Improved nerve regeneration scaffolds for accelerated regrowth

Abstract

An improved nerve regeneration scaffold is disclosed, which includes a plurality of modified nanotube yarn bundles disposed within a scaffold lumen. The modified nanotube yarn bundles have improved hydrophilicity and water absorption. They are separated by a distance to form channels corresponding to the diameter of the nerve fibers occupied by the regenerating nerve tissue. The walls of the channels have gaps between the yarn bundles to improve permeability. The scaffold supports the regrowth of individual nerve fibers with reduced inflammatory infiltration and rejection, reducing the potential for undesirable outcomes such as nerve pain or decreased nerve function.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 402,177, filed August 30, 2022, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates generally to improved devices with modified nanofiber yarns. Specifically, the disclosure relates to implantable medical devices based on functionalized nanofiber or nanotube yarns and yarn bundles for the regeneration of nerve tissue in regenerative medicine. This focuses on the regeneration of peripheral nerve tissue for long-gap injuries using oxidized nanofiber yarn bundle-based scaffolds. Additional applications include ex vivo neuronal cell growth and differentiation. [Background technology]

[0003] There has been much interest in the culture of neural cells, cell proliferation, and tissue regeneration. However, the general challenge remains the same because mature neurons do not divide as easily as other non-neuronal cell and tissue types. Propagation of primary cells obtained or dissociated from tissues harvested from the central nervous system (CNS) and peripheral nervous system (PNS) is even more challenging. Methods and devices for obtaining and maintaining effective cell cultures are important for neurological research and neurotoxicological testing.

[0004] Injury to the brain, spinal cord, or peripheral nerve tissue, especially long-gap nerve tissue loss due to severe tissue laceration and significant debridement, often leads to partial or complete loss of function. The highly desired complete functional recovery can be difficult to achieve, while complications or side effects inevitably occur under certain circumstances.

[0005] Unlike other tissue types, simply suturing the severed ends of a nerve together is often insufficient to promote complete repair and healing of injured peripheral nerve tissue. In particular, severing a portion of the nerve can limit the regeneration of nerve tissue to restore nerve function, resulting in little or partial recovery of lost nerve function in most cases. Recovery can also be accompanied by persistent nerve pain (commonly referred to as neuropathy or neuropathic pain), among other problems. In the case of long-gap injuries, bringing the proximal and distal ends of the severed nerve into close proximity is not an option. Consequently, excessive nerve stretching can lead to pinching and increased tension, potentially inducing pain. Many attempts and experimental techniques have been developed to improve nerve injury recovery. Among these, connecting the severed ends of nerve fibers with conduits or multichannel conduits allows cells, such as Schwann cells and pluripotent stem cells, to proliferate and differentiate to form connections between the severed nerve endings. Conduits also inhibit unwanted cells from surrounding tissues, such as fibroblasts, which often have faster replication rates and can occupy vacant space, limiting the regeneration of much-needed neural tissue.

[0006] Previous attempts have been made to make carbon nanotube yarns serve as structural and support materials for nerve regeneration scaffolds. Carbon nanotubes are generally hydrophobic, which as such is less than ideal for cells and tissues with amphiphilic bilayers. Summary of the Invention

[0007] According to aspects of the present disclosure, the following non-limiting exemplary embodiments or examples are provided.

[0008] Example 1 is a nerve regeneration scaffold comprising a tube having a first end and an opposite second end, the tube defining a lumen with a first diameter and a central axis, the tube comprising a biocompatible material and a plurality of nanofiber yarns within the lumen.

[0009] Example 2 includes the subject matter of Example 1, where the nanofiber yarn is modified.

[0010] Example 3 includes the subject matter of Example 2, where the modification is a strong oxidation process.

[0011] Example 4 includes the subject matter of Example 3, wherein the oxidation process involves an oxidizing gas or an oxidizing solution.

[0012] Example 5 includes the subject matter of any of Examples 2-4, wherein the modified nanofiber yarns have improved wettability and / or hydrophilicity compared to their wettability and / or hydrophilicity before the modification.

[0013] Example 6 includes the subject matter of any of Examples 2-5, wherein the modified nanofiber yarn elicits a weaker immune response compared to an unmodified nanofiber yarn.

[0014] Example 7 includes the subject matter of any of Examples 1-6, wherein the nanofiber yarn is a nanotube yarn or nanotube yarn bundle.

