Porous nerve guidance conduit based on bioabsorbable polymers incorporated with drugs for the repair of peripheral nerves, manufacturing method therefor and use of the polymers

Abstract

The present invention relates to a porous nerve guidance conduit produced using a combination of additive manufacturing and rotary jet spinning techniques from any bioabsorbable polymer, wherein the conduit can be incorporated or not with anti-inflammatory drugs. The nerve guidance conduit of the present invention can be used in the field of tissue engineering, in applications and preparations for medical purposes and, more specifically, to repair peripheral nerve injuries.

Description

[0001] POROUS NEURAL GUIDE CONDUCT BASED ON BIOABSORBABLE POLYMERS INCORPORATED WITH DRUGS FOR THE REPAIR OF PERIPHERAL NERVE, ITS MANUFACTURING METHOD AND USE OF THE POLYMERS Field of the Invention: [1] The present invention relates to a porous neural guide conduit, produced from the combination of additive manufacturing and rotospinning techniques from any bioabsorbable polymer, in which the conduit can be incorporated or not with anti-inflammatory drugs. [2] The area of ​​application of the neural guide conduit of the present invention is in the field of tissue engineering, in applications and preparations for medical purposes, more specifically, the neural guide conduit can be used in the repair of injuries to peripheral nerves. Fundamentals of the invention: [3] From the 1990s to the present day, there has been a significant increase in the use of the terms tissue engineering (TE) and scaffolds, which are increasingly linked to different applications, such as nerve regeneration,cartilage and bone. Tissue engineering can be said to be a relatively new research field, with a primary focus on restoring, maintaining, and improving tissue function through the combination of specific cells, scaffolds, and biological factors (SHIMOJO et al., 2018). Thus, tissue engineering aims to overcome the limitations of various aspects of medicine. [4] Scaffolds can be defined as three-dimensional matrices with a capacity similar to the structure and biological function of the extracellular matrix (ECM). That is, they must present properties of high surface-to-volume ratio, high porosity, and interconnectivity (LANGER, 1993). Thus, they enable nutrient transport, allow tissue proliferation, and possess mechanical strength, providing support and protection (BUJ-CORRAL, 2018). Furthermore, they can also function as carriers for cells, growth factors, drugs, and other bioactive molecules (AGRAWAL, 2001). [5] Therefore,The present invention considers scaffolds, the tube, and the polymeric fibers individually or together, since they are temporary structures for tissue regeneration, in the case of the present invention, nervous tissue. [6] Regarding peripheral nerves, it can be said that an organism is largely controlled by the nervous system, in which neurons, its fundamental unit, are responsible for coordinating and regulating bodily activities, which can be both voluntary and involuntary. [7] The nervous system is divided into the central (CNS), formed by the brain and spinal cord, and the peripheral (PNS), with the PNS bridging the gap between the CNS and the different tissues and organs of the body (HORCH, 2017). [8] According to LANGLEY (1852), the PNS has two components: sensory and motor. The sensory endings, consisting of sensory receptors at the extremities throughout the body,are responsible for transmitting information from the external environment and the body itself to the CNS. The information is then processed by the CNS, and the motor component then distributes it to the muscles. [9] The PNS is made up of peripheral nerves, which are formed by bundles of motor or sensory axons, which are surrounded by Schwann cells, whose function is to maintain the myelin sheath within the PNS (HORCH, 2017). The entire peripheral nerve is surrounded by a connective sheath that provides mechanical and trophic support, as it contains blood vessels (VIJAYAVENKATARAMAN, 2020). This tissue is divided into the epineurium, perineurium, and endoneurium.

[0010] The epineurium corresponds to the outermost layer that surrounds the entire nerve and is made up of veins, arteries, fat, and mainly a number of fascicles, which encompass the perineurium. Thus, the perineurium is a membrane that surrounds the bundle of nerve fibers,whose function is to maintain intrafascicular pressure, protect the fibers by forming a barrier, and assist in maintaining endoplasmic flow. And each nerve fiber (consisting of the axon surrounded by Schwann cells) is surrounded by the endoneurium. This sheath is the most sensitive layer, as it protects the neuron body from mechanical trauma and contains collagen and fibroblasts (GRAY, 1988; JONES, 2014).

[0011] Along with Schwann cells, there are other cells that also assist in axonal regeneration. These are macrophages, whose function is involved in the phagocytosis of fibers and disintegrating myelin, and satellite glial cells, which are involved in regulating the external chemical environment (HALL, 2005; HANANI, 2005). In turn, Schwann cells provide support within the tissue, as they produce an environment composed of cell adhesion molecules, extracellular matrix (ECM), integrins and neurotrophins (ALLODI; UDINA; NAVARRO, 2012; SCHMIDT; LEACH,2003).

[0012] In this context, Schwann cells are responsible for producing neural growth factors, which can be neurotrophic and neurotropic factors (laminin, fibronectin, and collagen). Examples of neurotrophic factors include NGF (nerve growth factor), BDNF (brain-derived neurotrophic factor), ciliary neurotrophic factor (CNTF), GDNF (glial cell-derived neurotrophic factor), and neurotrophin 3 and 4 (NT-3 and NT-4), which are extremely relevant in several aspects.

[0013] This relevance is due to the fact that it is necessary to maintain axon function, that is, its survival and growth. Studies show that gradients of neurotrophic factors can direct growth cones (BELLAMKONDA, 2019).

[0014] Peripheral nerve injury can be caused by mechanical, thermal, chemical damage, some degenerative disease or even some trauma caused by an accident, which can result in crushing, compression, stretching,partial or total nerve transection (ARSLANTUNALI et al., 2014; DANTAS, 2014). Such injuries can lead to the interruption of nerve impulses and a decrease or even total loss of neural communication in relation to sensory and motor nerves (LIM et al., 2017).

[0015] Initially, Seddon classified nerve injury into three major groups: neuropraxia, axonotmesis, and neurotmesis (SEDDON, 1941). Sunderland then subclassified these groups into five degrees of severity, based on the behavior of the axon.

[0016] In the first degree, called neuropraxia, the nerve is crushed and consequently nerve conduction is blocked, but its continuity remains (with slightly intact architecture). Therefore, in this degree, there is no motor injury, and only a biochemical injury is considered (FERNANDES, 2014).

[0017] While in grade II there is already loss of the axon in which the distal part undergoes Wallerian degeneration,without rupture of the connective tissue (endoneurium). This degree is known as axonotmesis, which is subclassified into degrees II, III, and IV. In this first stage, spontaneous recovery is still possible.

[0018] In degree III, axon loss also occurs, but there is also loss of the endoneurium, making the recovery of motor and sensory function delayed, since fibrosis can occur within the fascicles (ARSLANTUNALI et al., 2014).

[0019] Then, in degree IV, it progresses beyond the loss of the axon and endoneurium, there is also loss of the perineurium. Thus, in the last degree (neurotmesis), there is total loss of the nerve, with neuroma and Wallerian degeneration occurring, thus making recovery impossible without surgical intervention.

[0020] In the stages that occur in the degeneration of the peripheral nerve, initially the distal end is broken and consequently the proximal stump of the axon degenerates in a retrograde manner (GARCEZ,2015). Then, with the death of the cell body, the metabolism shifts its production to metabolites necessary for nerve regeneration.

[0021] Only after 48–96 hours or even weeks, Wallerian degeneration begins, in which the axon components are disintegrated, causing the collapse of the myelin sheath. Then, Schwann cells and macrophages proliferate rapidly to begin clearing the axonal debris and myelin.

[0022] The Schwann cells then align to form longitudinal bands known as bands of Bungner, whose function is to guide the regenerative axonal sprouts that have traveled through these channels (BELKAS; SHOICHET; MIDHA, 2004). These sprouts originated in the proximal stump and proliferated through gradients of neurotrophic factors. Only after they reach the distal receptor does reinnervation occur and they undergo myelination.

[0023] Therefore, axonal regeneration occurs,in which these axons reach the distal stump that was injured, causing functional reinnervation with the distal stump (KOENIG, 2009). Axonal growth of the proximal end is 1 to 2 mm / day under ideal conditions (ARSLANTUNALI et al., 2014).

[0024] However, although the peripheral nerve has this capacity for spontaneous regeneration, many factors can interfere and not be sufficient for regeneration to occur.

[0025] Such factors are the length of the injured nerve gap, the time elapsed between injury and repair, and the patient's age, which must be taken into account. For this reason, there is a growing search for other alternatives. Thus, treatments for nerve repair can be classified as surgical or non-surgical.

[0026] Non-surgical treatments consist of electrical stimulation (HAASTERT-TALINI et al., 2011), with steroids, magnetic stimulation (BANNAGA et al., 2002), laser phototherapy (ROCHKIND; GEUNA; SHAINBERG,2009) and neurotrophins. These methods even show positive results; however, for gaps greater than 3 cm, further study is needed (VIJAYAVENKATARAMAN, 2020). Non-surgical methods can also be combined with surgical methods as a combinatorial therapy (VIJAYAVENKATARAMAN, 2020). Regarding surgical methods, the most classic treatment is end-to-end repair through suturing of the nerve stumps, also known as neurorrhaphy (ARSLANTUNALI et al., 2014; FERREIRA; MATERIAIS, 2017). Suturing can be classified as epineural or fascicular in groups. The most common is epineural, but it does not guarantee good results, since there is poor alignment of the fascicles (GARCEZ, 2015). Furthermore, this technique is limited by the size of the gap, which can be a maximum of 1 cm, since for larger gaps (gap > 2 cm) saturation can cause tension in the nerve and inhibit regeneration (PABARI et al., 2010; STEED et al., 2011; VIJAYAVENKATARAMAN, 2020).

[0028] Thus,When neurorrhaphy is not possible, another possible alternative is nerve grafting. The graft can be an autograft, from the person's own nerve, an allograft, from another person with a high risk of rejection, or a xenograft from another species that, in addition to the risk of rejection, also presents a risk of contracting diseases.

[0029] Therefore, autografting has been considered the gold standard in treatments (LI et al., 2014; RAY; MACKINNON, 2010), since it presents better results in nerve regeneration when compared to other methods. One of its main advantages is the avoidance of immunological rejection, since it provides a microenvironment of the native nerve.

[0030] The nerves used are the sural nerve (lower part of the calf), the most commonly used, intercostal, peroneal, cutaneous, lateral thigh nerves, and others (NORKUS; NORKUS; RAMANAUSKAS,2005). Despite presenting satisfactory results, there are numerous disadvantages to applying the autograft.

