forming CNS neurons .

In one embodiment step (a) further comprises providing or forming a polymer scaffold in the lumen of the sheath, wherein the scaffold provides a support matrix for neuron growth. The scaffold is preferably a gel or hydrogel material.

The sheath formed in (a) is preferably tubular and preferably has a main length between a first end and a second end, wherein tissue in (b) is positioned at or adjacent the first end, with neuron growth, particularly axon growth, in (c) occurring towards the second end.

In some embodiments, prior to step (c) the method further comprises positioning tissue or factors capable of promoting the growth of neurons in the lumen of the sheath or in contact with the sheath. The tissue or factors may be positioned closer to the second end, relative to the positioning of the tissue or cells in (b) .

Tissue or cells in step (b) may be inserted through an open end of the sheath. The tissue or cells may be inserted in predetermined order or orientation. For example, embryonic tissue fragments may be used to encourage neuron growth towards an end of the sheath, by positioning such fragments at or near one end of the lumen of the sheath growth of neurons, and particularly their axons, between ends of the sheath can be encouraged. In another example, growth factors, peptides, proteins and/or nutrients may be similarly positioned to encourage axon growth between the ends of the sheath.

In some embodiments the sheath in (a) is preferably formed in the lumen of a cannula. For example, the sheath may be formed as a thin film polymer tube in the lumen of a tube, e.g. a cannula, by a method comprising dipping the tube in a polymer solution and evaporating solvent. In another example the sheath is formed as a thin film polymer tube in the lumen of a tube, e.g. a cannula, by a phase inversion method.

By forming the sheath in the lumen of a cannula the method provides for the preparation of a cannula suitable for direct medical use in the surgical implantation of the neural circuit conduit.

In a further aspect of the present invention a method of manufacturing an implantable neural circuit conduit is provided, comprising the steps of:

(a) forming a non-liquid polymeric elongate scaffold, the scaffold providing a support matrix for neuron growth;

(b) contacting the scaffold with tissue or cells capable of neuron formation;

(c) culturing the scaffold and tissue or cells under conditions in which neuron growth can occur, thereby forming a conduit having one or a plurality of neurons embedded in the scaffold.

The tissue or cells in (b) may be capable of forming CNS or PNS neurons, but are preferably capable of forming CNS neurons.

The scaffold is preferably made of a gel or hydrogel material. The scaffold formed in (a) is preferably tubular in shape. As described above, the scaffold may be formed as a cylindrical plug to provide a cylindrical conduit body.

The scaffold preferably has a main length between a first end and a second end, and tissue in (b) is positioned at or adjacent the first end, with neuron growth in (c) occurring towards the second end. In some embodiments, prior to step (c) the method further comprises contacting tissue or factors capable of promoting the growth of neurons with the scaffold.

The tissue or factors may be contacted with the scaffold closer to the second end, relative to the positioning of the tissue or cells in (b) .

Tissue or cells in step (b) may be arranged in the scaffold in predetermined order or orientation. For example, embryonic tissue fragments may be used to encourage neuron growth towards an end of the scaffold, by positioning such fragments at or near one end of the scaffold growth of neurons, and particularly their axons, between ends of the scaffold can be encouraged. In another example, growth factors, peptides, proteins and/or nutrients may be similarly positioned to encourage axon growth between the ends of the scaffold.

In some embodiments the scaffold in (a) is formed in the lumen of a tube, e.g. a cannula. By forming the scaffold in the lumen of a cannula the method provides for the preparation of a cannula suitable for direct medical use in the surgical implantation of the neural circuit conduit.

The present invention includes neural circuit conduits obtained by any of the methods described above.

The present invention also includes a cannula having a neural circuit conduit disposed in the lumen of the cannula, the neural circuit conduit obtained by any of the methods described above.

The present invention also includes a cannula having a neural circuit conduit disposed in the lumen of the cannula, the cannula obtained by any of the methods described above. The present invention also includes a syringe having a neural circuit conduit or cannula as described above.

The present invention also includes a sterotactic syringe apparatus comprising a neural circuit conduit, cannula or syringe as described above.

The present invention also includes a method of manufacturing a cannula having a neural circuit conduit disposed in the lumen of the cannula, the method comprising forming a neural circuit conduit according to any of the methods described above, wherein the neural circuit conduit is formed in the lumen of a cannula.

The present invention also includes a method of manufacturing a medical syringe having a cannula and a neural circuit conduit disposed in the lumen of the cannula, the method comprising forming a neural circuit conduit according to the method of any of the methods described above, wherein the neural circuit conduit is formed in the lumen of a cannula, and using the cannula in the assembly of a medical syringe.

The present invention also includes a method of manufacturing a surgical instrument for use in a method of surgical implantation, the instrument having a cannula and a neural circuit conduit disposed in the lumen of the cannula, the method comprising forming a neural circuit conduit according to the method of any of the methods described above, wherein the neural circuit conduit is formed in the lumen of a cannula, and using the cannula in the assembly of a surgical instrument .

In a further aspect of the present invention a method of stereotactically implanting material into biological tissue or a selected medium is provided, the method comprising: (a) providing a medical syringe mounted on a stereotactic apparatus, the medical syringe having:

(i) a single cannula, the cannula having a lumen extending longitudinally through the cannula forming an opening at one end adjacent a tip suitable for insertion into the tissue or medium;

(ii) a plunger element disposed in the lumen and extending towards an opposite end of the lumen;

(iii) a quantity of material to be implanted disposed in the lumen,

(b) inserting the cannula to a desired position in the biological tissue or selected medium, wherein prior to step (c) said quantity of material is positioned in the lumen of the cannula between said opening and said plunger element, such that an end of the plunger element is adjacent part of said material, the method further comprising:

(c) slideably withdrawing the cannula from the desired position over the plunger element whilst maintaining a constant position of the plunger element.

The use of a single cannula overcomes the problem of ^Λtissue pumping' associated with other implantation methods (as described above) .

In some embodiments the method is for the implantation of the material between a first and second location in the biological tissue or medium, the method involving inserting the cannula such that the region of the lumen containing said material passes through said first and second positions.

In some embodiments the method is for the implantation of the material between a first and second location in the biological tissue or medium, the method involving inserting the cannula such that said opening is positioned at the first position and the region of the lumen containing said material passes through said second position.

In some embodiments the method is for the implantation of the material between a first and second location in the biological tissue or medium, the method involving inserting the cannula such that said opening is positioned at the first position and the region of the lumen containing the interface between said plunger element and material is positioned at (e.g. proximal to or adjacent to) said second position.

In some embodiments the quantity of material has a length along the longitudinal axis of the lumen that is greater than or substantially the same as the distance between the first and second positions.

In preferred embodiments the stereotactic apparatus comprises a surgical implantation attachment having a frame comprising:

I. an adapter for attaching the surgical implantation attachment to the stereotactic apparatus;

II. a syringe support connected to the medical syringe; III. a control element configured to maintain a selected position of the plunger element in the lumen of the cannula, wherein the syringe support is mounted on a translation stage which is moveable to slide the cannula over the plunger element whilst the control element maintains the plunger element in the selected position.

In some embodiments the material is a neural circuit conduit and the method is a method of implanting the neural circuit conduit at a defined position in the tissue or medium to provide a neuron connection between first and second positions in the tissue or medium, in which the cannula is inserted such that the tip of the cannula is positioned at the first position and the lumen of the cannula passes through the second position, such that during (c) the neural circuit conduit is deposited in the tissue or selected medium so as to bridge the first and second positions.

In some embodiments the method is an in vitro method, which may be performed on a phantom model, or excised or non-living biological tissue, e.g. to assist surgeons in practising stereotactic implantation techniques.

In other embodiments the method may be performed on living humans or animals (i.e. in vivo), e.g. as part of surgical and/or therapeutic procedure.

In some embodiments the material is a neural circuit conduit (e.g. as described herein) and the method is for treating a neurological disorder comprising implanting the neural circuit conduit at a defined position in nervous system tissue (e.g. central or peripheral nervous system tissue) of a human or animal so as to provide a neural connection between first and second positions in the nervous system tissue.

Accordingly, the method may comprise implanting the neural circuit conduit in the brain of the human or animal. The method may be a method of treating a neurological disorder chosen from the group: Parkinson's disease, Huntingdon's disease .

In a further aspect of the present invention a surgical implantation attachment for a stereotactic apparatus is provided, the attachment having a frame comprising:

(a) an adapter for attaching the attachment to a stereotactic apparatus;

(b) a syringe support connected to a medical syringe the medical syringe having a single cannula, the cannula having a lumen extending longitudinally through the cannula forming an opening at one end adjacent a tip suitable for insertion into tissue or other matter, the medical syringe further comprising a plunger element disposed in the lumen and extending towards an opposite end of the lumen;

(c) a control element configured to maintain a selected position of the plunger element in the lumen of the cannula, wherein the syringe support is mounted on a translation stage which is inoveable to slide the cannula over the plunger element whilst the control element maintains the plunger element in the selected position.

In preferred embodiments the quantity of material to be implanted is positioned in the lumen between said opening and said plunger element, such that an end of the plunger element is adjacent part of said material.

The material is preferably non-liquid, more preferably solid or semi-solid. In some embodiments the material is a neural circuit conduit. In some embodiments the material comprises biological tissue, e.g. cells.

The plunger control element may comprise a retaining member configured to abut a part of the plunger element.

The implantation method and surgical implantation attachment described above may be used for implantation of neural circuit conduits or for implantation of other tissue, e.g. cells.

In a further aspect of the present invention a cannula is provided having a neural circuit conduit according to the present invention disposed in the lumen of the cannula. A method of manufacturing such a cannula is provided comprising forming a neural circuit conduit according to the present invention in the lumen of a cannula. Another method of manufacturing such a cannula is also provided comprising inserting a neural circuit conduit according to the present invention in the lumen of a cannula. The use of a neural circuit conduit according to any one of the preceding aspects in the manufacture of such a cannula is also provided. A sterotactic syringe apparatus comprising such a cannula is also provided.