[0015] Example 8 includes any subject matter of Example 7, wherein the nanotube yarn or nanotube yarn bundle comprises a carbon nanotube yarn or a boron nanotube yarn.

[0016] Example 9 includes the subject matter of Example 1, in which the nanofiber yarns form channels within the lumen inside the tube and gaps between two adjacent nanofiber yarns within the walls of the channels to guide nerve tissue regeneration in a direction parallel to the central axis of the tube, partially or completely filling each channel and gap.

[0017] Example 10 includes the subject matter of example 1, wherein the tube comprises a bioabsorbable material.

[0018] Example 11 includes the subject matter of Example 2, in which the modified nanofiber yarn has improved biocompatibility compared to unmodified nanofiber yarn.

[0019] Example 12 includes the subject matter of Example 1, wherein the nerve regeneration scaffold further comprises at least one foreign body-type multinucleated giant cell inhibitor.

[0020] The present disclosure will be further described in the following detailed description with reference to the several drawings, which are mentioned as non-limiting examples of preferred embodiments of the present disclosure, and in which like characters represent like elements throughout the several views of the drawings. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 is a schematic diagram of a nerve regeneration scaffold connected to a severed nerve fiber, according to an exemplary embodiment. [Figure 2A] FIG. 2 is a schematic diagram of a nerve regeneration scaffold with nanofibers inside the scaffold lumen as shown in FIG. 1, according to an exemplary embodiment. [Figure 2B] FIG. 1 is a schematic diagram of a cross-sectional view of a portion of a nerve regeneration scaffold, according to an exemplary embodiment. [Figure 3] 1 illustrates Sciatic Functional Index (SFI) measurements taken at predetermined intervals according to an exemplary embodiment. [Figure 4] 1 shows immunohistological staining of macrophage lineage markers in tissue, according to an exemplary embodiment.

[0022] The figures depict embodiments of the present disclosure for purposes of illustration only. Many variations, configurations, and other embodiments will become apparent from the detailed description that follows. Additionally, it is recognized that the figures are not necessarily drawn to scale or intended to limit the described embodiments to the particular configurations shown. For example, while some figures generally show straight lines, right angles, and smooth surfaces, actual implementations of the disclosed technology may have less-than-perfect straight lines and right angles, and some features may have surface topography or be otherwise non-smooth, given real-world limitations of the fabrication process. For simplicity, the figures are provided merely to illustrate example structures. DETAILED DESCRIPTION OF THE INVENTION

[0023] Through one or more of its various aspects, the embodiments and / or specific features or subcomponents of the present disclosure are intended to demonstrate one or more of the advantages specifically mentioned above and below.

[0024] overview Neurotmesis is the most severe type of nerve injury, resulting in the destruction of the nerve and its sheath, along with the transfer of nerve fibers and all supporting tissues. This results in proximal and distal nerve stumps and missing gaps, which can be as short as less than a millimeter or as long as several centimeters or more. Such injuries cause the destruction of sensory nerve fibers and corresponding loss of innervation, resulting in muscle paralysis and paresthesia due to weight loss. Furthermore, injured nerves generally do not recover without medical intervention. While the symptoms of neurotmesis can be treated with opioids and anti-inflammatory drugs, the restoration of destroyed nerve tissue and its associated functions can be assisted by surgical intervention. Common surgical approaches to promote recovery from nerve injury include suturing the separated nerve ends together or implanting autologous or allogeneic nerve grafts. Various implantable engineered "nerve regeneration scaffolds" are still under development.

[0025] Connecting damaged nerve ends together with sutures can have drawbacks. For example, suturing injured nerve ends together can result in unnecessary increased tension within the treated nerve due to the absence of a portion of nerve tissue. This tension can inhibit regrowth of the damaged nerve, increase the likelihood of scarring, and result in inadequate restoration of nerve function. Substitute nerve graft therapy can also have drawbacks. For example, the dissection and removal of a portion of donor nerve tissue can cause sensory loss, functional impairment, and neuropathic pain.

[0026] The use of hollow nerve regeneration scaffolds to promote regrowth of severed nerves is the subject of ongoing research. The scaffold is a biocompatible tube that is sutured to both ends (stumps) of an injured nerve. In some instances, the scaffold has multiple small channels or pathways that serve as guides for re-growing nerve fibers, while in other instances of the present disclosure, the scaffold is simply a hollow tube defining a single internal chamber. Regardless of the internal configuration, the purpose of the scaffold is to promote regrowth of nerve fibers and create an environment conducive to the restoration of sensory and muscle function. An exemplary nerve regeneration scaffold is shown schematically in Figures 1 and 2A-2B.