[0031] Among them are: i) morbidity at the donor nerve site; ii) possibility of sensory loss in the donor region; iii) potential for infection and neuroma formation; iv) restricted supply; v) incompatible size; vi) need for multiple surgical procedures and vi) limited to gaps of less than 5 cm (CHIONO; TONDA-TURO, 2015; DALY et al., 2012; KEHOE; ZHANG; BOYD, 2012; MOORE et al., 2009).

[0032] It is important to emphasize that the patient recovery rate is only 50% (PABARI et al., 2010), with less than 25% significantly recovering motor function and less than 3% recovering their sensitivity, according to Marquardt and Sakiyama-Elbert (LIN; MARRA, 2012; MARQUARDT; SAKIYAMAELBERT, 2013).

[0033] All these limitations drive the search for alternatives that enable the recovery of neural injuries that can be applied to larger injured areas,and that do not pose a risk of rejection for the patient. Thus, the neural guide conduit appears to be an alternative with great potential to meet these needs.

[0034] A neural guide conduit (NGC) can be defined as a cylindrical structure made from natural or synthetic polymers, that is, a tube-shaped object, with the main objective of causing the alignment of axonal sprouts, through topographical cues, to occur the reinnervation of the nerve and thus aid in its regeneration (VIJAYAVENKATARAMAN, 2020).

[0035] Furthermore, these tubes are approved by the FDA – Food and Drug Administration as tissue engineering devices developed to fill gaps in the injured nerve and provide structural and trophic support for it (DURGAM et al., 2010).

[0036] Its main advantages over conventional treatments are unlimited supply,reduced donor site morbidity rate and the need for only one surgery (SANTOS et al., 2017). For this reason, they are excellent candidates to replace them, in addition to being possible to manufacture tubes with specific designs, in order to meet the necessary requirements of an ideal tube and thus ensure the effectiveness of nerve regeneration. Such requirements are: biocompatibility, biodegradability, sufficient mechanical strength to provide structural support, permeability to ensure trophic support, internal structure that mimics the nerve architecture (biomimetic architecture), resources to longitudinally align axons, and addition of biomolecules that increase the nerve's growth and regeneration capacity (SALMORIA et al., 2016; VIJAYAVENKATARAMAN, 2020). Biocompatibility is an extremely important criterion, since it avoids immunological rejection, inflammation, and morbidity.In other words, the material is capable of performing its function without provoking any undesirable reactions in the body. Biodegradability, in turn, refers to the rate at which the tube will degrade within the body. It must present an optimal degradation rate sufficient for such application, being slow enough to allow cell growth, but fast enough to avoid the need for another surgery for removal and not impede the regeneration process (BOSWORTH; DOWNES, 2010; KEANE; BADYLAK, 2014; OLIVEIRA et al., 2004). Furthermore, the degradation products must not pose risks to the body or any reaction and must not cause significant swelling of the material (DOMINGUES, 2017). In addition to biodegradability, the tube must have mechanical properties compatible with the surgical process (suturing and manipulation), being resistant to compression and traction, as well as during patient treatment, in relation to its displacement and movement. However,It must also present a certain flexibility in order to avoid mechanical irritation.

[0042] Mechanical properties similar to human nerves present an elastic modulus ranging from 8 to 16 MPa, a breaking stress of 6.5 to 8.5 MPa, and a breaking elongation between 0.6 and 1.6 mm / day (CHIONO; TONDA-TURO, 2015). Thus, the conduit must provide structural and mechanical support corresponding to the native nerve (DOMINGUES, 2017).

[0043] Another support that must be guaranteed is trophic support through permeability. This promotes the transport of oxygen and nutrients into the canal and waste out. The average pore diameter and porosity must be small enough to prevent the entry of inflammatory cells and fibroblasts, but cannot be so small as to prevent the transport of nutrients and oxygen. In order to guarantee this requirement, the ideal parameters are from 5 to 20 µm up to a maximum of 30 µm in diameter and porosity of 60 to 80% (CHIONO; TONDATURO,2015; KEHOE; ZHANG; BOYD, 2012; KOKAI et al., 2009).

[0044] The porous tube design is necessary to meet this criterion. In this sense, the internal structure of the tube must mimic the nerve architecture and to meet this need, there are tubes with micro grooves, with porosity or even intraluminal channels or filled with intraluminal fibers.

[0045] In addition to topographic cues, it is also possible to provide biochemical cues through the addition of biomolecules such as neutrophil factors, growth factors or conductive substrates, or even biological cues offering supporting cells such as Schwann cells.

[0046] Thus, tubes are designed and manufactured with the purpose of providing topographic and biochemical cues. This is because the biggest challenge for a tube is to provide adequate alignment for the formation of Bungner Bands (DALY et al., 2012; MARQUARDT; SAKIYAMA-ELBERT,2013). Since the absence of topographical clues causes inadequate alignment and consequently hinders cell proliferation and the formation of the myelin sheath (DOMINGUES, 2017).

[0047] There are different tube models, and their microstructure significantly affects cell organization and proliferation, thus influencing nerve regeneration (GHORBANI; ZAMANIAN; NOJEHDEHIAN, 2017; WANG et al., 2015). Among the currently existing tubes, they can be hollow and non-porous; with or without grooves; porous; multichannel and with or without internal filling (VIJAYAVENKATARAMAN, 2020).

[0048] The primitive model was a cylinder with an empty internal space (lumen), which, despite being very easy to manufacture, has many limitations, such as low permeability of nutrients and oxygen, which hinders nerve regeneration.

[0049] Although the hollow model is easy and repeatable to manufacture,lacks topographical clues and does not allow the permeability of nutrients and growth factors (VIJAYAVENKATARAMAN et al., 2018).

[0050] In order to improve this limitation, modifications to the internal and external surfaces of the tubes have emerged. Examples of this include tubes with microgrooves, in which the grooves are expected to aid in neurons' alignment. However, this design has a more complex geometry, making its manufacture difficult.

[0051] Furthermore, although the tube with microgrooves favors directional alignment of neurons (NI et al., 2013; WANG et al., 2015), because it lacks pores, mass transfer is hindered. Thus, the porous tube emerges, facilitating the transport of mass and nutrients through the pores.

[0052] In turn, the porous tube also presents difficulties in its manufacture,regarding pore size control. This limitation can be addressed with 3D printing.

[0053] Its porous surface provides excellent trophic support, enabling efficient mass and nutrient transport. In addition to these models, the multichannel model with intraluminal channels has also emerged, which has been one of the most popular designs, with the work of Moore et al. (2006) being one of the pioneers. These channels have the ability to aid directional alignment. However, these designs lack reproducibility and repeatability. For this reason, tubes have emerged in which, instead of intraluminal channels, the lumen is filled with hydrogel or electrospun fibers.

[0054] Multichannel models (JEFFRIES; WANG, 2013) and with internal filling (HUANG et al., 2018) have also emerged, which can be with hydrogel or fibrous, both of which are explored to provide different topographic cues (VIJAYAVENKATARAMAN et al.,2018). An example of these models is the study by Huang et al. (2017), which obtained significant results from tubes with internal filling.

[0055] These models can be manufactured by different techniques, such as solvent casting (FREGNAN et al., 2016), gas formation (BHATI; BHATNAGAR, 2017), phase separation, freeze-drying (GHORBANI; ZAMANIAN; NOJEHDEHIAN, 2017), and electrospinning (VIJAYAVENKATARAMAN et al., 2019).

[0056] The hydrogel tube provides a favorable environment for cells in which it is possible to incorporate various biochemical and biological cues. However, there is an absence of topographic cues for directional alignment, and its fabrication has two steps. On the other hand, filling with fibers or so-called intraluminal guides,also have two manufacturing steps. However, their fibers function as guides for the directional alignment of neurons. Furthermore, it is interesting that the fibers are aligned rather than random, since aligned fibers provide even more topographical clues, favoring nerve regeneration, according to studies in the literature (KIM et al., 2008).

[0057] After insertion of the tube, it is expected that through it, the axons of the proximal nerve stump, from the injured site, will regenerate to the distal stump (BRUSHART, 2011). This process occurs in five phases: the fluid phase; the matrix phase; the cell migration phase; the axonal phase; and the myelination phase.

[0058] In the first phase, neurotrophic factors and extracellular matrix components are released from the plasma, composing the fluid phase that reaches a maximum concentration.

[0059] Then, in the matrix phase,A fibrin network is formed from ECM molecules. This network acts as a bridge through which Schwann cells begin to migrate, known as the cell migration phase. Along with it, endothelial cells and fibroblasts in the middle form a new network of cellular filaments. After this formation, the fibrin filaments decompose (BRUSHART, 2011).

[0060] It is in the fourth phase, the axonal phase, that Schwann cells begin to proliferate and align to form the band of Bungner, providing topographical cues suitable for neuron growth. The main function of this band is to guide axonal sprouts to the nerve end.

[0061] Finally, the myelination phase is characterized by the wrapping of specific Schwann cells around the axons, forming a myelin sheath. Only with myelinated axons is functional recovery possible (DIXON et al., 2011).2018).

[0062] It is important to emphasize that these phases follow the model of a mouse with non-critical injury and a spacing of 10 mm (DALY et al., 2012).

[0063] The choice of material to be used in a scaffold is one of the most important tasks in tissue engineering, since it directly influences the biocompatibility, biodegradability and mechanical properties required for the specific application (ARSLANYILDIZ; ASSAL; CHEN, 2016; YING; FUH; LU, 2019; ZHANG et al., 2019).

[0064] There are two main groups of materials used to manufacture tubes: natural and synthetic polymers. Among natural polymers, collagen, gelatin, thrombin, fibrinogen, cellulose, chitin, and chitosan stand out.

[0065] The main characteristic of natural polymers is their biocompatibility with cells, since they provide a biomimetic and favorable environment for them. On the other hand, they present very unsatisfactory mechanical properties. Conversely,Synthetic polymers have excellent mechanical properties, but very low cellular affinity. For this reason, mixing both types of polymers is a very interesting strategy and widely used in the literature (SIONKOWSKA, 2011).

[0066] Examples of biodegradable polymers of synthetic origin most used in the manufacture of tubes are: polycaprolactone (PCL), poly (lactic acid) (PLA), poly (L-lactic acid) (PLLA), polyurethane (PU) and poly (lactic acid-co-glycolic acid (PLGA). There are also non-biodegradable materials such as silicones, polystyrene, methacrylate-based hydrogel and polytetrafluoroethylene (ARSLANTUNALI et al., 2014).

[0067] Polycaprolactone (PCL) is one of the most promising materials and one of the most reported in studies for tube manufacturing, in addition to being widely used from the area of ​​bone regeneration to the regeneration of nervous tissue (LI; TAN, 2015).hydrophobic and with thermal properties of 350ºC (decomposition temperature) and -60ºC (glass temperature) (JIANG; ZHANG, 2013).