In a further aspect of the present invention a syringe is provided having a cannula, a plunger disposed within the lumen of the cannula and a neural circuit conduit according to any one of the preceding aspects disposed in the lumen between an end of the plunger and an end of the cannula. A method of manufacturing such a syringe is also provided comprising inserting a neural circuit conduit according to the present invention into the lumen of a cannula and using the cannula in the assembly of a syringe. The use of a neural circuit conduit according to any one of the preceding aspects in the manufacture of such a syringe is also provided. A sterotactic syringe apparatus comprising such a syringe is also provided. A method of manufacturing such a syringe is also provided, the method comprising inserting a neural circuit conduit according to any one of the preceding aspects into the lumen of a cannula of a syringe.

In a further aspect of the invention a method of implanting a neural circuit conduit is provided, the method comprising the steps of:

(i) inserting the cannula of a syringe into a tissue or selected medium, wherein the cannula has a lumen, a plunger disposed within the lumen and a neural circuit conduit according to the present invention disposed in the lumen between an end of the plunger and an end of the cannula;

(ii) whilst keeping the plμnger in a stationary position, sliding the cannula over the plunger thereby depositing the neural circuit conduit in the tissue or selected medium.

In a preferred embodiment the method is a method of implanting the neural circuit conduit at a defined position in the tissue or medium to provide a neuron connection between first and second positions in the tissue or medium, in which the cannula is inserted such that the tip of the cannula is positioned at the first position and the cannula passes through the second position, such that in (ii) the neural circuit conduit is deposited in the tissue or selected medium so as to bridge the first and second positions.

The implantation methods described herein may be performed in vitro or in vivo. For example, the method may be performed in vitro on a synthetic medium such as gelatin, or performed ex vivo on non-living human or animal tissue, or in vivo on a living human or animal.

In a further aspect of the present invention a method of treating a neurological disorder is provided, the method comprising implanting a neural circuit conduit according to the present invention at a defined position in a tissue of a human or animal so as to provide a neural connection between first and second positions in the tissue, the method comprising the steps of implanting a said neural circuit conduit such that a first end of the conduit is located at the first position and a second end of the conduit is located at the second position.

Preferably, the method comprises:

(i) inserting the cannula of a syringe into the nervous system of the human or animal, wherein the cannula has a plunger disposed within its lumen and a neural circuit conduit according to the present invention disposed in the lumen between an end of the plunger and an end of the cannula, wherein the cannula is inserted such that the tip of the cannula is positioned at the first position and the cannula passes through the second position;

(ii) whilst keeping the plunger in a stationary position, sliding the cannula over the plunger thereby depositing the neural circuit conduit in the tissue or selected medium so as to bridge the first and second positions .

In a preferred embodiment the method comprises implanting the neural circuit conduit in the brain of the human or animal. The neurological disorder may be chosen from from: Parkinson' s disease, Huntingdon's disease, stroke, cerebral infarction or ischaemia. The neurological disorder may be any loss of point-to-point neural connectivity in the PNS or CNS, which may, for example, result from traumatic injury.

In the aspects and embodiments described herein the human or animal to be treated may be a patient. The patient may be a non-human mammal, but is more preferably a human patient. The patient may be male or female.

In a further aspect of the present invention there is provided a stereotactic syringe apparatus having a frame and a syringe support mounted on the frame, the syringe support having: (i) a translation stage on which a medical syringe is mounted, the syringe having a cannula, the cannula having a tip at one end suitable for insertion into tissue or other matter, wherein the translation stage is moveable to position the cannula, the cannula having a plunger slideably disposed in the lumen of the cannula,

(ii) a plunger control element configured to control the position of the plunger in the cannula, wherein the translation stage is moveable to withdraw the cannula from the tissue or other matter independently of the plunger control element.

The translation stage is thus moveable to position the cannula and the plunger control element can maintain a constant position of the plunger during movement of the translation stage .

In the aspects and embodiments described herein, the plunger control element preferably comprises a retaining member configured to abut the plunger. The retaining member may be a stop against which the plunger may abut, the stop preventing outward movement of the plunger (i.e. out of the tissue or other matter) during outward movement of the cannula. The retaining member may therefore be fixed to the frame of the stereotactic apparatus independently of the translation stage. Adjustment means may allow for positioning of the retaining member independently of the translation stage.

In the aspects and embodiments described herein, the plunger may be formed by a single plunger element extending from the lumen of the cannula to an external position at which it interacts with the plunger control element. Alternatively, the plunger may comprise two or more mechanically linked components, e.g. a first component capable of interacting with the plunger control element in mechanical connection with a second component being a cylindrical element inserted in the lumen of the cannula and interacting with a neural circuit conduit. In some embodiments the first component may be a quantity of liquid, fluid or gel, e.g. mineral oil or culture medium, positioned in the lumen of the cannula adjacent the material to be implanted and in fluid-mechanical connection with a second component such as a solid element located in the lumen of the cannula, the solid element and liquid/fluid/gel together forming a plunger element capable of transmitting force against the material to be implanted.

A neural circuit conduit may be positioned in the cannula between the tip of the cannula and an end of the plunger.

The following numbered paragraphs contain statements of broad combinations of the inventive technical features herein disclosed: -

1. A solid or semi-solid implantable neural circuit conduit comprising an elongate polymer sheath and one or a plurality of neurons in the lumen of the sheath, wherein the conduit is permeable to molecules required for neuron growth.

2. An implantable neural circuit according to paragraph 1 wherein the neuron is a central nervous system neuron.

3. An implantable neural circuit according to paragraph 1 or 2 wherein the sheath comprises a thin film polymer tube.

4. An implantable neural circuit according to any one of paragraphs 1 to 3 wherein the sheath contains a polymer scaffold in which the neuron (s) are embedded.

5. An implantable neural circuit according to paragraph 4 wherein the scaffold is a gel or hydrogel.

6. An implantable neural circuit according to any one of paragraphs paragraph 1 to 5 wherein the conduit is tubular, the polymeric sheath forming walls of the tube.

7. A solid or semi-solid implantable neural circuit conduit comprising an elongate polymer scaffold and one or a plurality of neurons embedded in the scaffold, wherein the scaffold is permeable to molecules required for neuron growth. 8. An implantable neural circuit according to paragraph 7 wherein the neuron is a central nervous system neuron.

9. An implantable neural circuit according to paragraph 6 or 7 wherein the scaffold is a gel or hydrogel.

10. An implantable neural circuit according to any one of paragraphs 7 to 9 wherein the conduit is tubular.

11. An implantable neural circuit according to any one of paragraphs 1 to 10 wherein the polymer is a biodegradable polymer.

12. An implantable neural circuit according to any one of paragraphs 1 to 10 wherein the polymer is a biodegradable polymer chosen from the group of: alginate, agarose, collagen, chitosan, polycaprolactone, poly (DL-lactide-co-caprolactone) , poly (L-lactide-co-caprolactone-co-glycolide) , polyglycolide, polylactide, co-polymers and blends thereof.

13. An implantable neural circuit according to any one paragraphs 1 to 10 wherein the polymer is a non-biodegradable polymer.

14. An implantable neural circuit according to any one of paragraphs 1 to 10 wherein the polymer is a non-biodegradable polymer chosen from the group of: cellulose acetate; cellulose butyrate, polysulfone, polyurethane, polyacrylonitrile, sulfonated polysulfone, polyamide, polyacrylonitrile, polymethylmethacrylate, co-polymers and blends thereof.

15. An implantable neural circuit according to any one of paragraphs 1 to 14 wherein the conduit has a main longitudinal axis between a first end and a second end wherein the axon(s) of the neuron (s) predominantly extend substantially parallel to said axis.

16. An implantable neural circuit according to any one of paragraphs 1 to 15 wherein a majority of the axons in the conduit extend between said first and second ends.

17. An implantable neural circuit according to any one of paragraphs 1 to 16 wherein the conduit is non-immunogenic.

18. An implantable neural circuit according to any one of paragraphs 1 to 17 for use in a method of medical treatment.

19. An implantable neural circuit according to any one of paragraphs 1 to 17 for use in the treatment of a neurological condition.

20. An implantable neural circuit according to any one of paragraphs 1 to 17 for use in the treatment of Parkinson' s disease, Huntingdon's disease or Alzheimer's disease.

21. An implantable neural circuit according to any one of paragraphs 1 to 20 for use in providing a neuron connection between two positions in the brain of a human or animal.

22. A method of manufacturing an implantable neural circuit conduit, comprising the steps of:

(c) culturing the tissue and sheath under conditions in which neuron growth can occur in the lumen of the sheath, thereby forming a conduit having one or a plurality of neurons contained in the lumen of the sheath.

23. The method of paragraph 22 wherein the tissue or cells in (b) are capable of forming CNS neurons.

24. The method of paragraph 22 or 23 wherein step (a) further comprises providing a polymer scaffold in the lumen of the sheath, the scaffold providing a support matrix for neuron growth.

25. The method of paragraph 24 wherein the scaffold is a gel or hydrogel.

26. The method of any one of paragraphs 22 to 25 wherein the sheath formed in (a) is tubular.

27. The method of any one of paragraphs 22 to 26 wherein the sheath has a main length between a first end and a second end, and tissue in (b) is positioned at or adjacent the first end, with neuron growth in (c) occurring towards the second end.

28. The method of any one of paragraphs 22 to 27 wherein prior to step (c) the method further comprises positioning tissue or factors capable of promoting the growth of neurons in the lumen of the sheath or in contact with the sheath.

29. The method of paragraph 28 wherein the sheath has a main length between a first end and a second end, and tissue in (b) is positioned at or adjacent the first end, with neuron growth in (c) occurring towards the second end and said tissue or factors are positioned closer to the second end, relative to the positioning of the tissue or cells in (b) .

30. The method of any one or paragraphs 22 to 29 wherein the sheath in (a) is formed in the lumen of a cannula. 31. The method of any one of paragraphs 22 to 30 wherein the sheath is formed as a thin film polymer tube in the lumen of a cannula by a method comprising dipping the cannula in a polymer solution and evaporating solvent.

32. The method of any one of paragraphs 22 to 30 wherein the sheath is formed as a thin film polymer tube in the lumen of a cannula by a phase inversion method.

33. A method of manufacturing an implantable neural circuit conduit, comprising the steps of:

(b) contacting the scaffold with tissue or cells capable of neuron formation/

34. The method of paragraph 33 wherein the tissue or cells in (b) are capable of forming CNS neurons.

35. The method of paragraph 33 or 34 wherein the scaffold is a gel or hydrogel.

36. The method of any one of paragraphs 33 to 35 wherein the scaffold formed in (a) is tubular in shape, the scaffold forming a cylindrical conduit body.