[0027] Nerve regeneration scaffold The nerve regeneration scaffold 100 of FIG. 1 has a tube-shaped outer housing 102, an internal space or lumen, and bundles of nanofiber yarns 104 arranged to form a support framework, or pathways or tunnels, to guide regenerating nerve tissue axonally within the lumen along the length of the tube. In FIG. 1, nerve segments 101A and 101B are the result of injury or surgical transection of the original, intact nerve, and the nerve tissue segment between 101A and 101B has been removed. The nerve regeneration scaffold 100 is surgically placed between the opposing ends of the severed nerve segments 101A and 101B, and an appropriate anastomosis is performed to promote nerve fiber regrowth. The length of the scaffold is important for maintaining an appropriate tensile load between segments 101A and 101B for nerve fiber regeneration, depending on the need and circumstances. This length may be less than 1.0 mm to 10.0 mm, 10.00 mm to 20.00 mm, or 20 mm or more.

[0028] According to some examples described herein, the present disclosure provides a nerve regeneration scaffold including an outer housing made of a biocompatible material. The actual size of the outer housing, i.e., outer diameter and length, also varies depending on the regeneration requirements. The inner diameters at both ends of the outer housing may be the same or slightly different to suit the physiological characteristics and transient pathological characteristics of the involved nerve tissue according to various anatomical locations. In addition to a generally straight tube shape, the outer shape of the tube may vary to different curvatures according to the physiological needs when implanted.

[0029] The outer tubular housing, in some examples, is fabricated from a polymeric material, in some embodiments, the polymeric material is selected from, but not limited to, polyurethane, polyester, polycaprolactone (PCL), polylactic acid (PLA), polyethylene glycol (PEG), polytetrafluoroethylene (PTFE), polymethyl methacrylate (PMMA), ethylene-vinyl acetate copolymer (EVA), polydimethylsiloxane (PDMS), polyester polyurethane, polyether polyurethane, polysulfone (PS), polyethylene terephthalate (PET), or a combination of one or more of the foregoing.

[0030] In some other embodiments, the polymeric material selected is a biodegradable material selected from, but not limited to, PLA, PEG, or EVA.

[0031] The outer housing prevents surrounding cells, such as fibroblasts, from migrating into the scaffold lumen and causing scar formation, which would otherwise encroach on the lumen space because the surrounding cells replicate much faster than the regenerating neuronal cells.

[0032] Multiple nanofiber yarns or yarn bundles can be arranged inside the scaffold lumen and can be separated from each other by various distances. The nanofiber yarns can form multiple nanofiber yarn bundles prior to their arrangement within the lumen. These nanofiber yarns and / or nanofiber yarn bundles together form the walls of multiple tunnels within the lumen. The size of the tunnels corresponds to the size of the nerve fiber bundles, with subsets of the nanofiber yarns or yarn bundles forming individual virtual walls. Between the yarns and yarn bundles, there are gaps within the bundles for the exchange of nutrients and macromolecules, such as proteins and growth factors, and for the diffusion of metabolites, cellular decomposition products, and debris across the virtual walls.

[0033] In one embodiment, adjacent nanofiber yarns can partially support individual nerve fibers due to favorable dimensional separation between the nanofiber yarns, reducing the likelihood that the nerve fibers will experience tensile stress during regrowth.

[0034] In some examples of the present disclosure, the magnitude of tensile stress on regenerated nerve fibers is reduced compared to nerve fibers regenerated using nerve scaffolds that do not include nanofiber yarns. For at least these reasons, the likelihood of the undesirable outcomes of neuropathic pain or decreased nerve function is reduced when embodiments of the present disclosure are used as nerve regeneration scaffolds.

[0035] The light weight of the nanofiber yarn material and its high flexibility also enhance its compatibility and adaptability with biological tissues and organ systems.

[0036] Pristine nanotubes, nanotube yarns, and nanotube yarn bundles According to one of the embodiments of the present disclosure, nerve regeneration scaffolds include nanofiber yarns in their lumens.

[0037] Nanofibers are long, tube-shaped fibers with diameters in the nanometer range (about 1 nanometer to about 1 micrometer). They occur naturally, for example, in cellulose, collagen, keratin, and muscle fibers from plants, fibrinogen from mammals, and polysaccharides from many species.

