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    • 专利摘要:
    Modular device for nerve regeneration and manufacturing procedure. Modular device for nerve regeneration that, based on a hybrid structure based on materials of natural and/or synthetic origin and a glial support cell source (12), formed by one or more elementary modules (2) cylindrical of variable length, with one or more parallel flat or tubular bundles (4) of microfilaments (5) located in an inner lumen (3) of the cylindrical elementary modules (2), and fixed at the ends to fixation and suture structures (6). In the lumen (3), in addition to the supporting glial cells (12), axons can grow from end to end, in great lengths (tens of centimeters). The device can be applied in the reestablishment of neural connections in the central and peripheral nervous system, or other applications where a neural communication element of great length is necessary. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2818424A1
    申请号:ES202130065
    申请日:2021-01-27
    公开日:2021-04-12
    发明作者:Pradas Manuel Monleon；Ramos Cristina Martinez；Doblado Laura Rodriguez；Roca Fernando Gisbert
    申请人:Universidad Politecnica de Valencia；
    IPC主号:
    专利说明:

    [0001] MODULAR DEVICE FOR NERVOUS REGENERATION AND PROCEDURE
    [0003] OBJECT OF THE INVENTION
    [0005] The object of the invention is a modular device for nerve regeneration, based on the union of elementary modules forming modular neural cables made up of glial cells or glial cells and neurons inside cylindrical structures of natural and synthetic origin, for use in regeneration of neural tracts of the central nervous system (CNS) or peripheral nervous system (PNS).
    [0007] BACKGROUND OF THE INVENTION
    [0009] The loss of communication between the central nervous system (CNS) and the peripheral nervous system (PNS) is the cause of many disorders that compromise the quality of life of those affected. This loss of communication can be due to trauma, accidents, degenerative diseases, or other causes, such as birth problems. It can affect brain structures, such as the nigrostriatal tract in the case of Parkinson's disease, spinal cord, in the case of spinal cord injury, or nerves.
    [0011] There is currently no effective clinical therapy in the PNS for nerve injuries involving nerve lengths greater than 2 cm. In minor injuries, a nerve graft is used, usually from the patient himself (autograft), or from tissue from a cadaveric human donor (allograft). This alternative has the disadvantages of requiring an additional incision, loss of donor nerve function, size mismatch between donor and injured nerve, and limited availability of donor nerve.
    [0013] Allografts overcome several of the drawbacks of autografts, but require immunosuppression or decellularization to prevent immune rejection, as well as surgical intervention, rehabilitation, and the use of anti-inflammatories. Also, no have had satisfactory results when the lesions are greater than 2 cm in length.
    [0015] Therefore, a promising alternative has been the manufacture of natural and synthetic degradable polymeric structures as a new means for reconstruction and guided regeneration in the face of these injuries.
    [0017] Synthetic conduits have only been used successfully clinically for lesions less than 1 cm. Thus, both porous cylindrical collagen structures, whose ellipsoidal cavities have a preferential direction, can be found, as well as bundles of attached microfilaments of PLGA (poly (lactic-co-glycolic acid) and PCL (polycaprolactone), simulating the subfascicular structure of the endoneurium , although the most common is to use tubular conduits with one or more parallel through channels, which serve as a preferential pathway or guide to the axons, or mixed systems of the above. However, especially with regard to lesions in the CNS , the solutions proposed to date have not been successful enough.
    [0019] Document WO2007090102A2 refers to structures that include, among their possible configurations, conduits with a polymeric envelope that contain longitudinally aligned filaments inside them, made up of aliphatic polyesters such as PLA (polylactic acid), which act as scaffolding and guides for the regeneration of great lengths of nervous tissue.
    [0021] The structures are biohybrid in nature and can comprise, in addition to synthetic polymers, biomolecules useful for nerve regeneration, neural stem cells, Schwann cells and growth factors.
    [0023] The duct can reach a length of up to 50 cm and is longitudinally shaped as a single piece, which is manufactured using an electrospinning process. According to this procedure, a hollow polymeric structure with the required dimensions is first formed, which is subsequently filled with the fibers of the second polymeric material that are shaped to adapt to the size of the lumen of the conduit prepared in the first step in which it is have longitudinally aligned.
    [0024] Document WO2013 / 154780 describes devices in the form of conduits for the repair of peripheral nerves that are capable of reconnecting the nervous tissue, covering lengths greater than 3 cm. The conduits are made up of bioabsorbable polymeric hollow tubular modules or segments to which neurons, Schwann cells and growth factors can be added.
    [0026] These ducts lack fibers in their lumen that act as a guide for axonal growth, or as a connecting element between the segments. The segments present different conductivity and their assembly along the longitudinal axis gives rise to a conduit in which an electrical gradient and adequate growth factors can be established to stimulate axonal propagation, and with it, the repair of the lesion.
    [0028] The manufacture of the conduits comprises the use of a cylindrical mold in which a low conductivity polymer is introduced, on which a high conductivity polymer is placed, joining both polymers. This cylindrical or tubular material, and in which pores can be created, is removed from the mold, and the nerve cells can be seeded at a later stage.