37. The method of any one of paragraphs 33 to 36 wherein the scaffold has a main length between a first end and a second end, and tissue in (b) is positioned at or adjacent the first end, with neuron growth in (c) occurring towards the second end. 38. The method of any one of paragraphs 33 to 36 wherein prior to step (c) the method further comprises contacting tissue or factors capable of promoting the growth of neurons with the scaffold.

39. The method of paragraph 38 wherein the scaffold has a main length between a first end and a second end, and tissue in (b) is contacted at or adjacent the first end, with neuron growth in (c) occurring towards the second end and said tissue or factors are contacted with the scaffold closer to the second end, relative to the positioning of the tissue or cells in (b) .

40. The method of any one or paragraphs 33 to 39 wherein the scaffold in (a) is formed in the lumen of a cannula.

41. A neural circuit conduit obtained by the method of any one of paragraphs 22 to 40.

42. A cannula having a neural circuit conduit according to any one of paragraphs 1 to 20 or 40 disposed in the lumen of the cannula.

43. A syringe having a cannula, a plunger disposed within the lumen of the cannula and a neural circuit conduit according to any one of paragraphs 1 to 17 or 41 disposed in the lumen between an end of the plunger and an end of the cannula.

44. A sterotactic syringe apparatus comprising a syringe according to paragraph 43.

45. A method of manufacturing a cannula according to paragraph 41 comprising forming a neural circuit conduit according to any one of paragraphs 1 to 17 or 41 in the lumen of a cannula. 46. A method of manufacturing a syringe according to paragraph 42 comprising inserting a neural circuit conduit according to any one of paragraphs 1 to 17 or 41 into the lumen of a cannula and using the cannula in the assembly of a syringe .

47. Use of a neural circuit conduit according to any one of paragraphs 1 to 17 or 41 in the manufacture of a syringe, the syringe having a cannula, a plunger disposed within the lumen of the cannula, the neural circuit conduit disposed in the lumen between an end of the plunger and an end of the cannula.

48. A method of implanting a neural circuit conduit comprising the steps of:

(ii) inserting the cannula of a syringe into a tissue or selected medium, wherein the cannula has a lumen, a plunger disposed within the lumen and a neural circuit conduit according to any one of paragraphs 1 to 17 or 41 disposed in the lumen between an end of the plunger and an end of the cannula;

(iii) whilst keeping the plunger in a stationary position, sliding the cannula over the plunger thereby depositing the neural circuit conduit in the tissue or selected medium.

49. The method of paragraph 48 wherein the method is a method of implanting the neural circuit conduit at a defined position in the tissue or medium to provide a neuron connection between first and second positions in the tissue or medium, in which the cannula is inserted such that the tip of the cannula is positioned at the first position and the cannula passes through the second position, such that in (ii) the neural circuit conduit is deposited in the tissue or selected medium so as to bridge the first and second positions . 50. The method of paragraph 48 or 49 wherein the method is an in vitro method.

51. A method of treating a neurological disorder comprising implanting a neural circuit conduit according to any one of paragraphs 1 to 17 or 41 at a defined position in a tissue of a human or animal so as to provide a neural connection between first and second positions in the tissue, the method comprising the steps of implanting a said neural circuit conduit such that a first end of the conduit is located at the first position and a second end of the conduit is located at the second position.

52. The method of paragraph 51 wherein the method comprises: (i) inserting the cannula of a syringe into the nervous system of the human or animal, wherein the cannula has a plunger disposed within its lumen and a neural circuit conduit according to any one of paragraphs 1 to 17 or 41 disposed in the lumen between an end of the plunger and an end of the cannula, wherein the cannula is inserted such that the tip of the cannula is positioned at the first position and the cannula passes through the second position;

53. The method of paragraph 52 wherein the method comprises implanting the neural circuit conduit in the brain of the human or animal .

54. The method of paragraph 53 wherein the method is a method of treating a neurological disorder chosen from the group: Parkinson's disease, Huntingdon's disease, Alzheimer's disease .

55. A stereotactic syringe apparatus having a frame and a syringe support mounted on the frame, the syringe support having:

(i) a translation stage on which a medical syringe is mounted, the syringe having a cannula, the cannula having a tip at one end suitable for insertion into tissue or other matter, wherein the translation stage is moveable to position the cannula, the cannula having a plunger slideably disposed in the lumen of the cannula, (ii) a plunger control element configured to control the position of the plunger in the cannula, wherein the translation stage is moveable to withdraw the cannula from the tissue or other matter independently of the plunger control element.

56. The stereotactic syringe apparatus of paragraph 55 wherein the plunger control element comprises a retaining member configured to abut the plunger.

The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference. Brief Description of the Figures

Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

Figure 1.

Schematic illustration of dissection of explants of substantia nigra from embryonic day 12 Sprague-Dawley rat embryo.

Figure 2.

Growth of neural wire. (A) and (B) in vitro incubation of MFB and VM tissue tissue, scaffold and capillary are incubated all-in-one in vitro; (C) axonal growth of the neural wire is from VM tissue towards MFB tissue.

Figure 3.

In vitro growth of neural wire from dissociated VM cells.

Figure 4.

Schematic illustration of a hollow fibre spinning system.

Figure 5.

Illustration of exemplary apparatus for guiding a neural circuit conduit into the open end of a cannula and positioning neural circuit conduit between the plunger and tip of the cannula. This apparatus is suitable for assembling scaffolds that are not formed in the lumen of a cannula, such as those prepared by hollow fibre spinning.

Figure 6.

Photographic view of scaffold displacement accessory attached to a stereotaxic frame together with enlarged illustration of the scaffold displacement accessory. The accessory enables movement of the needle fixed to the Hamilton syringe while the plunger is in the fixed position. The stereotaxic frame is used for controlled movement of the whole syringe mounted in the displacement accessory, and the displacement accessory allows for precision movement of the needle.

Figure 7 •

Illustration of neural circuit conduit displacement from the needle. Retraction of the needle with the plunger position remaining constant leaves the conduit behind in the cavity formed by the needle.

Figure 8.

Cryosection showing cross section through a conduit implanted in brain of a rat.

Figure 9.

Illustration of ΞlectroForce® 3200 Series Test Instrument setup for measurements of needle friction within a phantom gel .

Figure 10.

Illustration of needle insertion into brain tissue. Brain tissue was positioned at a desired angle and needle insertion was made along a desired linear path, in the illustration this pathway links the striatum and substantia nigra.

Figure 11.

Friction profile for needle insertion into rat brain along the approximate path linking the striatum and substantia nigra. Arrows indicate needle friction changes at the various brain structure/layers .

Figure 12.

Friction profile measured during insertion of the needle into brain phantom made from 2% gelatin solution. Figure 13 .

Friction profile measured during insertion of the needle into brain phantom made from 3% gelatin. The lower part of the relaxation curve indicates the physiologically interesting range of needle friction.

Figure 14. (A) Diagram showing medical syringe apparatus prior to implantation. (B) Diagram showing medical syringe apparatus after implantation.

Figure 15. Diagram showing stereotactic apparatus including a surgical implantation attachment according to the invention.

Figure 16. Diagram showing a surgical implantation attachment according to the invention attached to a stereotactic apparatus .

Figure 17. Flow diagram illustrating the steps of neural conduit formation at one end of a cannula (A-D) , attachment of the cannula to a medical syringe (E-F) , attachment of the medical syringe to a stereotactic apparatus and implantation of the conduit into brain tissue (G) .

Detailed Description of the Invention

In order to provide a neural circuit capable of providing a neural connection between two points an implantable neural circuit conduit is provided.

The neural circuit comprises, or consists of, a support material and neuron(s) . The support material functions both to support neuron growth and to provide a carrier for transplantation of neurons. By controlling the direction of axon growth on the support a neural circuit can be created having a directionality or polarity that is preserved by the support and enables reconnection of neural centres following transplantation of the neural circuit.

The conduit is preferably formed as an elongate structure. This provides for directed growth of neurons, and more particularly of their axonal projections. By providing an elongate structure axon growth is generally restricted to directional growth along the main length of the conduit. Accordingly, in preferred embodiments the conduit has a first end and a second end with axonal projections of neurons generally extending between the first and second ends in the direction of a main length extending between those ends.

By providing an elongate conduit axon growth can be generally limited to growth along a main length of the conduit between its two short ends. The axons thus provide a neural circuit that is effective as a "wire" to connect the two short ends of the conduit.

Although in a preferred embodiment the main length of the conduit is greater than the width of the first and/or second ends, this is not an essential requirement of all embodiments of the conduit. For example, a conduit that is short in length between the first and second ends and broader at one or both ends than the conduit is long between those ends may be provided and may be useful to provide a short neural circuit between two relatively large anatomical points in the central nervous system.

The conduit has a support material arranged to support directed neuron growth. In one arrangement a sheath is provided that partially encapsulates neuronal tissue. The sheath is formed from a thin-walled polymeric tube that is open at one or both ends to receive cells or tissue capable of neuron growth. Prior to addition of neuronal tissue or cells the sheath has a lumen (i.e. a central space or cavity) in which neuronal tissue can be cultured to form the implantable neural circuit.

The conduit is designed to contain neurons in a manner in which they can continue to grow following implantation. This requires the conduit to permit entry of growth factors and nutrients required for neuron growth. This also requires the conduit to permit growth of axons beyond the confines of the conduit in order to realise the goal of re-connecting neural locations. This can be achieved by partial envelopment or encapsulation, e.g. where a tubular sheath is open to surrounding tissue/media at one or more ends. The conduit can therefore provide an enclosure in which neurons are contained for the purposes of directed neuron growth but wherein the enclosure also permits exchange of molecules with the surrounding environment and growth of axons beyond the ends of the conduit.

The conduit can therefore comprise, or consist of, a support material in which neurons are partially or entirely incorporated, integrated, embedded, or immobilised. The support material may partially envelope or encapsulate the neurons to provide a robust and mechanically strong conduit. The conduit may be provided as a capsule. However, the support material may allow for outward growth of axons beyond the confines of the conduit.

The support material is preferably biodegradable such that following implantation the support material biodegrades to leave only the implanted neurons.