[0038] Nanofibers may be synthetic, such as polymer yarns and nanotube yarns, including carbon nanotube (CNT) yarns or boron nitride nanotube (BNNT) yarns or yarn bundles.

[0039] Other synthetic nanofibers include, but are not limited to, conventional carbon fibers (CCFs), cup-stacked carbon nanofibers (also known as conical CNFs), platelet carbon nanofibers, and graphene fibers with different carbon allotropes.

[0040] Hybrid yarns or yarn bundles produced from natural sources and synthetic procedures are becoming more common in artificial tissues and organs.

[0041] Nanotube yarns are produced from synthetic nanotubes that are long, thin, and cylindrical with a high aspect ratio.

[0042] CNTs and BNNTs can be synthesized by standard methods. These methods vary depending on the selected end product, precursor, heat source, reaction time, reaction temperature, reaction atmosphere, catalyst, and support substrate. The most common methods include, but are not limited to, arc discharge, electrolysis, laser ablation, chemical vapor deposition (CVD), flame synthesis, and mechanothermal methods. Well-known CVD methods include plasma-assisted PE-CVD, aerosol-assisted CVD (AACVD), water-assisted WA-CVD, oxygen-assisted CVD, and catalytic CVD.

[0043] Conventional CVD methods utilize acetylene (C2H2), ethylene (C2H4), or other hydrocarbon gases as the carbon source, with a catalyst added to the reaction chamber at reaction temperatures ranging from 350°C to 1,000°C. Amorphous boron and iron catalysts are common choices for BNNT synthesis.

[0044] By spinning a nanotube suspension or twisting nanotube sheet(s) drawn from a nanotube forest, the nanotubes form nanotube yarns. Nanotube yarns can be twisted together to form multi-plied nanotube yarns or yarn bundles. The twisted nanotube yarns can have the same nanofibers or nanotubes (homogeneous) or different nanofibers or nanotubes (heterogeneous). Nanotube fibers and nanotube yarns and yarn bundles are hereinafter collectively referred to as nanotube yarn bundles.

[0045] Looping the yarn to form a spool and then cutting the spool open can be another convenient way to produce linear bundles. Van der Waals forces between the nanotubes aid in the formation of tight nanotube bundles.

[0046] Oxidized nanotube yarn bundles The surfaces of nanotubes or nanotube yarns, both CNTs and BNNTs, are hydrophobic and generally resistant to chemical alteration. They tolerate mild to moderate pH environments well and are not susceptible to enzymatic degradation. These stability characteristics are advantageous for in vivo applications as implants. Very strong oxidizing agents can modify the nanotube surface.

[0047] According to one embodiment of the present disclosure, the nanotube yarn bundles are oxidized by chemical treatment to reduce hydrophobicity and improve water absorption and wettability.

[0048] According to the present disclosure, an exemplary oxidizing agent can be an oxidizing gas or an oxidizing solution.

[0049] Exemplary strong oxidizing gases can be ozone or chlorine.

[0050] Exemplary strong oxidizing solutions can be strong acids or combinations of two or more strong acids, including, but not limited to, nitric acid, sulfuric acid, perchloric acid, and mixtures of nitric acid and sulfuric acid, sulfuric acid and potassium dichromate, and sulfuric acid and potassium permanganate.

[0051] After treatment, oxidized nanotube yarns or yarn bundles may lose intrabundle interactions between adjacent yarn bundles due to nanotube surface modification and reduced van der Waals forces. This loss is beneficial to the formation and stability of nerve regeneration channels within the scaffold lumen, preventing the channels from collapsing due to strong van der Waals forces. This facilitates the exchange of nutrients and metabolic waste products due to increased permeability of the channel walls.

[0052] The exemplary treated CNT yarn bundles have increased water absorption as shown in Table 1. [Table 1] Silicone tubing without CNT yarn served as a negative control. Three silicone tubes with pristine CNT yarn (untreated), ozone-treated, and acid-treated yarns were measured for weight gain after one end was immersed vertically in the same water bath overnight. Each silicone tube and CNT yarn within the same tube had substantially the same length. The silicone tube with ozone-treated yarn had a weight gain of 4.32%, and the silicone tube with acid-treated yarn had a weight gain of 6.68%. The silicone tube with untreated pristine CNT yarn had a weight gain of only 1.27%. The water retention capacity was 10 mg for the pristine CNT yarn silicone tube, 30 mg for the ozone-treated CNT yarn silicone tube, and 47 mg for the acid-treated CNT yarn silicone tube.