[0068] The mechanical properties vary from 200 to 440 MPa for the modulus of elasticity, 20 to 42 MPa for tensile strength and 120 MPa for compressive strength. Its molar mass varies from 5000 to 80000 Da, affecting crystallinity, since the lower the mass, the greater its crystallinity (DUMITRIU; POPA, 2013).

[0069] Polycaprolactone is obtained through ring-opening polymerization of ε-caprolactone.

[0070] There are several advantages in using Polycaprolactone (PCL) for manufacturing tubes, including excellent biocompatibility, with the FDA considering it one of the polymers that causes the least undesirable reactions (PAGGI, 2014; GONÇALVES, 2012); it is a biodegradable polymer, and the released products are non-toxic and, therefore, have low immunogenicity (LI; TAN,2015); it is a polymer approved by the FDA (GONÇALVES, 2012); with high solvent solubility and low temperature processability; it has good mechanical properties (LACKINGTON; RYAN; O'BRIEN, 2017); and presents adequate rheological properties that allow its use in 3D printing (WOODRUFF; HUTMACHER, 2010); it is very versatile for molding and is compatible with several drugs (DASH; KONKIMALLA, 2012) because it presents high permeability for medicines (LI; TAN, 2015); It presents excellent miscibility properties, low viscosity and mechanical compatibility with other polymers, being, therefore, an excellent candidate for the formation of blends (RAMOS, 2011).

[0071] In addition, another widely used material is polyurethane (PU).

[0072] PU can be synthesized from a polyol, a di- or multiisocyanate, and a chain extender, so that it presents hard and soft segments. Its structure provides different properties, such as toughness, durability,biocompatibility and biodegradability, high mechanical strength facilitating cell adhesion and migration, which makes it an excellent candidate for fiber production (FERNANDO; MCENERY; GUELCHER, 2016).

[0073] Furthermore, PU can present low cytotoxicity, high oxygen permeability, low interfacial tension and mechanical properties compatible with natural tissues (NAUREEN et al., 2021).

[0074] Thus, PU has a range of biomedical and pharmaceutical applications, as well as the development of scaffolds for both soft and hard tissues, being able to incorporate proteins, drugs and biomolecules in PU fibers (NAUREEN et al., 2021; PEREIRA RODRIGUES et al., 2020).

[0075] Another application is that PU can be obtained from renewable sources such as biomass (GABRIEL et al., 2017; WENDELS; AVÉROUS, 2021).

[0076] Regarding the techniques employed in the manufacture of the neural guide conduit,there is rotospinning and additive manufacturing.

[0077] The rotary jet spinning technique, also known as rotospinning (RJS), emerged as an alternative to overcome some limitations of electrospinning, these being the low productivity rate and the need for high voltage (BADROSSAMAY et al., 2014; STOJANOVSKA et al., 2016).

[0078] Furthermore, this technique has great potential because it is economical, because it presents safety during production since the use of high voltage is replaced by centrifugal force for fiber generation and because it still has high productivity, around 50 times greater than electrospinning (ROGALSKI; BASTIAANSEN; PEIJS, 2017).

[0079] The RJS device consists of a spinneret connected to a nozzle into which the polymer solution is inserted; a reservoir with two opposite poles coupled to the motor shaft with controllable rotation speed and a collector to collect the fibers (ZHANG et al.,2019).

[0080] The fiber production process basically involves three main steps: i) jet initiation, which induces a flow through the orifice; ii) jet extension and elongation, increasing the surface area; and iii) solvent evaporation to solidify the jet and consequently form the fibers (BADROSSAMAY et al., 2010; STOJANOVSKA et al., 2016).

[0081] In the first step, the polymeric material is injected into the spinneret at high speeds, in liquid or molten form, and is thus exposed to a viscous, gravitational force, surface tension, and centrifugal force. The latter force acts by pushing the polymer solution toward the orifices. Only when this force exceeds the surface tension and viscosity of the solution is the polymer jet expelled from the orifices. This force is directly dependent on the angular velocity of the chamber, the mass of the polymer, and the radius of the chamber.as indicated by Equation I: ^^ ^^ = ^^ ^^ 2 ^^ ^^ (Equation I)

[0082] According to the equation, when the angular velocity reaches a critical value, the centrifugal force overcomes the surface tension of the solution and thus the polymer jet is expelled from each nozzle. That is, as the angular velocity of the spinneret increases, a drop begins to form and only reaches the Taylor cone shape when the velocity exceeds a critical value, providing the initiation of the jet. Thus, after the jet exits, the second stage is initiated.

[0083] In turn, in the second stage, the elongation forces affect the jet when it leaves the orifice (in other words, when the fingertip is formed).

[0084] These forces are: inertia force, air resistance, drag force, Coriollis force, viscous force and centrifugal force (PADRON et al., 2013; ZHANG et al.,2019).

[0085] Thus, the jet travels a curved path, and under the resulting forces, the jet continues to be stretched. Then, it is elongated to a distance at which the forces reach equilibrium.

[0086] Simultaneously with the stretching process, the solvent evaporates from the polymer solution, causing the jet to solidify and contract. The evaporation rate can be determined by the volatility of the solvent, in which a very volatile solvent will make the fibers thicker, since it provides rapid solidification and consequently makes it difficult to extend the jet (BADROSSAMAY et al., 2010).

[0087] Then the third stage consists of fiber formation, in which the distance between the orifice and the collector is an essential parameter that influences the final result (STOJANOVSKA et al., 2016).

[0088] This is because if the distance is too small, there will not be enough time for complete evaporation and, therefore,will not form the fiber. Finally, the fiber reaches the collector where it is removed.

[0089] There are numerous parameters that influence fiber production from RJS. They can be divided into two categories: equipment parameters (nozzle angular velocity, nozzle diameter and direction, and distance from the nozzle to the collector); and solution parameters (surface tension, viscosity, solution concentration, and solvent evaporation rate).

[0090] Regarding equipment parameters, the angular velocity must exceed the critical value to overcome the surface tension and viscous force of the polymer solution. This is because with a sufficiently high angular velocity, the polymer solution will acquire sufficient centrifugal force to form the jet (STOJANOVSKA et al., 2018). However, the angular velocity must not be so high to prevent the fibers from disconnecting and forming beads (ZHANG et al., 2019).

[0091] Furthermore, the velocity influences the fiber diameter, that is,As the speed increases, the fiber diameter decreases. A typical example of this was demonstrated by the work of LU et al. (2013), in which they observed a reduction in the diameter of polyacrylonitrile (PAN) nanofibers with increasing speed.

[0092] Furthermore, the parameters related to the nozzle (its shape, direction, and diameter) affect the initial jet shape and fiber trajectory (FANG et al., 2016). There are several studies investigating how the orifice curvature affects the fiber diameter. One of them is the work of Padron et al. (2013), which investigates the most suitable angles for fiber production. While the nozzle diameter directly affects the flow of the polymer solution and the initial jet diameter (LU et al., 2013), for a small nozzle diameter, the flow rate will be lower and, therefore, makes nanofiber fabrication easier (ZHANG et al., 2019; ZOU, 2014). However, decreasing the diameter too much causes an increase in the viscous force of the solution. Thus,The nozzle diameter must be optimized for more suitable fiber production.

[0093] The distance from the nozzle to the collector is an essential parameter, since it determines the flight time of the polymer jet and, consequently, can affect the fiber morphology. Therefore, there is a minimum distance to provide sufficient time for solvent evaporation before reaching the collector (LU et al., 2013; ZHANG, ZM et al., 2019). Increasing this distance will increase the polymer jet's flight period and, consequently, may lead to a reduction in fiber diameter (ZHANG et al., 2018). However, if it increases too much, the fibers become contorted and brittle.

[0094] Regarding the solution property parameters, many intrinsic properties of the polymer solution significantly affect the fiber morphology and structure.

[0095] Such properties are: surface tension, rheological behavior, polymer molecular weight, solution concentration,solvent and additive.

[0096] In general, molecular weight, concentration, and solvent influence the fiber production process, modifying rheological behavior, especially viscosity, and surface tension (LU et al., 2013).

[0097] Surface tension acts mainly to reduce the surface area of ​​the jet, thus tending to transform it into droplets and consequently causing instability (LU et al., 2013; MAHALINGAM; EDIRISINGHE, 2013).

[0098] Since the intention in the RJS process is to stretch and lengthen the jet, increasing the surface area, its reduction is undesirable. For this reason, the centrifugal force is so necessary to overcome surface tension.

[0099] The concentration of the solution directly affects the surface tension; as the concentration increases, the surface tension of the polymer solution increases.

[0100] Furthermore, the concentration of a polymer solution affects the fiber diameter. According to studies,as the concentration increases, the fiber diameter increases (LU et al., 2013; ZHANG et al., 2019). Other parameters must also be taken into account, such as the solvent evaporation rate.

[0101] During the jet stretching process, solvent evaporation occurs simultaneously, affecting the fiber diameter (STOJANOVSKA et al., 2016). Therefore, when the evaporation rate is low, the solvent does not evaporate completely, and the fibers become a thin film, while for high rates, the fiber diameter increases (STOJANOVSKA et al., 2016).

[0102] Furthermore, the present invention also reveals tubes made by the additive manufacturing technique. In the late 1980s, additive manufacturing (AM), commonly known as 3D printing, was defined as a manufacturing process developed by the addition of successive layers of material,through information extracted from a 3D computational geometric design.

[0103] The main steps of 3D printing consist of: i) obtaining the 3D geometric model of the part from CAD design software generated in STL format; ii) slicing the 3D model, that is, obtaining the 2D contour lines that will define in each layer where the material will be added, thus providing the path for the printer; iii) manufacturing the part in the 3D printer; and iv) post-processing, which involves finishing and cleaning steps (VOLPATO, 2017).

[0104] One of the greatest advantages of this process is the production of customized scaffolds in different shapes and complexities, still with high precision and reproducibility (PATI et al., 2015).

[0105] It can be used with different materials, in addition to being fast when compared to other manufacturing processes. Limitations of this process include the surface finish of the part,which is inferior to conventional processes; the material can often be limited depending on the technology chosen.

[0106] Furthermore, considering large-scale application, additive manufacturing is still considered slow and expensive, but for biomedical and aerospace applications, which are highly customized, additive manufacturing has been growing increasingly (VOLPATO, 2017).

[0107] Regarding fused deposition modeling and process parameters, extrusion printing involves two main techniques: Fused Deposition Modeling (FDM) and the extrusion of liquid gelling material. This technique requires the use of a thermoplastic which is fed into the extruder head in the form of a polymer solution, or filament, to be molten.