In a preferred embodiment the cells or tissue capable of neuron growth are positioned at one end of the tube such that neuron growth occurs towards the opposing end. Further tissue or factors capable of stimulating or promoting neuron growth may be incubated with the polymeric tube and tissue capable of neuron growth. In order to stimulate growth of axons in a direction generally between the open ends of the tube the further tissue or factors may be placed at or towards the opposing end of the tube. A gradient of growth promoting factors may thereby be provided which stimulates growth of axons towards the opposing end.

Following in vitro culturing of the sheath and tissue capable of neuron growth in this manner for a period of time sufficient for axon growth to occur, a conduit is provided in which neurons are contained within the lumen of the sheath and in which the axons of the neurons are predominantly extending between the open ends of the tube.

In another embodiment the elongate conduit is formed by a sheath having a lumen containing a scaffold material on, and optionally through, which neuronal tissue can grow so as to become embedded in the scaffold.

Where the conduit comprises a sheath in which neurons are encapsulated, the sheath is preferably open at one or more ends, e.g. as a tubular sheath having open ends, such that nutrients and growth factors can be freely exchanged with the neuronal tissue contained within the sheath and such that following implantation axons can freely grow beyond the conduit to connect with adjacent neural tissues. The sheath can optionally be impermeable to molecules required for neuron growth. However, in preferred embodiments the sheath is permeable to exchange of small molecules and gases to facilitate exchange of nutrients and growth factors required for neuron growth. In some embodiments the sheath is impermeable to molecules that inhibit axon growth in the CNS or PNS.

The sheath is preferably formed from a polymer, co-polymer or combination (e.g. blend) of polymers, and can be a polymer film, polymeric porous semi -permeable membrane or dense membrane. The sheath can be manufactured to be: permeable, semi-permeable, non-permeable, porous, non-porous, biodegradable, non-bio-degradable .

In another preferred embodiment the elongate conduit is formed by an elongate body of a scaffold material on, and optionally through, which the neuronal tissue can grow so as to become embedded in the scaffold.

A gel or hydrogel polymer scaffold matrix is provided and is seeded with tissue or cells capable of neuron growth. Further tissue or factors capable of stimulating or promoting neuron growth may be incubated with the scaffold and tissue capable of neuron growth. In order to stimulate a uniform direction of axon growth the further tissue or factors may be placed at a position towards which axon growth is desired. A gradient of growth promoting factors may thereby be provided which stimulates growth of axons towards further tissue or factors.

The scaffold preferably provides a support matrix for neuron growth. Preferred scaffolds have a porous structure, which may be provided by a cross-linked polymer. The scaffold is preferably permeable to nutrients and growth factors required for neuron growth. In some embodiments the scaffoled is impermeable to molecules that inhibit axon growth in the CNS or PNS. The scaffold is preferably formed from a polymer, copolymer or combination (blend) of polymers. The scaffold can be manufactured to be bio-degradable or non-bio-degradable.

Scaffolds may be formed by crosslinking of liquid films of sodium alginate, chitosan, or other polysaccharides with suitable crosslinkers, e.g. calcium salts, polyacrylic acid, heparin. Alternatively scaffolds may be formed as a gel, fabricated by collagen or alginates, crosslinked using well established methods known to those skilled in the art. In preferred embodiments the scaffold or sheath is formed, or placed in, an elongate, e.g. tubular, mold. Tissue or cells capable of neuron growth are then seeded at one end of the elongate scaffold with the further tissue or factors capable of stimulating or promoting neuron growth at the opposing end. Axon growth between the ends of the elongate scaffold or sheath may thereby be predominantly promoted.

Suitable polymer materials for the scaffold or sheath formation include biodegradable/bioresorbable polymer which may be chosen from the group of: alginate, agarose, collagen, chitosan, polycaprolactone, poly (DL-lactide-co-caprolactone) , poly (L-lactide-co-caprolactone-co-glycolide) , polyglycolide, polylactide, polyhydroxyalcanoates, co-polymers and blends thereof, or non-biodegradable polymers which may be chosen from the group of: cellulose acetate; cellulose butyrate, polysulfone, polyurethane, polyacrylonitrile, sulfonated polysulfone, polyamide, polyacrylonitrile, polymethylmethacrylate, co-polymers and blends thereof. Copolymers of polycaprolactone, polyglycolide, polylactide allow for fine-tuning of the biodegradation rate, as well as permeability and mechanical properties of the conduit.

Collagen is a promising material for construction of neural circuit conduits due to its biocompatibility and favourable property of supporting cell attachment and function (U.S. Pat. No. 5,019,087; Tanaka, S.; Takigawa, T.; Ichihara, S. & Nakamura, T. Mechanical properties of the bioabsorbable polyglycolic acid-collagen nerve guide tube Polymer Engineering & Science, 2006, 46, 1461-1467) .

Neurons are nerve cells and normally have a cell body containing a nucleus, a single axon capable of conveying an electrical signals (action potential) and dendrites capable of delivering incoming signals. In any aspect of the present invention the neurons may be of any type, e.g. peripheral nervous system (PNS) type, or central nervous system (CNS) type. The central nervous system includes the brain and spinal cord. Most preferably neurons are of central nervous system type and more preferably are neurons normally found in the brain or suitable for implantation to the brain to correct a defect in the brain. They may be neurons found in, or suitable for implantation to, particular locations in the brain such as the substantia nigra, striatum, and globus pallidus, or any regions where circuitry can become disrupted or destroyed.

Neurons contained in the conduit are required to have axonal projections. In preferred embodiments neurons are therefore mature neuronal cells having an axon and cell body, as distinguished from immature neuron precursor cells capable of generating an axonal projection but not yet having done so. To bridge connections within the nervous system, cells contained within the conduit may obtain axonal projections before implantation or have the potential to acquire such projections before, during or after implantation, or have the ability to positively affect connections between regions of the brain and spinal cord. Cells, in this way, may either bridge connections via direct cell-cell contact in the host, or via the secretion of factors which cause a desired response in the host region receiving the implant.

Neurons may be grown from cells or tissue. Tissues and cells may be chosen from: cells or tissue from the central nervous system, primary cells, progenitor cells, adult stem cells (e.g. adult neural stem cells) and optionally embryonic stem cells (e.g. embryonic neural stem cells) . Purely by way of example, suitable cells and tissue may include: precursor cells, cell lines grown in vitro such as fibroblasts following their genetic transformation into embryonic type stem cells, cells derived from embryonic tissues, embryonic brain tissue fragments; embryonic or adult ventral mesencephalon cells (primary cells) , embryonic or adult lateral ganglionic eminence cells (primary cells) , embryonic or adult spinal cord cells (primary cells) , embryonic or adult cortical cells (primary cells); embryonic or adult neural stem cells, induced pluripotent embryonic or adult stem cells or any cell or cell line now or in the future which displays properties (e.g., electrical, secretory, biochemical properties) which allows them to restore a functional connection within the brain.

The neurons may be derived from human or non-human cells or tissue, e.g. human, non-human mammals, rabbit, guinea pig, rat, mouse or other rodent (including cells from any animal in the order Rodentia) , cat, dog, pig, sheep, goat, cattle, horse, non-human primate or other non-human vertebrate organism.

The mean axon length of neurons contained within the conduit will vary by the particular circuit which is being replaced and the animal in which this is being accomplished. As an example, the nigro-striatal circuit will span from 4mm in rodents to 25mm in humans. Other circuits, such as the striato-pallidal circuit will be much shorter (1-2 mm in rodent, , 3-5mm in humans) , while others may be substantial (such as bridges in the spinal cord which may be several (>10) centi-metres long) . Thus, preferably the mean axon length of neurons contained in the conduit is between lmm and 30mm. In different embodiments mean axon length will be chosen from one of: between lmm and 5mm, between lmm and 3mm, between 3mm and 5mm, between 5mm and 10mm, between 5mm and 7mm, between 7mm and 10mm, between 10mm and 15mm, between 10mm and 12mm, between 13mm and 15mm, between 15mm and 20mm, between 15mm and 17mm, between 17mm and 20mm, between 20mm and 25mm, between 20mm and 22mm, between 22mm and 25mm, between 25mm and 30mm, between 25mm and 27mm, between 27mm and 30mm. Preferably the conduit has a length of between about 3 to about 30 mm, more preferably between about 3 to 15 mm. The minimum conduit length may be one of 2, 3, 4, 5, 6, 7, 8, 9 or 10mm. The maximum conduit length may be one of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25m 26m 27m 28m 29 or 30 mm.

Preferably the conduit has a diameter or greatest width of between about lOOμm to about 600μm. The minimum conduit diameter may be one of lOOμm, 200μm, 300μm, 400μm or 500μm. The maximum conduit diameter may be one of 200μm, 300μm, 400μm, 500μm, 600μm, 700μm, 800μm, 900μm, lmm, 2mm or 3mm.

The main length of the conduit may be designed to correspond to the distance between the first and second anatomical positions. For example, when forming a neural circuit conduit suitable for providing a neural connection between the striatum and substantia nigra in an adult human a conduit length of about 25-30mm is required. Conduit length may be chosen from one of lmm-5mm, 5mm-10mm, 10mm-15mm, 15mm-20mm, 20mm-25mm, 25mm-30mm, 30miti-35mm, 35mm-40mm, or greater.

Tissue or factors capable of stimulating or promoting neuron growth may include embryonic brain tissue fragments normally associated with growth of the required neurons. By orienting the fragments in the conduit directed growth of neurons towards the fragments can be achieved.

Tissue or factors capable of stimulating or promoting neuron growth may also include trophic factors to enhance cell differentiation and regeneration, such as glial cell line- derived neurotrophic factor (GDNF) , laminin or laminin derived peptides (YIGSR) or other factors known to enhance nerve regeneration. These may be incorporated into the support material using well established methods. The support material surface or bulk of its structure may also be modified to enable binding of desired molecules. Known methods of bioconjugation chemistry allow for chemical modification of the polymer which may allow for coupling of bioactive molecules/factors supporting cell development.

In preferred embodiments the conduit is tubular thereby facilitating the delivery of the conduit to a desired position in a tissue or other medium via displacement/ejection from the cannula of a syringe. The cross-section of the conduit may be circular, i.e. providing a cylindrical conduit, but may be any convenient cross-sectional shape, e.g. square, triangular or other polygonal shape. The tubular shape of the conduit may be provided by forming a tubular sheath or scaffold. Where the conduit comprises a scaffold matrix the conduit preferably does not have a lumen but is formed as a solid/semi-solid plug of material.