    [0030] These conduits have an internal diameter of 1.5 mm and an external diameter of 1.8 mm. They are limited by a maximum regeneration length in SNP of 20 mm. Furthermore, the internal diameter is greater than that of many ribs, and said diameter cannot be modulated, making it not compatible with any type of rib.
    [0032] WO2018025186A1 describes a hollow tubular structure of silk fibroin for the regeneration of peripheral nerves. The synthesis procedure carried out allows to generate an increase in the viscosity of the material without increasing the concentration of silk. This makes it possible to create tubes of variable length, up to 10 cm, with improved mechanical properties to be implanted in areas where they are exposed to compressive forces, and where it is easier to create gradients of bioactive molecules.
    [0034] The structure obtained can further comprise a conducting agent, biodegradable polymeric material, therapeutic agents, growth factors and cells, such as stem cells or Schwann cells (SCs).
    [0035] Due to current limitations in the technique for treating and repairing CNS and peripheral nerve damage, new therapies require materials and factors, chemical or physical, that keep the implant active for several weeks. Therefore, new materials and methods are needed for the treatment of patients with lesions equal to or greater than 3 cm in length.
    [0037] DESCRIPTION OF THE INVENTION
    [0039] The modular device for nerve regeneration, the first object of the present invention, overcomes the drawbacks of the state of the art described, so that it allows progress in nerve regeneration in long neural tracts. The device is useful for the regeneration of neural tracts of the central nervous system (CNS) or the peripheral nervous system (PNS).
    [0041] It is a "biohybrid" device, as it comprises materials of natural and synthetic origin and is intended to house glial cells and / or neurons. It comprises an essentially cylindrical structure, made up of cylindrical elementary modules, of a combined material (natural and synthetic) that is degradable and biocompatible. The elementary modules are hollow, comprising inside a hole or lumen, inside which support glial cells (Schwann cells, oligodendrocytes) and axonal extensions of neurons are destined to lodge. From the union of one or more elementary modules, the cylindrical structure can reach a variable length between 0.6 cm and 90 cm approximately.
    [0043] Likewise, and to serve as support for glial cells and axonal processes of neurons, the device comprises one or more bundles of microfilaments, which can be parallel or branched, consisting of parallel microfilaments, distributed throughout the lumen of the elementary modules. In this way, it is the microfilaments that hold the different elementary modules together. Furthermore, as indicated, microfilaments serve as support for adhesion and guide during cell migration and axon extension, which can grow from end to end, in great lengths (tens of centimeters).
    [0045] These microfilaments are kept parallel to the longitudinal axis of the cylindrical structure thanks to their attachment to fixation and suture structures, formed by a material flexible polymeric, positioned in some extreme elementary modules, through which both the holding of the microfilaments and the subsequent suture of the device to the injured nerve are carried out.
    [0047] Likewise, the device can also comprise auxiliary fixing structures, for example, in the form of a ring and arranged in series, which are positioned in one or more of the interior lumens of the elementary modules. These auxiliary fixation structures help to maintain the cylindrical shape of the microfilament bundles in an aqueous medium, preventing the microfibers from collapsing by joining one another.
    [0049] Each of the elemental modules is formed by a porous material, in which a growth factor and / or drug release module can be incorporated for a controlled and localized release thereof. Furthermore, the device may additionally comprise means to allow electrical and / or magnetic stimulation of biological structures.
    [0051] The device can comprise a single cylindrical structure made up of a series of elementary modules arranged one after the other, inside which one or more bundles of microfilaments are arranged. Alternatively, it may comprise a cylindrical structure that in turn branches into at least two cylindrical structures. In this case, the beam also forks into two beams. Furthermore, the device can comprise two or more cylindrical structures that run in parallel and that, upon reaching a bifurcation, depart in different directions.
    [0053] When the device comprises several parallel or branched bundles, it can comprise a polymeric cylindrical sheath, which covers all the cylindrical structures.
    [0055] Also, if the device comprises a portion of the extracorporeal cylindrical structure, it can be covered by a cylindrical sheath of an elastomeric polymer. In this case, the device may comprise an extracorporeal pumping system that circulates biological fluids inside the sheath.
    [0057] The device is adaptable to the thickness, length and number of beams. In addition, it is intended to house cells, growth factors and drugs, among others.
    [0058] Another object of the invention is a process for manufacturing the modular device for nerve regeneration, described above, and comprising the steps of:
    [0059] a) preparation of micro or nanoparticles loaded with bioactive molecules,
    [0060] b) provision of a mold with grooves for the manufacture of the elementary modules, c) introduction of a polymeric material in the form of fiber in each of the mold grooves with structures at the ends to facilitate the centering of the fiber in each groove,
    [0061] d) preparation of solutions, for example, of hyaluronic acid (HA), with the micro or nanoparticles of step a) and stirring, in the presence of a crosslinking agent,
    [0062] e) injection of the solutions into the slots of the mold, obtaining a set of mold solutions that cross-links in situ,
    [0063] f) freezing of the mold-solutions set,
    [0064] g) lyophilization of the mold-solutions assembly obtaining the elementary modules of HA, h) extraction of the polymeric fiber, thus creating the lumen of the cylindrical structure in a centered position,
    [0065] i) swelling of the cylindrical structures in distilled water,
    [0066] j) insertion into the lumen of microfilaments, for example, of polylactic acid (PLA), attached at their ends to the fixation and suture structures made of flexible polymeric material, joining several elementary modules until reaching a desired size (from a few millimeters up to several centimeters), and
    [0067] k) when necessary, obtaining an external envelope to protect the device in ex vivo application.