[0108] Then, through pneumatic pressure,The material is extruded through the printer nozzle, hardening after being dispensed so that the next layer can adhere to the previous one. One of the limitations of this technique is the low resolution (around 200 µm) (PATI et al., 2015) and the requirement for high-viscosity inks.

[0109] The process parameters that affect 3D printing are pressure, temperature, and material type. While the design slicing parameters, such as printing speed, layer height, and nozzle diameter and type, also influence the final print result. The print head speed must be chosen according to the material's solidification time during extrusion. That is, it cannot be so fast that the first layer does not have time to dry, but it cannot be so slow that the next layer cannot adhere. Therefore, the bottom layer must be practically solidified to ensure the material's support.since the absence of this control can cause a collapse in the scaffold geometry (PEREIRA, 2014).

[0110] While pressure is related to the volumetric flow of the material, in which a higher pressure can increase the flow of the material, modifying its deposition and consequently the morphology of the scaffold.

[0111] An example of this is the porosity of the structure, which can be impaired when there is an increase in the amount of material deposited layer by layer (SÁNCHEZ; D'ÁVILA, 2014).

[0112] Finally, the layer height is strictly related to the nozzle diameter. Once the type of nozzle to be used (conical or straight) has been defined, the choice of its diameter is essential to set the layer height. The smaller the dimension of the scaffold produced, the use of smaller diameters (larger gauge) is recommended. The layer height should be proportional to the nozzle diameter.

[0113] Furthermore, scaffolds can be manufactured with or without the incorporation of drugs, for example,anti-inflammatories, which aid in the recovery of the injured nerve.

[0114] Ibuprofen (IBU) is classified as a non-steroidal anti-inflammatory drug (NSAID), thus presenting analgesic, antipyretic and anti-inflammatory properties (MILES, 2007).

[0115] It is generally used in the treatment of pain, fever, inflammation and also in rheumatoid arthritis, osteoarthritis and others (CRIELAARD et al., 2012; MILES, 2007). The molecular structure of IBU presents two isomers: (R)-IBU and (S)-IBU, and commercially it has a racemate of both isomers, with the (S) isomer being the only biologically active one (SHANKLAND et al., 1997).

[0116] The anti-inflammatory use of IBU in nerve regeneration can be seen as an essential key to its recovery. Since during Wallerian degeneration, in addition to blood-brain barrier dysfunction and brain reorganization,there is also an induction of a very acute inflammatory response from the release and production of cytokines that maintain an inflammatory state at the injured site (CAMARA-LEMARROY et al., 2012).

[0117] Especially the inflammatory mediator nitric oxide when in excess can be detrimental to regeneration (MCDONALD et al., 2007).

[0118] However, it is important to emphasize that this inflammation plays a crucial role in triggering the regenerative process, at the same time that if these inflammatory processes are not controlled they may prevent regeneration from occurring, as mentioned in different studies (BENOWITZ; POPOVICH, 2011).

[0119] Furthermore,There are studies using IBU to improve nerve regeneration. FU et al. (2007) demonstrate that the use of the anti-inflammatory drugs IBU and indomethacin promote axon regeneration through the inhibition of RhoA (Ras homolog family member A).

[0120] SALMORIA et al. (2016) developed tubes incorporated with 15% IBU, which can be used in bone regeneration and its inflammatory control.

[0121] It is estimated that IBU remains in the body for only around 2 hours, which entails the need for frequent use of this drug (DENG et al., 2016). Consequently, there will be many side effects such as stomach ulcers, bleeding, and other symptoms due to the high frequency of use of the drug.

[0122] Therefore, it is extremely important to look for a drug delivery system that ensures a controlled and on-target release profile, in order to obtain the desired therapeutic effect.

[0123] Among the existing administration systems,The use of micro and nanofibers has great potential for drug delivery (KAJDIČ et al., 2019; ZHAO et al., 2015). In addition to presenting specific nanoscale characteristics and functionality, they can be used in different administration routes (oral, topical, transdermal, and transmucosal) and also protect the drug from degradation before reaching the target in the body (HU et al., 2014; KAJDIČ et al., 2019).

[0124] Nanofibers can be classified according to the drug release mode, being immediate release, prolonged release, biphasic release, and stimulus-activated release.

[0125] For immediate drug release, nanofibers can be used with a simple and homogeneous structure in which the drug is added to the polymer or a mixture of polymers. Because it has a high surface-to-volume ratio, its contact area for dissolution is larger and consequently provides a more immediate release,in addition to having high porosity (BRAGHIROLLI; STEFFENS; PRANKE, 2014).

[0126] On the other hand, to obtain nanofibers with a more prolonged release, they must develop a more complex structure. One of the alternatives is core-shell nanofibers, which have multiple layers, with the outermost one acting as a barrier to control the drug release rate (ACEVEDO et al., 2018; GO et al., 2020). Or even, a sandwich-type fibrous mesh is also an excellent alternative successfully reported in the literature (MAO et al., 2021), as well as the incorporation of the drug in microspheres or control of granules in its structure to prolong drug release (LI et al., 2017; PARIS et al., 2015; TAYLOR et al., 2020).

[0127] In addition, there may be biphasic release or activated by pH or temperature stimulation and may also have an electroresponse (ELASHNIKOV et al., 2017; YU et al.,2016).

[0128] The literature reports the need for a more prolonged release for the delivery of ibuprofen in different applications, that is, to reduce the initial burst during drug release.

[0129] In the studies by Mao et al. (2020), a sandwich scaffold formed by electrospun PCL / PLGA nanofiber fibers incorporated with IBU was produced, in which the main objective with this type of scaffold was to reduce the initial burst of drug release to develop a barrier with high anti-adhesion capacity.

[0130] While in the studies by Taylor et al. (2020), a bilayer delivery system was developed, in which PLGA microspheres with IBU were sandwiched between two PCL nanofiber scaffolds, thus delaying IBU release by 70%.

[0131] Thus, the need to reduce IBU release is extremely relevant, since rapid release is detrimental to healing. Due to a faster than desired release,In the work of Paris et al. (2015), IBU was encapsulated in chitosone spheres and incorporated into a three-dimensional porous scaffold made of apatite / agarose for application in bone regeneration.

[0132] This showed a significant improvement in drug release, reducing the “burst effect,” in which complete drug release decreased from 3 hours to more than 2 days, making it more compatible with clinical application.

[0133] The fabrication of scaffolds for the recovery of injured nerves is widely studied in the literature; however, existing techniques have inherent limitations in their process, and they all share some problems such as the inability to control pore size, porosity, and interconnectivity; lower repeatability; and no multilayer structure.

[0134] The study by Wang, Y. et al. (2021), entitled “THE PROGRESS OF BIOMATERIALS IN PERIPHERAL NERVE REPAIR AND REGENERATION”, published in Journal of Neurorestoratology,reveals information about peripheral nerve repair and regeneration. The paper describes how these materials can be used as nerve repair membranes and nerve conduits in the field of peripheral nerve repair. Furthermore, it describes how several nerve conduits are functionalized with drug delivery capabilities for peripheral nerve regeneration. However, these studies present materials that are not customized based on the type of nerve injury, in addition to not presenting modulation of the fiber that incorporates the anti-inflammatory drug.

[0135] The paper by Rodrigues et al. (2019), entitled "Polyurethane Fibrous Membranes Tailored by Rotary Jet Spinning for Tissue Engineering Applications," published in the Journal of Applied Polymer Science,reveals the roto-spinning (RJS) process combined with a polymer as a strategy for designing RJS-polyurethane (RJS-PU) membranes produced with different microstructures. The document concludes that the combination of PU and RJS was effective for producing fibrous structures for tissue engineering applications, demonstrating good compatibility with the cultured osteoblastic cell line. Thus, this document focuses on the field of bone tissue engineering and does not demonstrate application in repair of peripheral nerve injuries. Furthermore, although this document presents aligned PU fibers, it does not mention drug incorporation, nor does it mention fiber modulation for controlled drug release or Schwann cell colonization.

[0136] Patent application BR 112020 025527 0 discloses the use of a copolymer composed of, inter alia, polycaprolactone used to produce membranes,3D structures and hollow tubes that can be used in biomedical devices for drug delivery and peripheral nerve repair and regeneration. The aforementioned document also describes the use of copolymers in regenerative medicine, more specifically as nerve guides. However, document BR 112020 025527 0 uses a copolymer, that is, a mixture of polymers, a single final polymer structure. Furthermore, the patent application requires that the copolymer be photopolymerized for chemical modification. The application also requires an additional step for pore formation, through the addition of porogenic agents. Although the patent application mentions the use of drugs, it is understood that this occurs through drug encapsulation, rather than the drug being incorporated into the polymer fibers.

[0137] US patent 10,405,963 B2 describes the characteristics of a process for manufacturing a 3D-printed tube.using polycaprolactone as a raw material in the manufacture of a neural conduit, associated or not with the use of drugs, to be allocated in an injured region of a neural pathway. However, the aforementioned patent US 10,405,963 B2 does not disclose polymeric fibers filling the tube containing an anti-inflammatory drug, with a larger surface area and aligned structure that promotes greater adhesion and proliferation of neural cells.

[0138] The article Vijayavenkataraman et al (2018), entitled “ELECTROHYDRODYNAMIC JET 3D PRINTED NERVE GUIDE CONDUITS (NGCS) FOR PERIPHERAL NERVE INJURY REPAIR”, published in Polymers (Basel), refers to porous three-dimensional neural conduits based on polycaprolactone, manufactured using electrohydrodynamic jet 3D printing (EHD-jetting), where the structure is in a layer-by-layer format. The document uses a polycaprolactone solution in glacial acetic acid at a concentration of 70% (w / v) and describes its use as a neural conduit. However,This document does not provide topographical clues for cells to adhere and proliferate. Furthermore, it requires an EHD Jetting apparatus that needs to be adapted to the printer used in this article. This adapted system does not provide printing precision because it is not pneumatic, so the pores lack precision. Furthermore, the article requires the installation of an electrical voltage source, which poses risks to the process / operator. Another disadvantage is the printing of a mesh that then needs to be folded, resulting in an additional processing step and contributing to the loss of precision and accuracy in the printed pores.