Preferably, the conduit has sufficient mechanical strength to remain intact during implantation and enable necessary manipulation of the conduit required for delivery to a tissue. Such manipulation may include insertion into the lumen of a cannula and/or displacement from the lumen of a cannula.

The polymeric material used to construct the conduit is preferably one or more of non-immunogenic, permeable or semipermeable to nutrients and growth factors, non-toxic.

The conduit may additionally contain therapeutic agents, e.g. drugs and/or drug releasing substances, which may be released into the nervous system after implantation.

In this specification reference to a cannula is to a tubular structure suitable for use in insertion to the human or animal body or to other tissue or matter. The cannula will preferably be cylindrical with a lumen (a central bore, cavity or channel) in which material to be inserted to the tissue or matter may be placed. The lumen may also receive a plunger slideable in the lumen which may act to assist in displacing material from the cannula into the tissue or matter. The cannula may form part of a needle or medical syringe. The cannula may be rigid or flexible and may be made from materials such as glass, plastics or metal, e.g. steel. The cannula may be sterilised before material is inserted or formed in its lumen. The cannula may form part of a needle.

Cannulas according to the present invention are described for use with a medical syringe. In this specification reference to a medical syringe is to an apparatus suitable for implantation of material and, at its simplest, has a cannula and plunger element receivable in the lumen of the cannula, and wherein the syringe is suitable for medical use, e.g. is capable of the necessary degree of sterilisation. A Hamilton syringe is one example of medical syringe design but medical syringes of a wide range of other designs may be provided in accordance with the above description.

For implantation, once the neural circuit conduit has been formed in the tip of the needle/cannula the conduit must be displaced from the cannula into the desired location. Using a solid bore plunger will exert pressure on the conduit such that it is pushed out. This may lead to damage of the conduit and is therefore less preferred, although remains possible. The use of a hollow plunger element is proposed. This allows for the conduit to be laid into position with less likelihood of damage to the conduit during implantation, e.g. between the neural centres that require re-connection.

A hollow plunger, e.g. glass capillary with hollow lumen, can be inserted into the needle lumen such that the wall of the hollow plunger rests on the wall of one end of the conduit (e.g. for a cylindrical conduit the wall of the plunger rests on the conduit circumferential wall at one end of the conduit) . The hollow plunger diameter is selected to closely- fit the needle internal diameter, which helps avoid application of pressure to the lumen or internal portion of the conduit. This helps minimise the pressure applied to the neurons contained in the conduit.

For surgical implantation, the needle is fitted to a medical syringe, e.g. Hamilton syringe, where the hollow plunger goes into the syringe and abuts against the syringe's own plunger element, which is maintained in a fixed position.

During displacement of the conduit, the needle and syringe is filled with culture medium which moves together with the hollow plunger at the same linear speed such that no additional pressure is applied to the conduit.

The present invention includes syringe apparatus having a cannula and a plunger. As described above, in some preferred embodiments the plunger is hollow, e.g. it is of tubular design configured to be slidable in the cannula but have a hollow lumen. The plunger may be suitably manufactured from glass or stainless steel. A hollow plunger is advantageous in that it minimises the pressure applied to the neural circuit conduit as the cannula is withdrawn over the plunger. The hollow plunger allows excess culture medium in or surrounding the conduit to flow into the lumen of the plunger and avoid a build up of pressure on the conduit that may be exerted by the plunger when acting on media trapped between the plunger and conduit. This could result in a conventional injection effect in which the cells are forced out of the cannula, rather than the desired displacement of the conduit into the cavity formed in the tissue by the inserted cannula. The capillary plunger preferably has thin walls so as to exert pressure only on the circumference of the conduit but not the neurons contained in the conduit. The plunger is made to closely fit the internal diameter of the cannula, in order to apply pressure only at the circumference of the conduit.

The conduit may preferably be formed in the lumen of a cannula by formation of the support material in the cannula and culturing of the neuron cells in the support material. Having formed the support material in the tip of the cannula the cannula with the support material inside is sterilized, e.g. using an alcohol solution. The lumen of the cannula can now be filled with a culture medium and cell suspension or tissue lump (fragment) aspired into the tip of the cannula and support material. The needle is then placed in a suitable vial containing culture medium and the cells are cultivated for a period of time to allow the cell to produce axons, e.g. 7 days .

Formation of the neural circuits continues until just before implantation surgery. At the moment of surgery the cannula is removed from the culture medium and the plunger, e.g. a glass capillary plunger, is carefully inserted so as not to damage the neural circuit. The plunger may be longer than the cannula such that it can extend into a Hamilton syringe such that the plunger forming part of the Hamilton syringe touches the glass plunger thereby mechanically connecting the two plunger components .

For implantation of other material (e.g. other cells), the material may be placed into the lumen of the cannula, or sucked into the lumen, by conventional techniques.

In some embodiments the plunger element may be partially formed by a liquid, fluid or gel (e.g. mineral oil, culture medium, or a sterilised medical solution, e.g. saline) placed in the lumen of the cannula and configured to abut the material to be implanted and capable of transmitting mechanical force from a second component of the plunger element .

The details of one or more embodiments of the invention are set forth in the accompanying description below including specific details of the best mode contemplated by the inventors for carrying out the invention, by way of example. It will be apparent to one skilled in the art that the present invention may be practiced without limitation to these specific details.

Three techniques have been developed: (a) the development of biocompatible and degradable scaffolds and sheaths which guide and support the neural circuit's growth (in vitro) and delivery (in vivo); (b) the generation of functional brain circuits in vitro; and (c) the establishment of a method and apparatus to implant the functional circuit into precise locations in the brain. Methodologies for neural circuit formation, scaffold and sheath fabrication and establishment of a surgical method and apparatus for easy and precise delivery of the neural circuit conduit into the desired position in the brain are described.

(a) Fabrication of tubular sheaths and scaffold

Selection of polymers for sheath or scaffold construction is based on several criteria including: biocompatibility, biodegradability, easy formation of desired structure, porosity, mechanical properties, e.g. tubular/capillary or gel shape or state.

Suitable biodegradable polymers include alginate, agarose, collagen, chitosan, polycaprolactone, polyglycolide, polylactide, poly (DL-lactide-co-caprolactone) , poly (L-lactide~ co-caprolactone-co-glycolide) , polyhydroxyalcanoates and their co-polymers as well as combinations of hyaluronic acid, chitosan and collagen. Non-degradable materials include cellulose acetate, cellulose butyrate, alginate, polysulfone, polyurethane, polyacrylonitrile, sulfonated polysulfone, polyamide, polyacrylonitrile, polymethylmethacrylate and their co-polymers To enhance nerve growth during culture and regeneration following implantation, trophic factors, such as glial cell line-derived neurotrophic factor (GDNF) , laminin or laminin derived peptides (YIGSR) may be incorporated into the sheath or scaffold using well established methods. The sheath or scaffold surface or bulk of its structure may be modified suitably to enable binding of the desired molecules. Known methods of bioconjugation chemistry allow for modification of polymer with chemical functional groups, which in the subsequent reactions are used for coupling of bioactive molecules/factors .

Phase inversion, extrusion and electro-spinning (Bini, T. B.; Gao, S.; Tan, T. C; Wang, S.; Lim, A.; Hai, L. B. & Ramakrishna, S. Electrospun poly (L-lactide-co-glycolide) biodegradable polymer nanofibre tubes for peripheral nerve regeneration Nanotechnology, 2004, 1459; M. Mulder, Basic Principles of Membrane Technology, Kluwer Academic Publishers, Dordrecht (1996) ISBN 0-7923-4247-X; Zhang, N.; Zhang, C. & Wen, X. Fabrication of semipermeable hollow fiber membranes with highly aligned texture for nerve guidance. J Biomed Mater Res A ; 2005, 75, 941-949) are exemplary techniques available for fabrication of the sheath or scaffold.

Two approaches for fabrication of tubular sheaths or scaffolds acting as the support for the neural circuit growth in vitro and the carrier when they are delivered to brain are described.

(i ) In situ conduit formation

The first approach involves formation of the sheath or scaffold in situ in the lumen of a needle or cannula that will be used for transplantation of the neural circuit. The internal wall of the cannula defining the lumen provides a mold for formation of the sheath. Following sheath formation, cells or tissue capable of neuron growth are aspired into the sheath and the whole cannula/needle with the sheath and cells are incubated for a time required for neuron growth and consequent generation of the neural circuit conduit. The cannula can then be directly used for surgical transplantation of the conduit.

Film formation, phase inversion and gelation techniques can be used to generate the tubular sheath or scaffold in situ in the lumen of the needle/cannula. This approach reduces the number of operations required to construct a conduit suitable for insertion into the brain, and in particular, reduces the risk of conduit damage that is associated with placing the conduit into a needle/cannula for transplantation.

(H) Construction of conduit followed by insertion into cannula

The second approach is to fabricate the conduit separately from the delivery needle/cannula and subsequently insert the conduit into the tip of the needle/cannula.

The neural circuit is grown in the sheath or scaffold for a culture period sufficient for neuron growth to occur. Conduits with matured neural circuits are then inserted into the needle/cannula tip before surgery.

Film formation

A tubular sheath can be produced by a film formation technique in which a required diameter tube is used as the mold and a polymer solution is dip coated on the tube. Once the solvent is evaporated, a coherent tubular film is formed along the tube. The thickness of the film can be tailored as desired.

For example, a polymer solution is aspired up to the desired length/distance into a needle or cannula and then displaced leaving a thin film of the polymer solution on the cannula walls, which is then solidified by drying or phase inversion induced by a non- solvent.

The tubular film can be detached from the wall of the mould tube, allowing the delivery of the tube to the brain. Non- porous sheaths can be readily formed by this technique.

Phase inversion

Porous scaffolds may be made by phase inversion from cellulose esters, e.g. cellulose acetate, cellulose butyrate, chitosan, polysulfone, polyurethane, polyacrylonitrile, sulfonated polysulfone, polyamide, polyacrylonitrile, polymethylmethacrylate .

Phase inversion is a technique for production of solid polymers and is a well known technique for production of hollow fiber dialysis or plasma separation membranes. It allows for manufacturing of porous, flexible membranes, which may be modified with required biologically active factors.