    [0069] In summary, the device and method of the present invention allow the manufacture of addressable structures for axonal regeneration of long length, of more than 10 cm, providing a more effective therapeutic alternative, and with fewer drawbacks and limitations than the use of graft of autologous tissue or cadaveric tissue.
    [0071] In addition, the modular design of the device provides a number of advantages from a functional point of view and its manufacturing process, namely:
    [0072] - The existence of elementary modules provides mechanically free sections throughout the entire cylindrical structure that increase the capacity of adaptation of the structure to any curvature, by being able to move and rotate one with respect to the other to a certain extent.
    [0073] - Faced with a single long tubular structure, which has no more openings than the two ends, the free sections provided by the elementary modules of the device increase the exchange of nutrients and cells with the environment, enabling, on the one hand, a greater survival of the cells transplanted inside and, on the other, an increase in metabolism necessary in the regeneration process.
    [0074] - It facilitates the creation of gradients of various types throughout the device, which promote regeneration. Thus, for example, elemental modules with different porosity, chemical composition of structural materials, loads of functional molecules or coatings can be incorporated.
    [0075] - During the manufacturing process based on short elementary modules, the possibility of defects being introduced decreases, thereby improving the process compared to the manufacturing of long ducts formed in a single segment.
    [0077] The device of the invention will have various impacts:
    [0078] - At a conceptual level, the implantation of neurocables that release factors and drugs can solve the problem of cell repopulation, facilitating the reconstruction of long pathways and creating a suitable niche to facilitate axonal growth, encompassing a multitude of irreparable injuries today.
    [0079] - Technological, since the development of biomaterials associated with cells and other substrates supposes a technological refinement aimed at clinical practice, revolutionizing nerve surgery by freeing it from its dependence on donors or cadaveric tissue banks.
    [0080] - Social, as it would mean an advance in cell therapies, impacting on an improvement in functionality in the affected people.
    [0081] - Economic, since it would open the way to new health products (artificial nerves), reducing health costs for hospitalization, surgeries, rehabilitation, etc.
    [0082] DESCRIPTION OF THE DRAWINGS
    [0084] To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, according to a preferred example of a practical embodiment thereof, a set of drawings is attached as an integral part of said description. where, with an illustrative and non-limiting nature, the following has been represented:
    [0086] Figure 1.- Shows a general view of the device.
    [0088] Figure 2.- Shows a partial longitudinal section of the device.
    [0090] Figure 3.- Shows a complete longitudinal section of the device.
    [0092] Figure 4.- Shows cross section of the device without auxiliary fixation structure (left) and with auxiliary fixation structure (right).
    [0094] Figure 5.- Shows a detailed view of the fiber ring and the fastening and suturing structure.
    [0096] Figure 6.- Shows a general view of a flat grouping of parallel microfilaments.
    [0098] Figure 7.- Shows a view of a device with nine modules in a curved position, without fixing at the ends.
    [0100] Figure 8.- Shows a general view of an embodiment of an association of two devices.
    [0102] Figure 9.- Shows a section of three devices of different diameters, contained within a protective outer casing.
    [0104] Figure 10.- Shows a view of an embodiment of the invention with a bifurcation of two cylindrical structures starting from a single cylindrical structure.
    [0105] Figure 11.- Shows a view of an embodiment of the invention with a bifurcation of two cylindrical structures starting from two cylindrical structures.
    [0107] Figure 12.- Shows a complete longitudinal section of the device with support cells inside after its in vitro culture.
    [0109] Figure 13.- Shows a complete longitudinal section of the device with support cells inside and a neuronal source at one end after its in vitro culture.
    [0111] PREFERRED EMBODIMENT OF THE INVENTION
    [0113] A preferred embodiment of the modular device for nerve regeneration, as well as its manufacturing process, is described below with the aid of Figures 1 to 13.
    [0115] Figure 1 shows a general view of the device, which in this embodiment comprises a single modular structure (1), of variable length between 0.6 cm and 90 cm, the modular structure (1) made up of elementary modules (2 ) cylindrical, made of a degradable and biocompatible natural and synthetic material. In the case of figure 1, the modular structure (1) comprises three elementary modules (2).
    [0117] As it appears in figure 2, which shows a partial section of the modular structure (1), the elementary modules (2) are hollow, comprising inside a hole or lumen (3), inside which they are intended to be housed. supporting glial cells (Schwann cells, oligodendrocytes) and axonal processes of neurons.
    [0119] Each elementary module (2) is a cylinder with dimensions between 0.5 cm and 2 cm in length, between 0.01 cm and 2 cm in internal diameter and between 0.05 cm and 3.5 cm in external diameter. From the union of one or more elementary modules (2) positioned in series or in parallel, the modular cylindrical structure can reach a variable length between 0.6 cm and 90 cm approximately.
    [0121] The elementary modules (2) can be made of different materials. It must be a biocompatible porous material that allows the exchange of fluids and nutrients between the internal and external part of the device. Also, it is preferable that the material is bioabsorbable or biodegradable, in this way it is not necessary to remove the device surgically when the regenerative process is complete.