[0139] US Patent 2013,110,138 B2 discloses a three-dimensional scaffold shaped from polymers, as well as its method of fabrication and use in the regeneration of nerve or spinal cord injuries. The document reveals that the scaffold can be curved into the shape of a conduit, in which the conduit has an external profile and an internal profile, and,Additionally, the conduit contains therapeutic agents, such as ibuprofen. However, the patent requires that the fibers be curved perpendicular to the conduit. Furthermore, the aforementioned patent requires the use of an electrical voltage source, and the polymer solution must conduct electrical current to be ejected by the system, which differs from the present invention. In turn, the present invention presents an external conduit (tube) customized according to the patient's lesion and with pores of the same diameters. The internal conduit can be filled with fibers aligned in any direction. Neither the use of electrical voltage nor a conductive solution is required. Furthermore, the fiber production speed / flow rate is much higher than that of the aforementioned patent. The fibers have larger diameters. Furthermore, the present invention proposes fibers with or without drug incorporation, that is,The drug is not on the outer surface of the conduit. This difference allows for a more prolonged and controlled drug release.

[0140] The article by Cheng et al. (2020), entitled "3D structured self-powered PVDF / PCL scaffolds for peripheral nerve regeneration," published in Nano Energy, presents porous scaffolds composed of PVDF / polycaprolactone (PCL), with applications in tissue engineering. However, the article focuses on electrical conduction properties and presents only the outer tube, without the topographical cues, and without mentioning the incorporation of anti-inflammatories.

[0141] The doctoral thesis by Rui Carlos Castro Domingues (2017), entitled "DEVELOPMENT OF POLY(LACTIC ACID) MEMBRANES FOR APPLICATION AS NEURAL GUIDE CONDUITS," presents the development of auxiliary implants for the treatment of peripheral nerve injuries. However, this document only discloses PLLA membranes in hollow fiber form,and does not incorporate drugs that aid nerve repair.

[0142] The article by Georgiou et al. (2013), entitled "ENGINEERED NEURAL TISSUE FOR PERIPHERAL NERVE REPAIR," published in Biomaterials, presents techniques in the field of tissue engineering, providing methods for the construction of aligned biomaterials. However, the article refers to a liquid material, a hydrogel, whose use promotes cell elongation. The article does not mention the fabrication of a rigid, customizable conduit that does not require additional steps to align the cells.

[0143] The article by Huang et al (2015), entitled “NERVE GUIDANCE CONDUITS FROM ALIGNED NANOFIBERS: IMPROVEMENT OF NERVE REGENERATION THROUGH LONGITUDINAL NANOGROOVES ON A FIBER SURFACE”, published in ACS Appl Mater Interfaces, presents fibrous neural conduits with or without grooves prepared by electrospinning, used in the recovery of peripheral nerves. However,This article presents only a tube-shaped membrane, without showing the aligned polymeric fibers or the incorporation of drugs.

[0144] The article by Jeong in Kim et al (2016), entitled “A CONTROLLED DESIGN OF ALIGNED AND RANDOM NANOFIBERS FOR 3D BI-FUNCTIONALIZED NERVE CONDUITS FABRICATED VIA A NOVEL ELECTROSPINNING SET-UP”, published in Scientific Reports, reveals aligned nanofiber scaffolds used in nerve regeneration. However, the article does not mention the use of drugs in the recovery of nerve injury, presenting only a tube used as a neural guidance conduit.

[0145] Rodrigo Acácio Paggi's (2014) thesis, entitled “MANUFACTURING OF POLYCAPROLACTONE / DRUG DEVICES BY EXTRUSION APPLICABLE IN PERIPHERAL NERVE REGENERATION” presents polycaprolactone (PCL) tubes produced by the extrusion technique, so it does not mention rotospinning or printing by additive manufacturing.

[0146] Both the article by Kim et al (2008),entitled “THE ROLE OF ALIGNED POLYMER FIBER-BASED CONSTRUCTS IN THE BRIDGING OF LONG PERIPHERAL NERVE GAPS”, published in Biomaterials, the article by Panahi-Joo et al (2016), entitled “DESIGN AND FABRICATION OF A NANOFIBROUS POLYCAPROLACTONE TUBULAR NERVE GUIDE FOR PERIPHERAL NERVE TISSUE ENGINEERING USING A TWO-POLE ELECTROSPINNING SYSTEM”, published in Biomedical Materials, and the article by Prabhakaran et al (2008), entitled “ELECTROSPUN BIOCOMPOSITE NANOFIBROUS SCAFFOLDS FOR NEURAL TISSUE ENGINEERING”, published in Tissue Eng Part A, present membranes in the shape of a tube, however they do not present the internal alignment of the polymeric fibers combined with the external tube and the incorporation of a drug.

[0147] The article by Qian et al. (2018), entitled “AN INTEGRATED MULTI-LAYER 3D-FABRICATION OF PDA / RGD COATED GRAPHENE LOADED PCL NANOSCAFFOLD FOR PERIPHERAL NERVE RESTORATION”, published in Nature Communications,presents a method integrated with 3D printing for the fabrication of scaffolds for nerve regeneration, however, it does not reveal fiber alignment or drug incorporation.

[0148] The article by Reid et al. (2013), entitled “LONG TERM PERIPHERAL NERVE REGENERATION USING A NOVEL PCL NERVE CONDUIT”, published in Neuroscience Letters, presents a conduit made of polycaprolactone for use in the repair of nerve injuries. However, the article does not mention the combination of an outer tube and aligned fibers, nor the incorporation of drugs in the neural guidance conduit.

[0149] The article by Wang et al. (2008), entitled “CREATION OF HIGHLY ALIGNED ELECTROSPUN POLY-L-LACTIC ACID FIBERS FOR NERVE REGENERATION APPLICATIONS”, published in Journal of Neural Engineering, presents aligned fibers produced by electrospinning made of PLLA. However,does not present the outer tube or the incorporation of drugs that aid nerve recovery.

[0150] The article by Xie et al. (2014), entitled “NERVE GUIDANCE CONDUITS BASED ON DOUBLE-LAYERED SCAFFOLDS OF ELECTROSPUN NANOFIBERS FOR REPAIRING THE PERIPHERAL NERVOUS SYSTEM”, published in ACS Appl Mater Interfaces, presents membranes manufactured by the double-layer rotospinning technique, to be used as neural guidance conduits, however, the aforementioned article does not mention the incorporation of the drug or the alignment of the internal fibers.

[0151] Thus, in general, the state of the art brings references only to the outer tube, without topographical clues or polymeric membranes in the form of hollow fiber and tube shape. Thus, no prior art document discloses a neural guidance conduit formed from a combination of polymers, which can be customized based on the nerve injury. Furthermore,The prior art does not reveal that these tubes can be incorporated with an anti-inflammatory drug whose prolonged release is more controlled, preventing damage to the nerve injury tissue.

[0152] Therefore, the need to develop technologies capable of being customized based on the injury, which promote the controlled release of drugs, and which can aid in the recovery of nerve injury is undeniable. Brief Description of the Invention:

[0153] Initially, it should be emphasized that the following description starts from the preferred embodiments of the invention, without being limited thereby.

[0154] The present invention describes a porous neural guide conduit, produced from the combination of additive manufacturing and rotospinning techniques from any bioabsorbable polymer, in which the conduit can be incorporated or not with anti-inflammatory drugs.

[0155] The area of ​​application of the neural guide conduit of the present invention is in the field of tissue engineering,in applications and preparations for medical purposes, more specifically, the neural guide conduit can be used in the repair of peripheral nerve injuries.

[0156] The proposed Neural Guide Conduit (CGN) is used in the repair of peripheral nerves and is porous, customized, bioabsorbable, with a drug that helps control the inflammatory process and with an aligned support that acts as a topographic track for cells to adhere and proliferate and for injuries of various sizes.

[0157] Therefore, the object of the present invention is the development of a CGN by additive manufacturing (AM) based on polycaprolactone (PCL) filled with aligned polyurethane (PU) fibers and incorporated or not with ibuprofen, produced using the rotospinning technique, for future applications in peripheral nerve regeneration.

[0158] Thus, the invention relates to a neural guide conduit, in which the conduit is shaped by the combination of polymeric fibers and a tube,in which the polymeric fibers are produced by the roto-spinning technique and the tube is produced by additive manufacturing, in which the tube may or may not contain the presence of a drug that aids in the recovery of the injured nerve.

[0159] Furthermore, the invention comprises the method of manufacturing a neural guide conduit, in which the method comprises the steps of (i) manufacturing the polymeric fibers by the roto-spinning technique, (ii) manufacturing the tubes by additive manufacturing, in which the tube is customizable according to the nerve injury; and (iii) introducing the polymeric fibers to the tube.

[0160] Finally, the invention also comprises the use of a polymeric solution in the manufacture of a neural guide conduit for the treatment and recovery of injured peripheral nerves,in which the conduit can be customizable according to the nerve to be recovered. Brief Description of the Figures:

[0161] To aid in the identification of the main characteristics of the composition of the present invention and its technical results and effects, the figures to which reference is made are presented, as follows:

[0162] Figure 1 shows the SEM image for PU, PU5I, PU10I and PU15I fibers.

[0163] Figure 2 shows the FTIR spectra of the PU membrane, PU10I and pure ibuprofen materials.

[0164] Figure 3 shows the ¹H NMR spectrum of the PU membrane.

[0165] Figure 4 shows the ¹H NMR spectrum of the pure IBU samples.

[0166] Figure 5 shows the NMR spectrum of, 1H in the PU5I sample.

[0167] Figure 6 shows the mass loss curves (A) of the PU membrane and PU5I produced.

[0168] Figure 7 shows the X-ray diffractogram of the PU membrane, PU5I, PU10I, and PU15I fibers.

[0169] Figure 8 shows the IBU release results for the PU5I, PU10I, and PU15I samples.

[0170] Figure 9 shows the effect of PU-Ibuprofen fibers on the cell viability of neuro2a cells.

[0171] Figure 10 shows the 3D modeling prepared for the production of the TSP, TP0.1, and TP0.4 tubes.

[0172] Figure 11 shows SEM images of the TSP tube at a 1 mm scale and 25X magnification.

[0173] Figure 12 shows the X-ray diffractogram of the PCL present in the produced tubes.

[0174] Figure 13 shows the stress-strain curve for the produced tubes (TSP and TP0.1).

[0175] Figure 14 shows the MTT test for TSP, TP0.1 and TP0.4 tubes and the statistical differences were indicated by * (p<0.01).

[0176] Figure 15 shows the final scaffold produced. Detailed Description of the Invention:

[0177] The present invention relates to a porous neural guide conduit, produced from the combination of additive manufacturing and rotospinning techniques from any bioabsorbable polymer, in which the conduit can be incorporated or not with anti-inflammatory drugs.

[0178] The area of ​​application of the neural guide conduit of the present invention is in the field of tissue engineering, applications and preparations for medical purposes, more specifically, the neural guide conduit can be used in the repair of injuries in peripheral nerves.

[0179] The present invention proposes the development of a CGN by additive manufacturing (AM) based on polycaprolactone (PCL) filled with aligned polyurethane (PU) membranes incorporated or not with ibuprofen (IBU).