For example, a cellulose acetate tubular sheath was prepared by the following phase inversion method from 10% cellulose acetate (Sigma) solution in acetone/methanol/glycerol mixture 3:1:1.

The sheath was formed within a glass cannula (later used for implantation of the neural circuit conduit) . The cannula had a 0.5mm external diameter and 0.1mm wall thickness. A 4 mm long sheath was formed in the tip of the cannula. A syringe was used to pull the polymer solution into the bore of the cannula up to 4 mm from the tip. The polymer solution was then pushed out of the cannula leaving a thin polymer solution film on the cannula lumen wall. The formed film was coagulated with water. The cannula and porous sheath membrane was stored in a wet state. Prior to culture of neurons in the sheath the cannula was sterilized in alcohol.

Gel formation

Gel formation techniques may be used for scaffold formation using suitable natural or synthetic polymers.

Embedding of tissue fragments/cells in a hydrogel for neuron growth during culture is a good alternative to the use of a more rigid tubular sheath. Biodegradable hydrogels are attractive scaffold materials. Following implantation of the neural circuit conduit in the brain the scaffold degrades to leave the neural circuit behind.

(b) In vitro construction of neural circuits

Primary/embryonic neural tissue or dissociated primary cells were used to pre-construct a nigro-striatal neural circuit which commonly degenerates in the adult human brain (e.g. in Parkinson's disease) .

Tissue capable of neuron growth:

Time-mated Sprague-Dawley rats (day of plugging designated as embryonic day 0) were given a terminal dose of Euthatol (Merial, UK) via an intraperitoneal (i.p.) injection at embryonic day (E) 12 and embryos (approximately 5mm crown-rump length) removed.

The brains were extracted from the embryos and placed in cold Hank's balanced salt solution (HBSS) . The entire mesencephalic-diencephalic tube (from the caudal notch at the mes-metencephalon boundary, to the rostral border of the immature diencephalon) was dissected and a longitudinal incision made along the dorsal midline (Figure 1) . Given that previous work illustrated that only a portion of this piece of embryonic brain contained cells that would eventually form the nigro-striatal circuit and cells that stimulate the growth of nigro-striatal neurons (Gates et al . , European Journal of Neuroscience, Vol.19, pp831-844, 2004), the explants were further dissected so that cells which would generate the nigro-striatal circuit (the dopaminergic cells of the substantia nigra) were dissected along with cells which have been shown to promote their growth (the medial forebrain bundle, MFB, cells) .

The tissue was bathed in a culturing solution consisting of 85% Neurobasal media (Gibco, UK) , 10% fetal calf serum (Gibco, UK), 4% 112 solution (consisting of 25% glucose - Sigma, IOOOU Pen-Strep - Sigma, 4OmM glutamine - Gibco, UK) , 1% B27 (Gibco, UK) , and 1% 5000U/ml Pen-Strep (Sigma) while transferring to glass capillaries containing conduit constructs.

Dissociated cells capable of neuron growth:

Cells from the E12 embryonic mesencephalon (i.e., natural stem cells which become nigro-striatal projection neurons) and MFB were dissected (as above) , separated, and incubated for 25 minutes at 37°C in an HBSS solution containing 0.1% trypsin, 0.05% DNase dissociation medium.

After dissociation via pipetting through lOOμl Gilson pipette tips, the cells were pelleted via centrifugation at 500rpm (on a table top centrifuge) for 5 minutes. The cell pellet was resuspended in cell culture media (as above for pieces of tissue) to reach a concentration of approximately 100,000 cells per microliter.

Placement of intact tissue / dissociated cells into sheath or scaffold Ti s sue :

To maintain directed growth, tissue is extracted so that neural cells which will form the nigro-striatal circuit (the ventral mesencephalon - VM dopamine neurons) are at one end of the piece of tissue, and the tissue which stimulates the growth of nigro-striatal neurons at the opposite end of the tube. To do this, the capillary / scaffold construct is placed on the end of a standard Hamilton syringe. The dissected piece of VM-MFB tissue is laid onto a metal spatula with the orientation of the VM and MFB regions noted. While drawing on the syringe plunger, the end of the capillary / scaffold construct is placed onto the edge of the tissue so that the growth promoting tissue (i.e., the MFB tissue) is aspirated into the capillary/scaffold construct first, followed by the portion of the neural tissue which forms the neural circuit (i.e., the VM tissue) . The tissue, scaffold and capillary are incubated all-in-one in vitro (either in media filled large Petri dishes, or in media filled 15-50 ml centrifuge tubes) for a period which allows for suitable growth of the neural wire (e.g., 1 week in the case of the nigro-striatal circuit from laboratory rats - see Figure 2) . Subsequently the grown neural circuit conduit is ready formed within the sheath or scaffold and the delivery cannula as an implantable conduit for implantation into CNS tissues.

Dissociated Cells

Dissociated cells suspended in culture medium or suitable gel are loaded into the capillary/conduit via syringe action. Prior to loading of the cells the whole cannula/needle and the sheath are filled with the culture medium or other medium (e.g. mineral oil) . Dissociated cells suspension is spotted onto a sterile Petri dish lid laid on a black backdrop (for visualisation) . Cells from the VM (or first from the MFB then VM) , are positioned in the scaffold via dipping one end of the unconnected capillary/conduit into the cell droplet and pulling it precisely into the sheath. VM cells can either be loaded alone, or in sequence with the MFB (i.e., the growth promoting MFB cells being loaded first, and the circuit producing cells second) . This helps promote directional growth of cells within the conduit (see Figure 3) .

Alternatively, dissociated cells are loaded into the capillary/conduit construct via passive capillary action. Dissociated cells are spotted onto a sterile Petri dish lid laid on a black backdrop (for visualisation) . Cells from the VM (or first from the MFB then VM), are positioned in the scaffold via dipping one end of the unconnected capillary/conduit construct into the cell droplet. Diffusion of cells into the construct can be controlled by the amount of time the construct is allowed to remain in the droplet (usually only a few seconds) . VM cells can either be loaded alone, or in sequence with the MFB (i.e., the growth promoting MFB cells being loaded first, and the circuit producing cells second) . This helps promote directional growth of cells within the conduit (see Figure 3) .

Formation of neural circuit conduit in the delivery cannula

In order to simplify the implantation procedure each phase of neural circuit conduit formation can be performed in the lumen of the cannula/needle that is to be used for the implantation. Thus, following the formation of the sheath or scaffold in the tip of the cannula lumen, cells or embryonic tissue fragments are inserted into the sheath or scaffold by suction and then cultivated for the necessary period of time.

Once the neural circuit conduit has been formed, the combined cannula and conduit can be stored for future use. The combined cannula and conduit thereby provide an independent product suitable for later implantation. Storage may be in sterile conditions at low temperature, e.g. about 4⁰C. Immediately before implantation a capillary plunger is inserted into the cannula and the cannula is fitted into the Hamilton syringe fitted to the implantation accessory (Figure 6) . A capillary plunger was used in order to facilitate insertion into the cannula without accidentally moving/displacing the cells from the sheath or scaffold. During insertion of the plunger the cannula tip is held against the base of a Petri dish and the capillary plunger inserted gently down toward the conduit.

Assembly of tubular neural circuit conduits into the delivery needle

Where the neural circuit conduit is not formed in the tip of the delivery cannula the conduit must be inserted into the lumen of the cannula to allow for implantation.

Referring to Figure 5, this can be achieved by providing a solid support on which a groove is formed, the groove having an approximately semi-circular cross section configured to mate with the exterior shape of the delivery cannula and being at least partially linear so as to receive and support one end of the linear cannula along at least part of its length. The tubular conduit is placed toward one end of the groove with the needle placed towards the opposing end. The conduit is then gently moved along the groove, using biological buffer as a lubricant if required, towards the tip of the cannula and into the lumen of the cannula. The groove is configured such that it forms a continuous surface with the cannula internal wall. A range of supports each having a different size of groove can be provided to accommodate different diameter needles .

(c) Implantation apparatus and method Implantation of the neural circuit conduit into the brain or other medium can be accomplished using a stereotaxic frame, which allows for precise orientation of the cannula and its positioning within the brain. Stereotaxic frames are used in neurological research and surgery for directing the tip of a cannula or needle to a desired location in the neurological tissue. They are commonly used in brain surgery. The frame firmly holds the head of the subject and provides for accurate three dimensional movement and positioning of a needle to be inserted in the brain.

Implantation of cells is normally achieved by the initial sucking of a required volume of a cell suspension into the glass capillary, filled with mineral oil, and attached to a Hamilton syringe. Following the insertion of the needle into the desired position in the brain, the cell suspension is pushed out by the mineral oil serving as a convenient plunger.

In contrast, implantation of neural circuit conduits, which after implantation are intended to span between and link desired points in the brain, requires precision implantation apparatus. Proper implantation does not involve simple injection of the conduit as might be required for delivery of a liquid cell suspension. As the conduit is intended to bridge two locations in the tissue it is required to be laid out by implantation between those locations. An accessory for a stereotaxic frame was developed which allows for controlled precise implantation of the scaffold in the desired position.

The apparatus allows a needle to be inserted into a desired position and then retracted in a manner which leaves the scaffold in place in the cavity formed by the delivery needle (i.e., laid into the tissue and not injected) . This allows precision implantation of the conduit between anatomical locations without damaging or compressing the conduit, especially when it is made from soft hydrogel. Referring to Figure 6, the accessory comprises a translation stage (601) on which a medical syringe (602), e.g. a Hamilton syringe, is mounted. The translation stage has a micrometric screw (603) controlling movement of the stage towards or away from the implantation tissue (604) . The micrometric screw typically allows control of the needle position to within 0.1mm. The medical syringe, e.g. Hamilton syringe, has a plunger element (606) . A needle/cannula is provided for implantation of the conduit and comprises a distinct hollow plunger element configured to co-operate with the medical syringe plunger element (606) to allow implantation of the neural circuit conduit. Thus a two-part plunger arrangement is provided, having a first plunger element formed in the medical syringe and a second hollow plunger arrangement in the lumen of the needle/cannula. The two plunger elements being mechanically connected. A retaining member (605) limits movement of the plunger (606) disposed in the lumen of the delivery needle/cannula by abutting one end of the plunger, or its controlling part. The retaining member limits the extent of plunger withdrawal from the lumen and enables the position of the plunger (606) to be maintained constant during implantation. If required the retaining member can fix the plunger position, i.e. preventing any movement of the plunger. Movement of the retaining member is independent of the translation stage. The retaining member can be fixed to the stereotaxic frame. Having moved the translation stage such that the needle is in the required position for implantation the retaining member can be locked in position preventing movement of the plunger (606), whilst the translation stage can be moved relative to the retaining member to withdraw the needle. Such withdrawal results in withdrawal of the cannula of the needle over the plunger which is held in constant position in the lumen of the cannula. A neural circuit conduit positioned at the tip of the needle between the tip and plunger is thereby laid into position in the tissue and into the cavity formed by the cannula.