    [0123] Suitable biocompatible materials for elemental modules (2) include synthetic polyesters and natural polymers, forming a porous structure such as: hyaluronic acid (HA), cross-linked HA, cross-linked methacrylated HA, cross-linked chitosan, alginate, collagen, gelatin, agarose cross-linked, polylactic acid (PLA), polycaprolactone (PCL), polyethylene glycol, poly (glycerol sebacate), etc.
    [0125] The elementary modules (2) are manufactured by using a mold or by electrospinning techniques or by any other technique that allows this morphology.
    [0127] As indicated, each modular structure (1) is destined to viablely house in the inner lumen (3) both supporting glial cells (Schwann cells, oligodendrocytes) and axonal extensions of neurons located at their ends, with neuronal connectivity. functional end-to-end device.
    [0129] Likewise, and to serve as support for glial cells and axonal processes, the device comprises one or more bundles (4), which can be parallel, as is the case in figure 2, or branched, made up of microfilaments (5) parallel, distributed by the lumen (3) of the elementary modules (2), with a length between 0.6 cm and 90 cm and a diameter of each microfilament between 0.001 cm and 0.1 cm.
    [0131] In this way, it is the microfilaments (5) that hold the different elementary modules (2) together. In addition, the microfilaments (5) serve as support for adhesion and guidance during cell migration and axon extension, which can grow from end to end, in great lengths (tens of centimeters). The microfilaments (5) drive the end-to-end axonal extension of the device, stimulated by the support cells seated on the microfilaments (5).
    [0133] In figure 3 a section of the bundles (4) is shown, in which it is observed how the microfilaments (5) are kept parallel to the longitudinal axis of the modular structure (1) thanks to their attachment to some fixation and suture structures (6), of a flexible polymeric material, positioned in elementary modules (2) ends, through which both the fastening of the bundles (4) and the subsequent suture of the device to the injured nerve are carried out.
    [0135] As shown in Figure 2, and in detail in Figure 4, the device can also comprise auxiliary fixing structures (7) in the form of rings arranged in series, and which are positioned in one or more of the lumens (3 ) interiors of the elementary modules (2), and that contribute to maintaining the cylindrical shape of the bundles (4) of microfilaments (5) in an aqueous medium, preventing them from collapsing by joining each other. Specifically, in the left part of figure 4 a section of an elementary module (2) without auxiliary fixing structure (7) is shown, and in the right part a section of an elementary module (2) with auxiliary fixing structure ( 7).
    [0137] Figure 5 shows a more detailed view of the microfilaments (5) attached to the fixation and suture structures (6). The microfilaments (5) can be made of different materials, preferably degradable synthetic polyesters (polylactic acid (PLA), polylactic-coglycolic acid (PLGA), poly-£ -caprolactone (PCL), polypyrrole (PPy), carbon nanotubes (CNTs) , silk fibroin, poly (3,4-ethylenedioxythiophene) (PEDOT), polyaniline (PANI), etc.) or from carbon fibers or conductive metals such as gold or platinum, or a combination thereof.
    [0139] The way in which the microfilaments (5) are arranged within the inner lumen (3) or lumen of the elementary modules (2) of the modular structure (s) (1) can be variable: in the form of flat parallel microfilament groupings (5) joined at the ends by a flexible polymeric material or in the form of microfilament cylinder bundles (5) held at each end by rings of flexible polymeric material. For a better understanding of the invention, the manufacturing processes of the parallel microfibers (5) joined at both ends are described below.
    [0141] To obtain the flat groups of parallel microfilaments (5), which are shown in a detailed view in Figure 6, a certain number of the microfilaments (5) (600 microfilaments (5)) are grouped and arranged in a parallel manner. next to each other on a flat surface, such as a glass plate. In order to maintain this arrangement, a flexible polymeric material dissolved below and above the microfilaments (5) is placed at both ends, which, when solidifying, holds the filaments together in a flat and parallel arrangement.
    [0142] To obtain a bundle (4) of microfilaments (5) in cylinder mode, like the one that appears in figure 2, they are first arranged in the form of flat parallel groups of microfilaments (5), like those in figure 6 , and subsequently they are wound around a rod of suitable diameter in order to obtain the cylindrical shape without the microfilaments (5) losing their parallel arrangement.
    [0144] A flexible polymeric material is then applied dissolved into a ring of the desired length and allowed to dry. Once dry, the rod is extracted and the microfilament cylinder (5) is obtained. The number of microfilaments (5) that make up the cylinder can vary depending on the diameter of the individual microfilaments (5) and the diameter of the bundle (4) of microfilaments (5) to be generated.
    [0146] The cylindrical microfilament bundle (5) is inserted into the lumen (3) of the conduit, from end to end of the elemental module (2). The microfilaments (5) are the common element to all the elementary modules (2), which are positioned side by side, with the microfilament bundle (5) passing through their lumens or holes (3).
    [0148] The modular structures (1) composed of several elementary modules (2) have at both ends some fixing and suturing structures (6) of a flexible polymeric material that, in addition to maintaining the cylindrical arrangement of the bundle (4) of microfilaments (5 ), prevent excessive separation between elementary modules (2) and allow the manipulation of the modular device (1) as a single entity.