[0053] Examples of neural scaffolds As indicated above, some example nerve scaffolds of the present disclosure include a plurality of modified yarns and / or yarn bundles fabricated from a variety of materials, including, but not limited to, synthetic polymer fibers, nylon fibers, carbon nanofibers, carbon nanotubes, or boron nitride nanotubes.

[0054] In one embodiment, the plurality of nanotube yarn bundles 104 can include true-twist multi-ply and single-ply nanotube yarns, untwisted multi-ply and single-ply nanotube yarns, and false-twist multi-ply and single-ply nanotube yarns. The nanotube yarns provide a smooth and nanoporous surface onto which nerve fibers can grow. The nanotube yarns can also provide scaffolding and mechanical support for re-growing nerve fibers. Taken together, these nanotube yarn characteristics render the re-growing nerve fibers and the regenerating nerve as a whole less susceptible to tensile or compressive stresses that can cause neuropathic pain or adversely affect nerve fiber growth.

[0055] While this disclosure primarily refers to oxidized carbon nanotube yarn bundles, it will be understood that modified carbon nanotubes and nanotube yarns and BNNT nanotubes and nanotube yarns and bundles share many of the same advantageous characteristics and are included within the general term "nanotube yarn bundles."

[0056] The nanotube yarn bundles applied herein primarily function as structural components of the overall regeneration scaffold and the walls of individual channels for nerve fiber regeneration. Neuronal tissue and nerve fibers can grow within and along these channels (sometimes referred to as "regeneration" or "regrowth"). They also have additional advantages because they are electrically conductive and biocompatible, the latter of which will be described below. When connecting two severed nerve segments, the nanotube yarn bundles can provide a conductive pathway for the transmission of sensory and motor signals from one end of the severed nerve to the other. The presence of an electrophysiological signal conductive pathway can further improve nerve tissue regrowth and enhance the recovery of nerve function, providing additional benefits beyond those generally expected for regenerated nerves. In some instances, the application of nanotube yarn bundles within a nerve scaffold is believed to be able to restore function to the same nerve level before injury, depending on the regenerated nerve. In some other instances, nanotube yarn bundles, and particularly single-yarn false-twisted carbon nanofiber yarns, yarn bundles, or modified yarn bundles, can provide a topographically nanoporous surface onto which nerve fibers can regrow extensively and promote regeneration at low tensile stresses. Taken together, the features presented herein provide an effective solution to the unmet clinical need of long-gap peripheral nerve injury repair.

[0057] An example of a neural scaffold of the present disclosure is shown schematically in Figures 2A and 2B.

[0058] In FIG. 2A, the nerve scaffold 100 includes a tube 102 and a plurality of nanotube yarn bundles 104.

[0059] FIG. 2B exemplarily illustrates a cross-sectional view of a portion of a nerve regeneration scaffold, which does not include a tube 102. Advantages of the nerve scaffold 100 include providing gaps or channels defined between adjacent nanotube yarns and multiple yarn bundles. The structural arrangement of the nanotube yarn bundles 104 provides multiple channels for the regeneration of nerve fibers or nerve tissue, one of which is shown as feature 108. A channel has a minimum of three nanotube yarn bundles to form a wall, or more specifically, a substantial wall with gaps between the nanotube yarn bundles. Feature 106 represents one of the gaps in the wall of the channel. Adjacent nanotube yarn bundles are two nanotube yarn bundles that form a single substantial wall with no third yarn bundle between them, as shown by α1 in FIG. 2B. One of the adjacent nanotube yarn bundles can be part of more than one wall for the other corresponding channel. For a channel containing at least four nanotube yarn bundles against its substantial wall, pairs of nanotube yarn bundles separated by at least another nanotube yarn bundle at a maximum distance are defined as being diagonally positioned, this maximum distance being the diagonal distance shown as α2 in FIG. 2B.

[0060] The gap 106 allows for the movement of macromolecules and metabolites within the channel (not shown) and the branching of new nerve fiber branches from the new nerve fiber track.

[0061] The channels 108 are configured and dimensioned to encourage nerve growth. In the case of customized or individualized nerve regeneration scaffolds, the channel size is selected according to the needs of the particular physiological location and desired implantable scaffold.