The translation stage is fixed to a stereotaxic frame and its general position is regulated by the settings of the stereotaxic frame which are adjusted to suit the subject or tissue. The translation stage enables precise movement of the needle fitted to the 10 μl Hamilton syringe, by the means of the micrometric screw. The micrometric screw moves the translation stage, to which the Hamilton syringe is clamped, thereby enabling movement of the needle into or out of the tissue .

As described above, the syringe plunger can be extended using a glass capillary having an external diameter marginally smaller than the internal diameter of the needle and long enough to reach to the tip of the needle when pushed by the Hamilton syringe plunger.

Referring to Figure 7, having inserted the cannula to the desired location, a neural circuit conduit (701) mounted in the tip of the cannula (702) between the open end of the cannula and the hollow plunger (703) can be implanted by withdrawing the cannula of the needle over the stationary hollow plunger. Turning the micrometric screw moves the translation stage, and attached needle in the upward direction whilst the retaining member prevents movement of the plunger elements in the upward direction such that the plunger elements are held stationary. The cannula is thus drawn over the conduit and hollow plunger leaving the conduit in position in the tissue. After inserting into the desired position within the brain, the syringe is retracted and thus the scaffold is precisely implanted into the cavity formed by the needle . The conduit can thereby be accurately positioned to extend between two points "A" and "B" in the tissue. By inserting the needle such that its length passes through point B and the tip of the needle is at point A, withdrawal of the cannula over the plunger deposits the elongate conduit between the two points . A neural circuit conduit can be constructed to have a length corresponding to the distance between two anatomical points and thereby provide a neuron circuit that is an effective bridge between the two points.

The brain phantom system for circuit implantation

Surgical implantation of neural circuit conduits into brain tissue is a complex procedure. Practitioners require the opportunity to practice without wasting valuable brain tissue. An in vitro system (a "phantom model") that is not reliant on animal tissue was developed to allow practitioners to practice and research the procedure.

A phantom model of brain tissue, especially when it is made of transparent material e.g. gel, greatly facilitates the development of many techniques that are required for surgical implantation and allows continuous observation of the surgical procedure. This not only allows one to eliminate animal experiments for developing a surgical technique but can give feedback on the friction forces involved in the implantation procedure .

A phantom was made from gelatine, which offers a very simple transparent gel preparation procedure and easy modification of its mechanical properties by the simple variation of the concentrations of gelatine solutions. In order to establish the required mechanical properties of the phantom gel, initial measurements of the friction of the needle inserted into rat brain were made using an ElectroForce® 3200 Series Test Instrument (Figure 9) . Three gelatine concentrations 1%, 2% and 3%, were chosen.

First, to determine the friction profile of brain tissue a freshly isolated adult rat brain was placed in a conical bottom vessel enabling one to set the brain at the required angle. The brain was positioned at the desired angle and the needle was inserted vertically along the desired path linking the striatum and substantia nigra (Figure 10) . A 5 mm diameter needle was forced into the half-brain along the insertion path intended for conduit implantation: between the striatum and substantia nigra. The friction profile is shown in Figure 11. The speed of the needle movement was set for 0.25mm/s and penetration depth up to 8 mm.

In general, measurements made using the phantom gels show friction profiles characteristic of homogenous structures, where a steady increase in friction is observed until the gel top layer is punctured. The following relaxation curve indicates the friction resistance of the needle wall during penetration of the gel . The measured load during insertion into a 2% gelatine gel was found to be in the range of the loads obtained for the rat's brain (Figure 12) . A 3% gelatine gel created much higher measured loads, although the needle friction after puncture of the gel top layer appeared to be within the range of the friction resistances observed in real brain tissue, in particular the lower part of the relaxation curve (Figure 13) .

A 1% gelatine gel appeared to be too soft to produce measurable results. Both 2% and 3% gelatine gels appear to be suitable as brain tissue phantoms for modelling and the development of the scaffold implantation into the brain.

Considering that the brain tissue phantom is designed for analyzing and modelling insertion of the scaffold over the whole delivery path the 3% gelatine gel was chosen because it provides a good representation of the friction measured during needle insertion into real brain tissue.

Surgical Implantation Apparatus and Method of Implantation

In accordance with the above, the inventors have provided a surgical implantation method useful for surgery on live humans and animals as well as on non-living tissue, e.g. in practising surgical techniques.

The method involves the implantation of material into biological tissue or other matter by laying out the material between first and second positions. This allows a precise implantation of the material and the method causes a minimum of damage to the biological tissue or matter into which the material is being implanted.

The method and apparatus is suitable for use in implanting the neural circuit conduits described herein, and for use with a cannula in which such a conduit is formed. However, the method is also useful for implantation of other material, e.g cells (including non-neuronal cells such as somatic cells or stem cells (e.g. adult, embryonic, induced pluripotent cells) which may be human, mammalian or non-human) , other tissue or other organised material, or surgically implanted medicaments (e.g. bolus implants) and other non-organised material.

The method involves the insertion of a single cannula into the biological tissue or other matter. The cannula will normally form part of a medical syringe having a tip or stylet used to puncture the tissue or matter. Prior to insertion the cannula preferably has a quantity of the material to be implanted positioned in the lumen adjacent the opening of the cannula that is inserted into the biological tissue or other matter. A plunger element is also located in the lumen and is positioned adjacent a part of the tissue or matter distal to the opening. The plunger element is mechanically connected to a plunger system of the medical syringe. The plunger element may comprise several components arranged to transmit a force applied by the surgeon to the plunger actuator on the medical syringe to the material contained in the lumen of the cannula.

By providing a single cannula in which the plunger element and material to be implanted are pre-positioned, and are maintained in that position during insertion, insertion of the cannula has minimal impact on the tissue or matter. This is because none of the tissue or matter in which the material is being implanted can be sucked into the lumen.

Once the cannula is in position the material is laid out into the tissue or matter by withdrawing the cannula over the plunger element whilst maintaining the plunger element in a substantially fixed position. The material can thereby be placed between a first position at, or adjacent, the opening of the lumen and a second position through which the lumen of the cannula passes. The distance between the first and second positions is determined by the length of the quantity of material. This length is preferably about the same as the distance from the opening of the cannula to the terminal part of the plunger element adjacent the material.

Referring to Figure 14A, a medical syringe is illustrated having a cannula 1402 with a quantity of material (e.g. a neural circuit conduit) 1401 positioned between the opening of the lumen (at the insertion tip of the syringe) and one end of a first plunger element 1403. The first plunger element is in mechanical communication with a second plunger element 1404. The position of the plunger elements is controlled by an actuator 1405 abutting a plunger control element 1406. In Figure 14B the cannula has been withdrawn over the first plunger element by a distance corresponding to arrows 1407 and 1408. The position of the neural circuit conduit has not changed, but it is no longer in the lumen of the cannula. This action allows for the passive implantation of the neural circuit conduit at the implantation site.

Referring to Figure 15, a stereotactic apparatus is shown attached to a surgical implantation attachment according to the present invention. The surgical implantation attachment is shown in Figure 16 having an adapter allowing releasable attachment to the frame 1601 of the stereotactic apparatus. The medical syringe is mounted on a syringe support which in turn is mounted on a translation stage permitting movement of the cannula 1402 towards and away from the plunger control element 1402 and enabling the cannula to slide over the plunger element whilst maintaining the position of the plunger element (s) .

Figure 17 is a flow diagram illustrating the steps of neural conduit formation at one end of a cannula (A-D) , attachment of the cannula to a medical syringe (E-F) , attachment of the medical syringe to a stereotactic apparatus and implantation of the conduit into brain tissue (G) .

A medical grade cannula is optionally sterilised prior to formation of the polymer sheath/scaffold in the lumen at one end of the cannula. The scaffold/sheath is then formed in the tip of the cannula, e.g. using a phase inversion method, and is then sterilised (A-B) . The lumen of the cannula is filled with culture media, the sheath/scaffold is seeded with neurons and the cannula is cultured for several days (C-D) . Following growth of neurons for a desired period of time, or to a desired length, a plunger element is inserted in the cannula and the cannula is assembled as part of a medical syringe (E- F) . The syringe is then attached to a stereotactic apparatus using the surgical implantation attachment described above. The plunger element in the lumen of the cannula is positioned to abut the scaffold/sheath and to mechanically communicate with the syringe plunger. The implantation apparatus is mounted on an arm of a stereotaxic frame and used in a method of implantation, as described above (G) .

Examples

Example 1 - Preparation of cellulose acetate sheaths.

A polymer solution was prepared by dissolving 10 g of cellulose acetate (Eastman CA-398-10 USP grade) in a mixture 87g of acetone and methanol in ratio 3:1 respectively. The polymer solution was sucked into the tip of a glass needle/cannula to the length of about 5 mm, using a syringe. Casting solution was displaced from the needle and the formed polymer film was coagulated by immersing the needle in water. The polymer tube was washed extensively and the needle with the tube in its tip is stored in a wet state in a water or alcohol solution. In case of the need for prolonged storage the tube may be immersed in 10-25% glycerol solution for 2-12h and then dried.

Example 2 - Preparation of polysulfone sheaths.

15 g polysulfone (Udel polysulfone P3500 Natural 11, AMOCO, molecular weight 45000) was dissolved in 80 g of a mixture of dimethylacetamide and polyvinylpyrrolidone in the ratio 6:2 and processed as in Example 1.

Example 3 - Preparation of chitosan sheaths

Chitosan solution was prepared at a concentration of 2% in a solution of 2% acetic acid in water. Sheaths were prepared by forming the chitosan films as in Example 1, by coagulation in a 3% NaOH water solution. Formed sheaths may be additionally- stabilized by crosslinking with 0.4% glutaraldehyde containing 0.05 M H₂SO₄ for 15 min. Where prolonged storage is required the tube and sheath may be immersed in 10-25% glycerol solution for 2-12h and then dried.