    [0150] In the case of the modular structures (1) of larger dimensions in terms of their internal diameter, a slightly conical fixing and suturing structure of a flexible polymeric material is positioned in the elementary modules (2), shown in detail in the figure. 5, to improve the introduction of the distal and proximal end of the injured nerve and facilitate the suture to said structure.
    [0152] Modular manufacturing to span a long length, versus building a single conduit spanning that long length, minimizes failures in the manufacturing process. In addition, the modular design provides free sections along the entire length, which increase the ability to adapt the total length to any curvature, by being able to move and rotate one with respect to the other to a certain extent, as reflected. in figure 7. That is, the flexural strength of the modular structures (1) is much shorter than that of the same length obtained from a single piece, which makes it more flexible and adaptable to variable curvatures.
    [0154] As shown in figure 8, the device of the invention can comprise two or more modular structures (1) arranged in parallel, which in turn can comprise a plurality of bundles (4) of microfilaments (5) of variable diameter. Each beam (4) extends continuously over the entire length of the device, in such a way that a continuous internal architecture is generated (with the exception of the areas between elementary modules (2)), longitudinal and aligned that promotes neural regeneration, and which in turn can be included within a protective outer envelope (9), as shown in figure 9.
    [0156] When the device comprises several bundles (4) in parallel or branched, it can comprise a cylindrical sheath (9), as shown in Figure 9, which covers each of the bundles (4). The cylindrical sleeve (9) can be made of the same materials as the elemental modules (2): synthetic polyesters and natural polymers, forming a porous structure such as: hyaluronic acid (HA), cross-linked HA, cross-linked methacrylated HA, cross-linked chitosan , alginate, collagen, gelatin, cross-linked agarose, polylactic acid (PLA), polycaprolactone (PCL), polyethylene glycol, poly (sebacate glycerol), etc., or it can be made of inert polymers such as silicone, polytetrafluoroethylene (PTFE, Teflon) or polyacrylates.
    [0158] Likewise, the device can comprise an extracorporeal modular structure portion (1), covered by a cylindrical sheath of an elastomeric polymer, such as silicone or polyacrylates. In this case, the device comprises a connection to an extracorporeal pumping system that circulates biological fluids inside the sheath.
    [0160] The internal architecture when there are several modular structures (1) increases the surface for the adhesion of the SCs that are part of the device. This architecture allows to contain many more SCs than the device with a single modular structure (1) (with a single bundle (4) of microfilaments (5) in its lumen (3)). The greater number of SCs represents an improvement in the regenerative process.
    [0162] The internal architecture of several modular structures (1) can provide greater control over the direction of growth of different groups of nerve fibers in regeneration. A single nerve can contain thousands of nerve fibers or axons. Through the use of multiple internal fibrillar structures, each axon or a group of axons can be guided during the regenerative process through an individual modular structure (1) or a group of modular structures (1).
    [0164] Also, as detailed in Figure 10, a branching architecture can be created if desired. For example, in the simple model of a bifurcated nerve, two distinct groups of bundles (4) of internal microfilaments (5) can be provided within the internal lumen (3) of the device.
    [0166] As shown in Figure 10, at a first end of the device (proximal), the bundle (4) of microfilaments (5) has a single proximal end for connection with the proximal nerve stump. At a second (distal) end, specifically in an elementary bifurcation module (10), they are separated into two distinct groups of bundles (4), so that two distinct distal ends of the device are provided. Each of these ends connects to a separate branch of the distal nerve stump.
    [0168] Alternatively, as reflected in figure 11, the device can comprise two modular structures (1) that run in parallel, and which, upon reaching elementary separation modules (11), divide in different directions.
    [0170] For its part, a second object of the invention is the device manufacturing process, which is a modular and sequential process. To achieve the desired length, the corresponding number of elementary modules (2) are assembled, passing through their interior the bundle (4) of continuous microfilaments (5) joined at the ends by holding and suturing structures (6).
    [0172] Next, the supporting glial cells (12) are seeded at different points inside each elementary module (2), which are cultured in vitro until they form a continuous covering of the internal face of the elementary modules (2), reaching continuity, as shown in figure 12.
    [0174] As shown in figure 13, after culturing the glial cells (12), neuronal sources can be seeded at one end of the device and, where appropriate, at different points along its length (13). as neural precursors, explants of dorsal root ganglia, etc. The whole can then be cultured in vitro until the desired axonal extension is obtained.
    [0176] Each of the elemental modules (2) is made up of a porous material that allows the incorporation of bioactive substances from one or more growth factor and / or drug release modules (8) for a controlled and localized release of the The same, as shown in figure 1, either in the matrix of the elemental module (2) itself, or encapsulated inside micro or nanofibrils, micro or nanoparticles, liposomes, or other methods of encapsulation of these substances. Said particles or fibrils can have a hydrophilic character like gelatin or hydrophobic like PLA, or cross-linked gelatin, depending on the character of the molecule to be loaded on them, and variable sizes up to the order of tens of microns.