[0180] The aligned membranes act as topographical cues, favoring the proliferation of neural cells, and the incorporation of IBU helps control the inflammatory process. Additive manufacturing technology is used to produce the conduit, and rotospinning is used to produce fibers.

[0181] The CGN model chosen in the present invention proposes to meet the need for transporting nutrients and growth factors inside the conduit through the pores, using a promising technique for repeatability in their production.

[0182] Furthermore, the present invention also proposes filling with aligned fibers that are capable of promoting directional alignment for the neuron, thus providing topographical cues for it. It also has mechanical properties similar to those of the peripheral nerve.

[0183] Thus, the proposed CGN is porous, customized, bioabsorbable, with a drug that helps control the inflammatory process and with an aligned support that acts as a topographic track for cells to adhere and proliferate and for lesions of various sizes.

[0184] The present invention explores a technique that overcomes existing barriers in the literature: additive manufacturing. Since additive manufacturing has great potential to control the manufacturing process, based on computer modeling, it is thus possible to optimize the parameters of the peripheral nerve manufacturing process.

[0185] Furthermore, the additive manufacturing technique offers compatibility with different types of materials, from nanomaterials to synthetic polymers and biomaterials (DIXON et al., 2018).

[0186] Furthermore, the additive manufacturing technique allows the neural guide conduit to be modeled based on the peripheral nerve injury, which provides better recovery.

[0187] In turn, the present invention proposes the production of the tube from the polymer poly ε-caprolactone (PCL), a biodegradable polymer with excellent biocompatibility, approved by the United States Food and Drug Administration (FDA).

[0188] Poly ε-caprolactone (PCL) has good mechanical properties (LACKINGTON; RYAN; O'BRIEN, 2017), rheological properties that allow its use in AM (WOODRUFF; HUTMACHER, 2010); being very versatile to mold and compatible with several drugs (DASH; KONKIMALLA, 2012).

[0189] In addition to the development of PCL tubes, the present invention also proposes aligned fibers for their internal filling, using the rotospinning technique. Rotospinning allows the production of aligned fibers with the same desired microstructure, and with even greater advantages, such as greater speed, high efficiency and productivity, and lower risk.

[0190] Aligned fibers are desired since they mimic the orientation of the native endoneurium of peripheral nervous tissue, thus promoting the formation of Bungner bands, Schwann cell migration and axon elongation (HUANG et al., 2015; WANG et al., 2015; XIE et al., 2014).

[0191] Furthermore, the present invention proposes that the produced fibers may or may not incorporate drugs, since the fibers are excellent candidates to act as a drug delivery system.

[0192] Thus, the present invention proposes a scaffold produced by the association of a tube and polymeric fibers that meet the need for transporting nutrients and growth factors inside the tube through the pores, from a promising technique for repeatability in their production.

[0193] The invention proposes filling with aligned fibers that are capable of promoting directional alignment for the neuron, thus providing topographical clues for it.

[0194] Thus, the scaffold of the present invention meets the main needs reported in the literature, being a very promising biomaterial to ensure peripheral nerve regeneration.

[0195] An advantage of the present invention is in relation to the incorporation of a drug in the scaffold. Regarding the incorporation of the drug, the present invention provides a better release of the drug, since the drug is retained within the microfiber that fills the printed tube, and as the microfiber is degraded, the drug is gradually released.

[0196] There is also the advantage that the drug is incorporated in an aligned elastomeric polymer, which acts as a topographic track for cells to adhere and proliferate.

[0197] Another advantage of the present invention is that the pores of the printed tube facilitate cell adhesion, tissue regeneration and drug administration.

[0198] Furthermore, because the drug is retained in porous microfibers, its degradation and administration is faster than the drug that is incorporated into a 3D printed structure, due to the larger surface area.

[0199] Therefore, in one embodiment of the present invention, scaffolds were developed from two distinct techniques: polymeric fiber fabrication and tube fabrication.

[0200] In one embodiment of the present invention, the polymeric fibers are produced using the rotospinning technique (RJS). The fibers are made from a polyurethane and chloroform solution, which may or may not contain the addition of an anti-inflammatory. In an additional embodiment of the invention, the incorporated anti-inflammatory is ibuprofen (IBU), which, at the correct dose, controls the exacerbated inflammation that may occur at the injury site.

[0201] The fibers are produced by preparing the polymeric solution and, subsequently, processing the fibers.

[0202] Thus, in one embodiment of the present invention, a PU solution is prepared at a concentration of 2 to 14%, preferably 9% (w / v), in solvent, in which the solvent is chloroform (called PU fibers). This concentration takes into account the viscosity of the solution (PEREIRA RODRIGUES et al., 2020), being adapted for the rotospinning technique. In additional embodiments, other polymeric fibers are prepared by adding 2.5% to 30%, preferably 5, 10 and 15% by mass (w / w) of ibuprofen. The solutions are subjected to ultrasonic agitation for 1 hour, or until completely dissolved, at room temperature and left to rest for 24 hours.

[0203] After this rest, each solution is poured separately into the RJS reservoir, a conical reservoir with four 1 mm diameter holes.To manufacture the fibers, the rotation is set to 6000 rpm, or another rotation that allows fiber extrusion, causing the polymer solution to be extruded through the reservoir holes, and from the evaporation of the solvent, the fibers are formed.

[0204] In another embodiment of the present invention, the tubes are produced from a polycaprolactone (PCL) and dichloromethane solution, using the additive manufacturing (AM) technique, also known as 3D printing.

[0205] For this, it was first necessary to perform 3D modeling of the tubes, then the preparation of the polymer solutions, and finally, 3D printing.

[0206] Thus, in one embodiment of the present invention, a PCL solution in dichloromethane is prepared with a concentration of 0.7 g / mL to 1.5 g / mL, preferably 0.8 g / mL, in which the dissolution occurs without stirring, and then the solution is left to rest for 48 hours, for later printing.

[0207] For processing the tubes, a bioprinter is used, in which the tubes are produced by pneumatic pressure extrusion technology.

[0208] First, it is necessary to model the tubes, according to the peripheral nerve injury. In additional embodiments of the invention, poreless tubes, tubes with pores of 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm can be elaborated, however, without limiting the scope of the invention.

[0209] In one embodiment of the present invention, the tubes can be customizable according to the injury in the nerve to be regenerated.

[0210] In one embodiment of the invention, it is possible to vary the shape of the pores. Thus, in one embodiment of the present invention, the tube can be round or square.

[0211] In one embodiment of the invention, it is possible to vary the value of the inner diameter of the tube. Thus, in one embodiment of the present invention, the tube has an internal diameter of 1.0 to 2.0 mm.

[0212] In one embodiment of the invention, it is possible to vary the value of the external diameter of the tube. Thus, in one embodiment of the present invention, the tube has an external diameter of 1.5 to 3.5 mm.

[0213] Furthermore, in one embodiment of the present invention, it is possible to vary the number of pores in the tube. Thus, in one embodiment of the present invention, the number of pores can vary in the horizontal position and in the vertical position.

[0214] Furthermore, in one embodiment of the present invention, it is possible to vary the spacing between the pores in the tube and even their sequence. Furthermore, in one embodiment of the present invention, the pores can be in an alternating or sequential arrangement.

[0215] In one embodiment of the invention, it is possible to vary the value of the pore diameter. Thus, in one embodiment of the present invention, the pores have a diameter of 0.05 to 0.5 mm.

[0216] In one embodiment of the present invention, the tube manufacturing parameters such as printing speed, layer height, temperature and pressure used can be adjusted according to the 3D modeling of the tube. Thus, in one embodiment of the present invention, the printing speed can vary from 0.8 to 2.0 mm / s, preferably 1.0 mm / s. Furthermore, in one embodiment of the present invention, the height of the printed layer can vary from 0.1 to 0.3 mm, preferably the height of the printed layer is 0.1 mm. Furthermore, in one embodiment of the present invention, the temperature at which the tube is printed can vary from 25 to 100°C, preferably the tube is printed at room temperature, of about 28°C. Furthermore, in one embodiment of the present invention, the tube is printed using a pressure that can vary from 103.4 to 813.6 KPa, preferably, the tube is printed at a pressure of 744.6 KPa.

[0217] In one embodiment of the present invention, the tubes are filled with polymeric fibers, in which the polymeric fibers are introduced into the tubes with the aid of a needle.

[0218] The present invention additionally comprises the use of polymeric solutions in the manufacture of neural guide conduits for the treatment and recovery of peripheral nerve injury.

[0219] Thus, by proposing the combination of polymers for the manufacture of the conduit, the present invention promotes a neural guide conduit that modulates the rates of conduit degradation, drug release and peripheral nerve repair, factors that are major challenges in obtaining the ideal product. Examples: Example 1

[0220] In one example of the invention, polymeric fibers were prepared and evaluated for their morphological characterization (SEM), chemical and thermal characterization (FTIR, NMR, XRD, TGA), biological evaluation (MTT), and drug release (UV-vis).

[0221] The analyzed results were statistically checked by one-way ANOVA, followed by Tukey's post-hoc test, using BioStat software version 3.5 for Windows. Differences were considered statistically significant when probabilities were <0.05 (*p<0.05) and <0.01 (*p<0.01).

[0222] To characterize the fibers, a polyurethane (PU) solution in 9% (m / v) chloroform, called PU fibers, was prepared. In addition to this solution, other solutions were prepared by adding 5, 10, and 15% by mass (m / m) of ibuprofen (IBU), called PU5I, PU10I, and PU15I, respectively.

[0223] The solutions were subjected to ultrasonic agitation for 1 hour at room temperature and then left to rest for 24 hours.

[0224] The solutions were poured separately into the roto-spinning reservoir (RJS) with four 1 mm diameter holes, in which the rotation was set to 6000 rpm.After extrusion of the solution through the orifices and evaporation of the solvent, fibers were formed. The aligned fibers, adhered to the reservoir, were collected.

[0225] The morphological characterization of the produced fibers was performed using a scanning electron microscope (SEM), in which the samples were coated with gold. The results are shown in Figure 1.

[0226] It is possible to observe that aligned and continuous fibers were obtained for all samples, confirming that the reduction in viscosity of the solutions incorporated with IBU was not sufficient to affect fiber production.

[0227] In the PU5I and PU10I samples, some particles were distributed across the surface of the fibers, suggesting they are drug crystals. These results were not observed in PU15I. This suggests that in the fibers with 5 and 10% IBU, the IBU-polymer interaction is lower, and therefore the drug crystals are more adhered to the surface.While the one with the highest IBU concentration suggests a greater interaction and therefore a uniform incorporation of the drug into the polymer, obtaining a homogeneous system and, therefore, resulting in the absence of crystals on the fiber surface.