Example 4 - Preparation of capillary membranes (hollow fibers) by phase inversion.

A polymer solution was prepared as in Example 1 or 2. Referring to Figure 4, the spinning solution was pumped into a ring shaped orifice of a spinneret (internal diameter of the ring: 0.15 - 0.3 mm, external diameter of the ring 0.20 - 0.5 mm) . The spinneret was placed 2 cm above a coagulating bath (water) . The core liquid, water, is pumped through the tube positioned in the centre of the orifice. The nascent hollow fibre moves through the coagulating bath from the outlet of the coagulating bath. The hollow fibre is guided to the washing bath and is wound on a suitable wheel.

Example 5 - Preparation of polycaprolactone sheaths

Polycaprolactone (SIGMA, 440744) sheaths were synthesised from a 20% solution in chloroform. The polymer film was formed in the needle as in Example 1, but solidified by drying in ambient temperature for 12 h and finally under vacuum for 2h.

Example 6 Preparation of polyurethane sheath

Polyurethane (Tecoflex SG60D) tubing with internal diameter 0.45 mm and wall thickness 0.05 mm was prepared by extrusion. The 3 to 8 mm long tubing is cut in order to conduct culture of cells within the lumen of the tube.

Once the cell culture phase is complete, the constructs are assembled into the delivery needle using the assembly apparatus shown in Figure 5. Brain implantation occurs according to the techniques described herein.

Example 7 - Preparation of collagen gel scaffolds and cultivation of neural cells

Gels were prepared by mixing collagen (0.5%), Tris-HCl (pH D8.0, 50 mM) , CaCl₂ (2.5 iriM) , DL-dithiothreitol (1 itiM) , and TG in ratio 1:5000 to collagen at 4⁰C in a 15 ml centrifuge tube. Equal volume of cells suspension IxIO⁶ cells/ml in DMEM containing penicillin (50 Iϋ/itiL) , streptomycin (50 g/rtiL) was added. Gels were aspired to the needle and cultivated for 7 days .

Example 8 - Implantation of neural circuit conduit in rat brain

A 4 mm long semipermeable cellulose acetate neural circuit conduit was formed in the tip of a glass needle/cannula. The needle was mounted on a modified stereotactic frame and the conduit implanted into both live rat brain and fixed rat brain. The implantation region was selected to extend between two regions of the brain we were attempting to re-connect, the substantia nigra and striatum. These regions can be accessed by following coordinates derived from an atlas of the rodent brain .

Sectioning of the brain after implantation of the conduit showed the conduit to be properly inserted at the desired location. Tissue cross-sections clearly show the implanted conduit and neuron tissue contained in the conduit (Figure 8) .

Claims

Claims :

1. A method of manufacturing an implantable neural circuit conduit, comprising the steps of:

2. The method of claim 1 wherein the sheath in (a) is formed in the lumen of a cannula.

3. The method of claim 1 or 2 wherein the tissue or cells in (b) are capable of forming CNS neurons.

4. The method of any one of claims 1 to 3 wherein step (a) further comprises providing a polymer scaffold in the lumen of the sheath, the scaffold providing a support matrix for neuron growth.

5. The method of claim 4 wherein the scaffold is a gel or hydrogel .

6. The method of any one of claims 1 to 5 wherein the sheath formed in (a) is tubular.

7. The method of any one of claims 1 to 6 wherein the sheath has a main length between a first end and a second end, and tissue in (b) is positioned at or adjacent the first end, with neuron growth in (c) occurring towards the second end.

8. The method of any one of claims 1 to 7 wherein prior to step (c) the method further comprises positioning tissue or factors capable of promoting the growth of neurons in the lumen of the sheath or in contact with the sheath.

9. The method of claim 8 wherein the sheath has a main length between a first end and a second end, and tissue in (b) is positioned at or adjacent the first end, with neuron growth in (c) occurring towards the second end and said tissue or factors are positioned closer to the second end, relative to the positioning of the tissue or cells in (b) .

10. The method of any one of claims 1 to 9 wherein the sheath is formed as a thin film polymer tube in the lumen of a cannula by a method comprising dipping the cannula in a polymer solution and evaporating solvent.

11. The method of any one of claims 1 to 9 wherein the sheath is formed as a thin film polymer tube in the lumen of a cannula by a phase inversion method.

12. A method of manufacturing an implantable neural circuit conduit, comprising the steps of:

(b) contacting the scaffold with tissue or cells capable of neuron formation;

13. The method of claim 12 wherein the scaffold in (a) is formed in the lumen of a cannula.

14. The method of claim 12 or 13 wherein the tissue or cells in (b) are capable of forming CNS neurons.

15. The method of any one of claims 12 to 14 wherein the scaffold is a gel or hydrogel.

16. The method of any one of claims 12 to 15 wherein the scaffold formed in (a) is tubular in shape, the scaffold forming a cylindrical conduit body.

17. The method of any one of claims 12 to 16 wherein the scaffold has a main length between a first end and a second end, and tissue in (b) is positioned at or adjacent the first end, with neuron growth in (c) occurring towards the second end.

18. The method of any one of claims 12 to 17 wherein prior to step (c) the method further comprises contacting tissue or factors capable of promoting the growth of neurons with the scaffold.

19. The method of claim 18 wherein the scaffold has a main length between a first end and a second end, and tissue in (b) is contacted at or adjacent the first end, with neuron growth in (c) occurring towards the second end and said tissue or factors are contacted with the scaffold closer to the second end, relative to the positioning of the tissue or cells in

(b) .

20. A neural circuit conduit obtained by the method of any one of claims 1 to 19.

21. A cannula having a neural circuit conduit disposed in the lumen of the cannula, the neural circuit conduit obtained by the method of any one of claims 1 to 19.

22. A cannula having a neural circuit conduit disposed in the lumen of the cannula, the cannula obtained by the method of claim 2 or 13.

23. A syringe having a neural circuit conduit or cannula according to any one of claims 20 to 22.

24. A sterotactic syringe apparatus comprising a neural circuit conduit, cannula or syringe according to any one of claims 20 to 23.

25. A method of manufacturing a cannula having a neural circuit conduit disposed in the lumen of the cannula, the method comprising forming a neural circuit conduit according to the method of any one of claims 1 to 19, wherein the neural circuit conduit is formed in the lumen of a cannula.

26. A method of manufacturing a medical syringe having a cannula and a neural circuit conduit disposed in the lumen of the cannula, the method comprising forming a neural circuit conduit according to the method of any one of claims 1 to 19, wherein the neural circuit conduit is formed in the lumen of a cannula, and using the cannula in the assembly of a medical syringe .

27. A method of stereotactically implanting material into biological tissue or a selected medium, the method comprising:

(a) providing a medical syringe mounted on a stereotactic apparatus, the medical syringe having:

(iii) a quantity of material to be implanted disposed in the lumen,

28. The method of claim 27 wherein the stereotactic apparatus comprises a surgical implantation attachment having a frame comprising:

29. The method of claim 27 or 28 wherein the plunger element comprises a first component comprising a quantity of liquid, fluid or gel positioned in the lumen of the cannula adjacent the material to be implanted and in contact with a second component, wherein force applied to the second component is transmitted to the first component, the first and second components together forming a plunger element capable of transmitting said force against the material to be implanted.

30. The method of any one of claims 27 to 29 wherein the material is a neural circuit conduit and the method is a method of implanting the neural circuit conduit at a defined position in the tissue or medium to provide a neuron connection between first and second positions in the tissue or medium, in which the cannula is inserted such that the tip of the cannula is positioned at the first position and the lumen of the cannula passes through the second position, such that during (c) the neural circuit conduit is deposited in the tissue or selected medium so as to bridge the first and second positions .

31. The method of any one of claims 27 to 30 wherein the method is an in vitro method.

32. The method of any one of claims 27 to 30 wherein the material is a neural circuit conduit and wherein the method is for treating a neurological disorder comprising implanting the neural circuit conduit at a defined position in nervous system tissue of a human or animal so as to provide a neural connection between first and second positions in the nervous system tissue.

33. The method of claim 32 wherein the method comprises implanting the neural circuit conduit in the brain of the human or animal .

34. The method of claim 33 wherein the method is a method of treating a neurological disorder chosen from the group: Parkinson's disease, Huntingdon's disease, Alzheimer's disease .

35. The method of any one of claims 32 to 34 wherein the neural circuit conduit is a solid or semi-solid implantable neural circuit conduit having an elongate polymer sheath and one or a plurality of neurons in the lumen of the sheath, wherein the conduit is permeable to molecules required for neuron growth, or a solid or semi-solid implantable neural circuit conduit comprising an elongate polymer scaffold and one or a plurality of neurons embedded in the scaffold, wherein the scaffold is permeable to molecules required for neuron growth.

36. The method of claim 35 wherein the neurons are central nervous system neurons.

37. A surgical implantation attachment for a stereotactic apparatus, the attachment having a frame comprising:

(a) an adapter for attaching the attachment to a stereotactic apparatus;

(c) a control element configured to maintain a selected position of the plunger element in the lumen of the cannula, wherein the syringe support is mounted on a translation stage which is moveable to slide the cannula over the plunger element whilst the control element maintains the plunger element in the selected position.

38. The surgical implantation attachment of claim 37 wherein a quantity of material to be implanted is positioned in the lumen between said opening and said plunger element, such that an end of the plunger element is adjacent part of said material .

39. The surgical implantation attachment of claim 37 or 38 wherein the plunger element comprises a first component comprising a quantity of liquid, fluid or gel positioned in the lumen of the cannula adjacent the material to be implanted and in contact with a second component, wherein force applied to the second component is transmitted to the first component, the first and second components together forming a plunger element capable of transmitting said force against the material to be implanted.

40. The surgical implantation attachment of any one of claims 37 to 39 wherein said material is non-liquid.

41. The surgical implantation attachment of any one of claims 37 to 40 wherein said material is a neural circuit conduit.

42. The surgical implantation attachment of any one of claims 37 to 41 wherein said material comprises biological tissue.

43. The surgical implantation attachment of any one of claims 37 to 42 wherein the plunger control element comprises a retaining member configured to abut the plunger.