    [0178] In addition, the device can additionally comprise means for electrical stimulation, such as one or more electrodes in contact or not with electroconductive microfilaments for electrical stimulation of the device, being able to be housed both in the lumen (3) of the elementary module (2) and on the outside. In turn, the device can comprise coils around it that allow magnetic stimulation thereof. Likewise, the use of electroconductive microfilaments that form closed circuits also allows electrical stimulation of the device without the need for electrodes.
    [0180] Therefore, a process for manufacturing the modular device for nerve regeneration, described above, and comprising the steps of:
    [0181] a) preparation of micro or nanoparticles loaded with bioactive molecules, which can comprise different polymers, such as gelatin, hyaluronic acid, polylactic acid, polyglycolic acid,
    [0182] b) provision of a mold with grooves to contain the elementary cylindrical modules (2),
    [0183] c) introduction of a polymeric material in the form of fiber in each of the slots of the mold, with structures at the ends to facilitate the centering of the fiber in each slot
    [0184] d) preparation of solutions, for example, of hyaluronic acid (HA), with the micro or nanoparticles of step a) and stirring, in the presence of an HA crosslinking agent, which can preferably be selected from the group consisting of divinylsulfone, ethers such as 1,6-hexanediol diglycidyl ether or polypropylene glycol diglycidyl ether, diepoxides such as 1,2,7,8-diepoxyoctane or 1,3-butadiene diepoxide, and water soluble carbodiimides. Preferably, the crosslinking agent is divinylsulfone.
    [0185] e) injection of the solutions into the slots of the mold, obtaining a set of mold solutions that cross-links in situ,
    [0186] f) freezing of the mold-solutions set,
    [0187] g) lyophilization of the mold-solutions set obtaining microporous tubular matrices of HA,
    [0188] h) extraction of the polymeric fiber or fibers, thus creating the lumen (3) of the modular structure (1) in a centered position.
    [0189] i) swelling of the modular structures (1) in distilled water,
    [0190] j) insertion into the lumen (3) of microfilaments (5), for example, made of polylactic acid (PLA), attached at their ends to fixation and suture structures (6) made of flexible polymeric material, joining several elementary modules (2) to a desired size (from a few millimeters to several centimeters), and
    [0191] k) when necessary, obtaining an external envelope to protect the device in ex vivo application.
    [0193] EXAMPLES
    [0195] The procedure for manufacturing an elementary module (2) is detailed below. A polytetrafluoroethylene (PTFE) mold with 1.5mm wide channels is used. In each channel a 0.4 mm diameter fiber of a hydrophobic material, PCL, is introduced using PTFE washers with a diameter that fits the channel of the mold to keep it centered, to serve as a negative of the internal diameter of the elemental module (two).
    [0197] A 5% hyaluronic acid (HA) solution is prepared in the presence of 0.2M sodium hydroxide (NaOH) and excess divinylsulfone (DVS) (in a DVS: HA molar ratio of monomer units of 9:10) as crosslinking agent. This solution is injected into the channels of the PTFE mold. Once the solution has gelled, the mold is placed in a Petri dish to avoid evaporation and cooled to -20 ° C.
    [0198] The template-solution assembly is then lyophilized for 24 h at 20 Pa and -80 ° C to generate HA microporous matrices due to water sublimation. The dry product is obtained, from which the polycaprolactone fiber (PCL) is extracted, thus creating the hollow internal lumen (3). Finally, they are hydrated and cut to a length of 6 mm to obtain the different elementary modules (2) that will make up the modular structures (1).
    [0200] By way of example, for the serial assembly of 3 elementary modules (2) of 6 mm in length, as in the device shown in figures 1-3, or of 9 elementary modules (2) as is the case in the figure 7, 120 microfilaments (5) of polylactic acid (PLA) of 10 µm diameter each are inserted into the lumen (3). To do this, each of the microfilaments (5) are aligned to generate a beam (4) that passes between the elementary modules (2). In this way the assembly of "n" number of elementary modules (2) can be achieved.
    [0202] A device with 3 elementary modules (2) could cover a short lesion. A device with 9 elementary modules (2) could cover a long injury without the need for large nerve supplies, such as brachial plexus surgery or oncological surgery, even consider the reconstruction of spinal connections avoiding scarring areas or establishing new connections between the central nervous system (CNS) and peripheral nervous system (PNS).
    [0204] For fixing the ends of the device, alternative arrangements of microfilaments (5) are proposed, such as the parallel flat groups of microfilaments (5) of Figure 6, joined at the ends with a flexible polymeric material, or microfilament cylinders (5) held at each end by a ring made of flexible polymeric material.
    [0206] The device of the present invention is not limited only to the repair of PNS nerves but also to structures based on axonal tracts of the CNS such as the spinal cord, optic nerve and other tracts.
    [0208] In the modular design of the device, the free sections along the length greatly facilitate the exchange of nutrients and cells with the environment, thus enabling, on the one hand, the greater survival of the cells transplanted inside, and, Moreover, the higher speed of metabolism with the necessary environment in the regeneration process (both very difficult in a single long tubular structure concept, which has no more openings than the two ends).
    [0210] For example, for the culture of Schwann cells (SCs) as a source of supporting glial cells (12) inside the modular structure (1) formed by 3 elementary modules (2) of HA cross-linked with DVS of 6 mm of length each, with an internal diameter of 400. ^ m internal diameter each. The elementary modules (2) house a bundle (4) of 120 PLA microfilaments of 10. ^ M diameter each.