[0228] Chemical characterization was performed through Fourier transform infrared (FT-IR) spectroscopy by attenuated total reflectance (ATR). 16 scans were performed from 650 to 4000 cm. -1 , with a resolution of 4 cm -1 , at room temperature. From the FTIR analysis it was possible to characterize the PU, PU5I, PU10I and PU15I fibers, and pure ibuprofen. Figure 2 shows the characteristic FTIR peaks of the PU membrane materials, PU10I and pure ibuprofen.

[0229] The most intense IBU bands are at 2955, 1711 and 1231 cm -1which correspond respectively to the asymmetric CH3 stretching, C=O stretching and CC stretching. However, these bands cannot be distinguished due to the overlapping of the polymer signals. The presence of IBU is confirmed in the sample by the peak at 850 cm -1found in the PU10I spectrum, corresponding to out-of-plane CH deformation, which is characteristic of a 1-4 substituted aromatic ring and is not observed in the PU membrane.

[0230] Confirmation of the drug by a more refined technique such as hydrogen nuclear magnetic resonance spectroscopy is necessary. Therefore, in addition to FTIR analysis, the fibers were also characterized by hydrogen (1H) nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR spectra were recorded on a Bruker Nuclear Magnetic Resonance Spectrometer - model avance II 500 MHz, at room temperature.

[0231] For this characterization, the samples to be analyzed were dissolved in deuterated chloroform and transferred to a 5 mm tube, using the following conditions: acquisition time of 3.18 seconds and a spectral window of 20.6 ppm. TopSpin software (version 4.1) was used to obtain the spectra.3 - Bruker) and the chemical shifts (δ) are given in parts per million, referenced to the residual CDCl3 peak as internal references. The ¹H NMR spectra of the samples are presented in Figures 3, 4 and 5. All assignments related to the hydrogen peaks present in the IBU are presented in Table 1. Table 1: Attribution of the hydrogen signals of the IBU structure. Identified structure of Ibuprofen:

[0232] The signals designated as d and e are attributed to the hydrogen of the CH present in the aromatic rings, the hydrogens present in the methyl group are designated by a and g, while the signal represented by c corresponds to the hydrogen of the CH2 between the methyls and the aromatic ring, highlighting the confirmation of the IBU present in the samples.

[0233] It should be noted that the detailed structure of the PU is not known, since the manufacturers do not provide the type of polymer in question, only stating that it is based on an aliphatic polyether. For this reason, the assignment of signals 1 to 6 (whose shifts were around 4; 3.5; 1.7; 1.4 and 1 ppm) becomes uncertain. However, it is evident that the signals referring to the PU present in the spectrum of Figure 3 are repeated in the spectra of PU5I (Figure 5), with the additional signals characteristic of the IBU whose shifts were 7.4; 7.1; 3.7; 2.5; 1.5 and 1 ppm, confirming the presence of the drug in the sample.

[0234] The fibers were also evaluated for their thermal characterization. Thermal characterization was performed using thermogravimetric analysis (TGA) using a Shimadzu TGA-50M equipment. For this purpose, 15 to 20 mg of sample was used in an inert nitrogen atmosphere with a flow rate of 50 mL / min and a heating rate of 10°C / min, from 25°C to 600°C.

[0235] The mass loss curve, as well as the first derivative, are shown in Figure 6. PU presents two main stages of mass loss: the first varies between 270 and 460°C, in which thermal degradation of the rigid PU segments occurs, and the second between 460 and 570°C, which corresponds to the flexible segments. Both stages are observed for the PU membrane and PU5I samples (Figure 6), but a slight temperature reduction is noted in the second stage.

[0236] For IBU, two endothermic peaks are expected: the first at 77°C, which corresponds to the melting point of the IBU most characteristic of the racemic one, but was not observed in the spectrum, and the other peak that corresponds to the boiling point of IBU at 250°C, which was observed.

[0237] In addition, the fibers were characterized by X-ray diffraction, which identifies the crystalline fraction present. The polymeric samples were inserted into the sample holder in the form of membranes and for the IBU sample, the powder was seeded on the glass adhered by a layer of silicone grease (Shin Etsu, HIVAC-G). The equipment used was the Panalytical X-ray Diffractometer, equipped with CuKα radiation emission (λ = 1.540598 Å), at room temperature at 40 KV and 30 mA, with Soler aperture (0.5 and 1). Diffraction patterns were collected at a scan rate of 5° < 2θ < 60°, for 10 seconds, step of 0.008.HighScorePlus software (Malvern Panalytical version 5.1) was used to generate the diffractogram. The results can be found in Figure 7.

[0238] As the polymer is an amorphous compound, it does not present a well-defined peak and therefore an amorphous halo is observed in the PU membrane diffractogram, with the 2θ angle varying around 15 to 30°. In turn, the IBU presents characteristic peaks at 2θ = 6.18°; 12.26; 16.68° and 20.40°. Furthermore, it is possible to note that the peaks appear gradually as the IBU mass in the PU fibers increases, due to the detection limit of the equipment (Figure 7).

[0239] Furthermore, the fibers were characterized regarding drug release. The fibers were weighed at around 15 to 16 mg in triplicate and previously dried under vacuum for 24 hours. They were then inserted into a Foucault tube and 20 mL of PBS solution was added to ensure the sink condition (saturation).Then, they were incubated in a refrigerated chamber with orbital shaking (Shaker) (MA830, Marconi) at 37°C with a rotation speed of 150 rpm. At certain times of 3, 6, 9, 12, 15, 30, 60, 120, 180, 240, 480, 720 and 1440 min, 4 mL of the solution was removed and replaced with an equal volume of fresh PBS. Then, for each interval, the absorbance reading at the wavelength of 273 nm was performed on an ultraviolet-visible spectrophotometer (Q 898U, Quimis). The calibration curve with known IBU concentration was made from 5 dilutions (0.05; 0.10; 0.20; 0.30 and 0.50 mg / mL) and obtained an R² of 0.9992. The results were reported as a cumulative percentage of IBU released. It is important to emphasize that all calculations took into account the actual amount of IBU in the fibers, that is, the process yield.

[0240] To determine the yield, the drug was quantified.For this, 50 mg of the sample was weighed in triplicate and 20 mL of PBS was added, maintaining high agitation (300 rpm). On different days, aliquots were removed (and replaced with fresh PBS) and the absorbance at 275 nm in the UV-VIS was measured until it became constant, which reached the maximum amount of drug in that sample.

[0241] Thus, it was possible to observe from the release curve (Figure 8) that the proportions of IBU incorporated in the fibers resulted in around 40 to 55% release during the first minutes, with no significant difference between them. However, it is observed that while the PU5I and 10I samples release up to 98% of IBU, PU15I only releases up to 85%.This difference suggests that for a much lower percentage of IBU (up to 10%), the drug is weakly absorbed on the surface of the fibers, resulting in a greater drug release. While for the 15% IBU, which has 3 times more IBU than PU5I, hydrogen bonds occur between the IBU-polymer, keeping the drug more trapped in the fibers and therefore not completely released. This result corroborates the SEM images found.

[0242] Therefore, both PU5I, 10I, and 15I samples showed burst-type release in the first period and then remained relatively slow until becoming complete after 1440 min.

[0243] The burst-type release profile can lead to toxicity for the patient in this type of application, therefore, in the literature there are different alternatives to achieve a more controlled release, thus promoting a sustainable release over a period of time.

[0244] Furthermore, in vitro cell culture is also evaluated.For this analysis, mouse neuroblast cells (neuro2a) are used to perform biological tests considering the neural application of the fibers. The Neuro2a cell line was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin (Life Technologies, Inc., Carlsbad, CA, USA) in a humidified atmosphere containing 5% CO2 at 37°C.

[0245] Furthermore, to characterize the fibers, their biocompatibility was evaluated, based on the invention, using the MTT assay with neuro-2a cells. The cytotoxicity of the produced fibrous and tubular scaffolds was verified by a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Neuro-2a cells were cultured in 96-well plates at a density of 7.5x103 cells per well in 100 μL of medium for 24 hours at 5% CO2 and 37°C.The scaffolds (discs with an average diameter of 5 mm) were sterilized overnight with ultraviolet (UV) light. They were then added to the wells containing the cells. Subsequently, both the scaffold-treated cells and the control (without ibuprofen – PU Membrane) were incubated for 24, 48, and 72 hours. After this period, the culture media were removed, and MTT (0.5 mg / mL in PBS) was added to the cells, which were incubated for 3 hours in a humidified atmosphere containing 5% CO2 at 37°C. Then, the growth medium was discarded, and 100 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the MTT. Absorbance was determined at 570 nm using a scanning spectrophotometric multiwell plate reader (F5 Microplate Reader, Molecular Probes). Experiments were performed in triplicate and repeated three additional times (independent assays). The same was done for the TSP, TP0.1 and TP0.4 tubes.Figure 9 shows the results of the MTT assay for 24, 48, and 72 hours with PU5I, 10I, 15I fibers, and the control (PU membrane). The statistically significant differences between PU5I, PU10I, and PU15I fibers and the control (PU membrane) were indicated by * (p < 0.01). From the results, it is possible to observe that at 24h and 48h, as the amount of IBU incorporated into the fibers increases, there is a statistically significant reduction in cell viability (p < 0.01) compared to the control sample. It was seen that, at all times, they presented an average of 300, 600, and 750 µg / mL of IBU, respectively for PU5I, PU10I, and PU15I. Therefore, these concentration values ​​corroborate the literature that promote a reduction in cell proliferation.

[0247] However, when it reaches 72 hours, we observe another phenomenon. It is noted that cell proliferation increases for all samples, including the control, in relation to the 48h time.It is also possible to observe that PU15I increases cell proliferation even more than PU5I and PU10I. This phenomenon suggests two possibilities: at 24 and 48 hours, cell cycle arrest may occur, associated with drug adaptation, and then cell proliferation returns at 72 hours. Or, the IBU may stimulate cell differentiation, and therefore, cell proliferation initially reduces at 24 and 48 hours, allowing the cells to differentiate, and then resumes at 72 hours. Example 2

[0248] In another example of the invention, tubes were prepared by additive manufacturing and evaluated for morphological characterization (SEM), chemical and thermal characterization (XRD), biological evaluation (MTT), and mechanical testing (tensile test).

[0249] The results were verified through statistical analyses performed using one-way ANOVA, followed by Tukey's post-hoc test. BioStat version 3 software was used.5 for Windows. Differences were considered statistically significant when probabilities were <0.05 (*p<0.05) and <0.01 (*p<0.01).