    [0212] The modular structure (1) formed by the elementary modules (2) of HA is sanitized and pre-conditioned, with its lumen (3) occupied by 120 microfilaments (5) of PLA of 10. ^ M each in a flow cabinet Laminate, by means of two successive washes with a 70% ethanol solution. The samples are washed with 50% and 30% ethanol solutions. They are then washed with deionized water and conditioned with culture medium before seeding in the modular devices (1).
    [0214] On the other hand, the SCs are cultured in flasks until confluence, at 37 ° C, 5% CO 2 , in a culture medium for SCs. All experiments are performed with cells in passage 4 to 6.
    [0215] These glial support cells (12) are seeded at different points inside the modular structure (1) at a density of 27,000 cells / mm2 with the help of a pipette. Wait 30 min before adding culture medium for SCs.
    [0217] After 5 days of in vitro culture, the SCs form a layer-like structure along the internal lumen (3) of each elemental module (2) continuously, and also grow on the bundle (4) of microfilaments (5) of PLA in the direction marked by them, even in the intermodular areas where the microfilaments are no longer covered by the elementary modules (2) of HA.
    [0219] Furthermore, it is possible to use neuronal sources to study axonal growth in modular structures (1) in co-culture with supporting glial cell sources (12). For example, after 5 days culture of the SCs in the modular structure (1), a dorsal root ganglion explant of neonatal rats can be seeded at one end of each modular structure (1) as a source projecting neurons ( 13).
    [0220] The whole is then cultured in vitro for an additional 21 days, obtaining an axonal extension that covers the entire length of the modular structure (1), even with these axons traversing the intermodular areas between different elementary modules (2). The axonal extension was greater than in the devices in which there were no pre-seeded SCs, showing how this element is fundamental for a more efficient axonal growth.
    [0222] In long-term lesions, regeneration is hampered by the long period of time required for regeneration, the rapid appearance of a glial scar, and the lack of chemical and / or electrical stimuli. Electrical stimulation stimulates axonal extension after nerve injury.
    [0224] For example, flat groups of parallel microfilaments (5) or cylinders formed by microfilaments (5) of PLA can be coated with an electroconductive polymer such as polypyrrole (PPy). For this, one of the different techniques that can be used is polymerization in situ.
    [0226] As a preliminary step, the groups of microfilaments (5) are immersed in deionized water under compression and applying a fixed vacuum until they stop floating in order to achieve the introduction of water into the spaces between microfilaments (5) in order to obtain a homogeneous coating of all microfilaments (5), not just the most superficial ones. Next, each microfilament grouping (5) is placed inside a polypropylene tube with an aqueous solution of pyrrole monomer (Py) and sodium para-toluenesulfonate (pTS) that acts as a dopant, followed by the application of ultrasound to Allow the microfilament cluster (5) to saturate with the Py / pTS solution.
    [0228] Clusters of microfilaments (5) are incubated with shaking at 4 ° C. Then, an aqueous solution of ferric chloride (FeCh) is added and incubated with shaking at 4 ° C to achieve oxidative polymerization and the deposition of PPy on the PLA substrates. The PPy-coated substrates are washed with shaking deionized water and then sonicated in deionized water to remove all PPy residues not adhering to the microfilaments (5). Finally, the microfilament clusters (5) are dried in a desiccator with a fixed vacuum at 40 ° C.
    [0229] Additionally, the modular structure (1) can be functionalized by loading one or more bioactive substances in each elemental module (2) with the same or different concentration. In this way, it is possible to create gradients of various types that favor regeneration. The creation of gradients in a non-modular structure could not be done by having to functionalize with one or more substances, but with the same concentration.
    [0231] For example, functionalized devices with bioactive molecules in each elemental module (2) with the same or different concentration could be created by incorporating them inside using polylactic-coglycolic acid (PLGA) microparticles loaded with a growth factor that favors axonal extension, such as for example nerve growth factor (NGF) at a concentration of 10 ng / mL.
    [0233] PLGA microparticles are prepared by the water-in-oil-in-watert (W / O / W) method. An aqueous solution with the NFG is prepared and added to a solution of PLGA in dichloromethane obtaining the first emulsion (W / O). This emulsion is added dropwise into a solution of polyvinyl alcohol in an aqueous solution of sodium chloride, obtaining the second emulsion (W / O / W). The W / O / W emulsion is stirred at room temperature to allow evaporation of the solvent and finally the microparticles are collected by centrifugation.
    [0235] Elementary modules (2) can be designed with more or less porosity, with different chemical composition of the structural materials, with more or less charges of bioactive molecules or surface coating, with different seeded cell types, etc. so that there is a gradient between some elementary modules (2) and others when forming part of a device with a unique modular structure (1), giving this added functionality to the device as a whole.
    [0237] Bioactive substances can be incorporated into the modular structure (1) by adding the drug to the polymer solution before injection into the mold if the drug is soluble in the selected solvent. This will result in the dispersion of the drug throughout the modular structure (1) and each elemental module (2). Alternatively, an emulsion of the drug and a drug solvent can be added to the polymer solution before injection, resulting in the drug being retained in the pores of the modular structure (1).
    权利要求:
    Claims (15)
    [1]
    1. - Modular device for nerve regeneration, comprising:
    - one or more elementary modules (2) essentially cylindrical, hollow and porous, arranged one after the other according to a modular structure (1) with an interior lumen (3),
    - one or more bundles (4) of parallel microfilaments (5), arranged in the lumen (3) of the modular structure (1), intended to serve as a support for an axonal supporting cellular component, and
    - Fixation and suture structures (6) positioned in some extreme elementary modules (2) and connected to the microfilaments (5), intended to fix the microfilaments (5) in a desired position and to serve as platforms where to proceed to suture a nerve to be regenerated.
    [2]
    2. - The device of claim 1, further comprising auxiliary fixation structures (7) in the form of rings arranged in the lumen (3) of the modular structure (1) and to which the microfilaments (5) are fixed, keeping them parallel.
    [3]
    3. - The device of claim 1, further comprising one or more growth factor and / or drug release modules (8) embedded or encapsulated for a controlled and localized release inside the elemental modules (2).
    [4]
    4. - The device of claim 1, further comprising electrical and / or magnetic stimulation means connected to the modular structure (1).
    [5]
    5. - The device of claim 1, comprising a first modular structure (1), at least one elementary bifurcation module (10) positioned at one end of the first modular structure (1) and a second and third modular structures (1) connected to the elementary branch module (10).
    [6]
    6. - The device of claim 1, comprising two or more first parallel modular structures (1), two or more elementary separation modules (11) positioned at one end of the first parallel modular structures (1), and two or plus second parallel modular structures (1) connected to the elementary separation modules (11) arranged in a non-parallel manner.
    [7]
    7. - The device of claim 1, wherein the modular structure (1) comprises a bundle (4) of microfilaments (5) in the lumen (3), the modular structure (1) being covered by a cylindrical sheath ( 9).
    [8]
    8. - The device of claim 1, comprising a portion of the extracorporeal modular structure (1), covered by an extracorporeal cylindrical sheath.
    [9]
    9. - The device of claim 8, further comprising a connection to an external physiological fluid pumping system.
    [10]
    10. - The device of claim 1, in which the elementary modules (2) are made of a natural and / or synthetic material, degradable and biocompatible.
    [11]
    11. - The device of claim 10, in which the elemental modules (2) are made of a biocompatible material selected from synthetic polyesters and natural polymers.
    [12]
    12. - The device of claim 11, in which the elemental modules (2) are made of a material selected from hyaluronic acid (HA), cross-linked HA, cross-linked methacrylated HA, cross-linked chitosan, alginate, collagen, gelatin, cross-linked agarose, polylactic acid (PLA), polycaprolactone (PCL), polyethylene glycol and poly (glycerol sebacate).
    [13]
    13. - The device of claim 1, in which the microfilaments (5) are made of a material selected from degradable synthetic polyester, carbon fibers, conductive metals and a combination thereof.
    [14]
    14. - The device of claim 13, in which the microfilaments (5) are made of a material selected from polylactic acid (PLA), polylactic-coglycolic acid (PLGA), poly- £ -caprolactone (PCL), polypyrrole (PPy ), carbon nanotubes (CNTs), silk fibroin, poly (3,4-ethylenedioxythiophene) (PEDOT) and polyaniline (PANI).
    [15]
    15. - Manufacturing process of the modular device for nerve regeneration of any of the preceding claims, comprising the steps of:
    - preparation of micro or nanoparticles loaded with bioactive molecules, - arrangement of a mold with grooves to contain the elemental modules (2), - introduction of a polymeric material in the form of fiber in each of the slots of the mold, with structures at the ends to facilitate the centering of the fiber in each slot,
    - preparation of solutions, with the micro or nanoparticles and stirring, in the presence of a crosslinking agent,
    - injection of the solutions into the mold grooves, obtaining a set of mold-solutions that cross-links in situ,
    - freezing of the mold-solutions set,
    - lyophilization of the mold-solutions set, obtaining the elementary modules (2) that make up the modular structure (1),
    - extraction of the fibers, thus creating the lumen (3) of the modular structure (1) in a centered position,
    - swelling of the cylindrical structures in distilled water,
    - insertion into the lumen (3) of the microfilaments (5), and
    - optionally, obtaining an external envelope (9) to protect the device in ex vivo application.
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    同族专利:
    公开号 | 公开日
    ES2818424B2|2021-08-13|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    ES2528192T3|2001-08-02|2015-02-05|Collagen Matrix, Inc.|Implant device for nerve repair|
    WO2007090102A2|2006-01-27|2007-08-09|The Regents Of The University Of California|Biomimetic scaffolds|
    WO2013154780A1|2012-04-12|2013-10-17|Wake Forest University Health Sciences|Design of a conduit for peripheral nerve replacement|
    WO2016097448A1|2014-12-16|2016-06-23|Universitat Politècnica De València|Biohybrid for the use thereof in the regeneration of neural tracts|
    WO2018025186A1|2016-08-01|2018-02-08|Association For The Advancement Of Tissue Engineering And Cell Based Technologies & Therapies A4Tec - Associação|Nerve guidance conduits, methods of production and uses thereof|
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