[0250] Therefore, to characterize the tubes, a polycaprolactone (PCL) solution in dichloromethane with a concentration of 0.8 g / mL was prepared based on tests and adjustments made to the protocol proposed by the equipment manufacturer. Dissolution occurred in a few hours without stirring, and the solution was left to rest for 48 hours for subsequent printing. A bioprinter with pneumatic extrusion technology was used to process the tubes.

[0251] The modeling of the produced tube structure was done with Autodesk Fusion 360 software. Three tubes were designed: poreless (TSP), with 0.1 mm pores (TP0.1), and with 0.4 mm pores (TP0.4), in STL format. The modeling of the tubes can be seen in Figure 17.

[0252] For the fabrication of the tubes, the printing speed was set to 1.0 mm / s, and the layer height was set to 0.1 mm. The temperature was set to room temperature (28°C), and the pressure was set to 744.6 kPa. The printing processing parameters are summarized in Table 2. Table 2: Optimized conditions for printing the tubes. Optimized conditions for 3D printing of tubes. printing Pressure 108 psi

[0253] After production, the tube was filled with polymeric fibers, for this, the fiber was cut to the size of the tube and with the help of a needle these fibers were introduced inside it.

[0254] The morphology of the produced tubes was verified by SEM images, as in Example 1, and the TSP tube is shown in Figure 11. The tubes with the TSP, TP0.1 and TP0.4 geometries were successfully processed and observed from different perspectives, providing information on the outer and inner diameter of the tubes and pore diameters. Individual measurements were carried out and the results are shown in Table 3. Table 3: Measured values ​​of the produced tubes.

[0255] From the SEM images and measurements performed, it is observed that, in the TP0.1 tube, diameters of 0.2 mm were obtained, however, it was desired to obtain diameters of 0.1 mm. While the TP0.4 tube presented regular pores and the pore diameter obtained was faithful to the modeled one. It is possible to see irregularities in the pores. The PCL tube was characterized by X-ray diffraction, and its diffractogram is shown in Figure 12. It is possible to identify two diffraction peaks, the one with the highest intensity at 2θ equal to 22.1° and the lowest at 24.4°.

[0256] In addition, the tubes were evaluated for their mechanical properties. The mechanical test was performed, in triplicate, on the TSP, TP0.1 and TP0.4 tubes. A texture analyzer (TAXT plus Stable Micro Systems) equipped with a 10 mm diameter delrin probe, set to a pre-test and post-test speed of 1.00 mm / s and a test speed of 0.5 mm / s, was used.The trigger force was 0.5 N, and a 5 kg load cell with a 10% strain target mode was used to evaluate the tensile stress and deformation of the tubes hydrated in phosphate-buffered saline (PBS) pH 7.4, 30 minutes before starting the tests. From the stress-strain graph, the tensile stress value can be obtained, which is the maximum tensile strength at break—that is, the point reached before breaking (y-axis) and the deformation corresponding to this point (x-axis). And the Young's modulus is obtained by the ratio of stress to strain. These properties are important to evaluate since they are essential for maintaining the tube's shape over the long term and provide a stable space for internal nerve regeneration. The results can be found in Table 4 and Figure 13. Figure 13 presents the stress-strain curve for the tubes produced.Table 4: Tensile strength, deformation and Young's modulus values ​​of TSP, TP0.1 and TP 0.4 tubes.

[0257] From the stress-strain curve (Figure 13), it is possible to observe that the materials presented low Young's modulus and, therefore, have predominantly elastic properties. The elastic modulus of the tube without pores was higher than that of the porous tubes TP0.1 and TP0.4, respectively.

[0258] This result was already expected, since the absence of pores would make the material more rigid and less elastic. Thus, the Young's modulus of all the tubes obtained fits the typical value of a nerve (0.58 ± 0.16 MPa) (BORSCHEL et al., 2003; YU et al., 2020). The tensile strength stresses of the porous tubes are comparable to the typical tension of a nerve (2.7 ± 0.97 MPa) (BORSCHEL et al., 2003), while the tension of the TSP presented values ​​higher than that of the nerve, being sufficient to mechanically resist the applied tension.

[0259] The results demonstrate that all tubes presented mechanical properties comparable to those of the peripheral nerve and, therefore, in this regard, they can act as a tube.

[0260] MTT assays were performed on tubes TSP (control sample), TP0.1, and TP0.4 to evaluate their cell viability with neuro-2a cells (Figure 14). In the first 24 hours, a lower cell viability was observed for tube TP0.4 in relation to the control (p<0.001).

[0261] However, from 48 to 72 hours, this tube had a significant increase in viability and equaled the control sample and the one with the smallest pore. Meanwhile, tube TP0.1 had similar viability to the control, indicating that the presence of small pores does not alter cell proliferation at all times. Therefore, in general, both tubes, regardless of the presence and size of the pores, will be viable for the cell of interest.Example 3

[0262] The present invention also evaluated the manufacture of scaffolds by obtaining polymeric tubes filled with aligned PU fibers and incorporated with ibuprofen. For this, the polymeric fibers were cut to the length of the tubes and added to the tube with the aid of a needle. Figure 15 shows the images obtained by SEM of the PCL-based tube incorporated with the polymeric fibers aligned inside.

[0263] It is possible to verify that a tube filled with fibers was obtained. From the results obtained previously, all the tubes produced are suitable for future applications as a tube.

[0264] Those skilled in the art will value the knowledge presented herein and will be able to reproduce the invention in the presented embodiments and in other variants, covered by the scope of the attached claims.

Claims

1 / 5 CLAIMS 1. Neural Guidance Conduit, characterized in that the conduit is formed by the combination of: (i) polymeric fibers; and (ii) tube, in which the polymeric fibers are produced by the rotospinning technique and the tube is produced by additive manufacturing.

2. Neural Guidance Conduit, according to claim 1, characterized in that the polymeric fibers are produced from a polymeric solution of polyurethane (PU) and solvent, in which the PU is in a concentration of 2 to 14% (m / v), preferably 9% (m / v), and, optionally, in which the solvent is chloroform.

3. Neural Guide Conduit, according to claim 1 or 2, characterized by the fact that the polymeric solution optionally comprises the addition of a drug, preferably in which the drug is an anti-inflammatory, more preferably in which the drug is ibuprofen. 4.Neural Guide Conduit according to claim 3, characterized in that the ibuprofen is present in an amount of 2.5 to 30%, preferably 5 to 15% by weight of the polymeric solution. Neural Guide Conduit according to any one of claims 1 to 4, characterized in that the polymeric fibers have a diameter of 1.0 mm to 4.0 mm, preferably 2.00 to 4.00 mm. 2 / 5 6. Neural Guide Conduit, according to claim 1, characterized by the fact that the tubes are produced from a polymeric solution of polycaprolactone (PCL) and solvent, in which the PCL is present in an amount of 0.7 to 1.5 g / mL, more preferably 0.8 g / mL, in solvent, and, optionally, in which the solvent is dichloromethane.

7. Neural Guide Conduit, according to claim 6, characterized by the fact that the tube can be customized as to its shape, internal and external diameter, presence or absence of pores, pore diameter, distance between the pores and arrangement of the pores, according to the type of lesion.

8. Neural Guide Conduit, according to any one of claims 1 to 7, characterized by the fact that the polymeric fibers are located inside the tube. 9.Method of manufacturing a Neural Guide Conduit, as defined in any one of claims 1 to 8, characterized by the fact that it comprises the steps of: (i) manufacturing the polymeric fibers by the roto-spinning technique, (ii) manufacturing the tubes by additive manufacturing, in which the tube is customizable according to the nerve injury; and (iii) introducing the polymeric fibers into the interior of the tube.

10. Method of manufacturing a Neural Guide Conduit. 3 / 5 according to claim 9, characterized in that the manufacture of the polymeric fibers comprises the steps of preparing the polymeric solution and manufacturing the fibers by rotospinning, in which the preparation of the polymeric solution comprises: (a) preparing a polymeric solution of polyurethane (PU) and solvent, in which the PU is in a concentration of 2 to 14% (m / v), preferably 9% (m / v), and, optionally, in which the solvent is chloroform; (b) optionally, incorporating a drug in a concentration ranging from 2.5 to 30%, preferably from 5 to 15% (m / m) based on the weight of the polymeric solution, preferably in which the drug is an anti-inflammatory, more preferably in which the anti-inflammatory is ibuprofen; (c) agitation of the polymer solutions in ultrasound for 60 to 120 minutes, preferably 60 minutes at room temperature, and rest preferably for 24 hours; (d) in which, after the rest, the fibers are manufactured by roto-spinning;wherein the manufacture of fibers by roto-spinning comprises: (a) version of the solution in the roto-spinning equipment adjusted with 1 mm diameter holes and rotation adjusted to 6000 rpm; 4 / 5 (b) extrusion of the polymer solution; (c) evaporation of the solvent; (d) recovery of the polymer fibers.

11. Method of manufacturing a Neural Guide Conduit, according to claim 9 or 10, characterized by the fact that the polymer fibers have a diameter of 1.0 mm to 4.0 mm, preferably of 2.00 to 4.00 mm. 12.Method of manufacturing a Neural Guide Conduit, according to any one of claims 9 to 11, characterized in that the manufacture of the tubes by additive manufacturing comprises 3D modeling of the tubes, preparing the polymeric solutions and printing the tube by additive manufacturing, in which the 3D modeling of the tubes is customized based on the type of peripheral nerve injury; and in which the preparation of the polymeric solutions comprises: (a) dissolving polycaprolactone in a solvent, in which the polycaprolactone is in an amount of 0.7 to 1.5 g / mL, preferably 0.8 g / mL, and the solvent is dichloromethane; (b) resting the solution for 24 to 72 hours, preferably 48 hours; in which 3D printing parameters such as printing speed, temperature, pressure and layer height are adjusted according to the 3D modeling of the tube.

13. A method of manufacturing a Neural Guide Conduit according to any one of claims 9 to 12. 5 / 5 characterized by the fact that the polymeric fibers are cut to the desired size, and subsequently introduced into the tube with the aid of a needle.

14. Use of a polymeric solution, characterized by the fact that it is in the manufacture of a neural guide conduit, as defined in any one of claims 1 to 8, for the treatment and recovery of injured peripheral nerves.

15. Use, according to claim 14, characterized by the fact that the polymeric solution comprises polymers selected from polyurethane and polycaprolactone.

16. Use, according to claim 14 or 15, characterized by the fact that the conduit optionally comprises a drug, preferably in which the drug is an anti-inflammatory, more preferably in which the drug is ibuprofen.

17. Use, according to any one of claims 14 to 16, characterized by the fact that the conduit is customized according to the injured nerve to be recovered.

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