[0062] The nanotube yarn bundles 104 (the fabrication of which is described in U.S. Patent Application No. 16 / 353,608, incorporated herein by reference in its entirety) within the tube 102 in FIGS. 1 and 2A are shown as being parallel to one another and unevenly distributed within the tube 102. It will be understood that this is for convenience of illustration only. Rather, the nanotube yarns are assembled into the nanotube yarn bundles 104 to occupy a portion of the lumen of the tube 102 and may be substantially aligned with one another, but not necessarily precisely parallel to one another. In many embodiments, this general alignment of the nanotube yarn bundles within the nanotube yarn bundles 104 is sufficient to define channels and gaps between the nanotube yarn bundles that roughly correspond to the cross-sectional diameter of a nerve fiber. The channel diameters are generally between 5 μm and 10 μm in diameter, and in some examples, between 8 μm and 15 μm, or between 5 μm and 20 μm. Even in situations where the nanotube yarn bundles may cross each other or be misaligned, being at least partially continuous along the length of the tube 102 (i.e., more than 10% or more than 20% along the length of the tube 102) and having a spacing within a range of about 5 μm to 15 μm in diameter is sufficient to promote nerve regeneration so as to minimize the aforementioned tensile forces. The distance between adjacent nanotube yarn bundles can be 2 μm to 8 μm in some examples, 5 μm to 15 μm in other examples, or 2 μm to 15 μm.

[0063] The tube 102 can perform any of several functions. In some instances, the tube 102 separates an area from severed terminations into which nerve fibers can regrow. This area is also ideal for transplantation of neuronal or stem cells. Another function of the tube 102 is to help protect existing nerve tissue and its terminations, as well as the nerve fiber regeneration process, from physical damage or other perturbations that could otherwise reduce the growth rate or continuity of the re-growing nerve. The tube 102 further shields the area, preventing fast-replicating fibroblasts in the surrounding tissue from occupying the area intended for slower nerve regeneration. The tube 102 also defines an interior space into which carbon nanofiber yarns can be arranged and configured to have a density and arrangement that promotes regeneration of nerve fibers closer to their original native structure, as described herein.

[0064] The tube 102 can be fabricated from biocompatible and / or bioabsorbable materials. Examples of these materials include silicone, which has the added advantage of being flexible rather than rigid, such as plastic materials. This flexibility facilitates attachment of the ends of the tube 102 to the severed nerve segments 101A and 101B via sutures or surgical adhesives, such as BioGlue®. Other examples of biocompatible materials that can be used for the tube 102 include, but are not limited to, poly(methyl methacrylate), poly(tetrafluoroethylene), polyethylene, polyglycolide, polycaprolactam, poly(lactic-co-glycolic acid), polylactic acid, poly(glycerol sebacate), polysialic acid, polyethylene glycol, polyurethane, collagen, chitosan, silk, and alginate, among others. In another set of embodiments, the tube can be made from a carbon material, such as carbon nanotubes, in the form of wrapped sheet(s). The nanotubes within the sheet can have a random orientation or an aligned end-to-end orientation when drawn from the nanotube forest relative to the planar orientation of the sheet surface. Nanotube sheet tubes 102 can exhibit very low mechanical stiffness while exhibiting excellent durability and long lifespans. In some embodiments, the tubes 102 can be fabricated from biological materials, including heterologous blood vessels. In certain embodiments, the tubes 102 can contain pristine (unmodified) or modified (oxidized or functionalized) nanotube yarn bundles that are the same as or different from the nanotube yarn bundles inside the tube lumen.

[0065] In some other examples, the length of the nerve regeneration scaffold can be in any of the following ranges: 1.0 mm to 1.5 mm, 1.0 mm to 1.0 cm, 2.0 mm to 5 mm, 1.0 cm to 2.0 cm, 1.5 cm to 2 cm, 2.0 cm to 5 cm, 3.0 cm to 8.0 cm, or 1.0 cm to 15.0 cm. The inner diameter of the nerve regeneration scaffold is sized so that a tube 102 can be placed over the severed end of the nerve and sutured or otherwise connected thereto, as shown in FIG. 1. In some examples, the diameter of the lumen can be in any of the following ranges: 0.5 mm to 2.0 mm, 1.0 mm to 4.0 mm, 1.0 mm to 5.0 mm, or 3.0 mm to 10.0 mm.

[0066] The multiple nanotube yarn bundles 104 have lengths and outer diameters that are similar in size to or smaller than the length and inner diameter of the tube 102. This reduction in size of either the length or outer diameter can be 2%, 5.0%, 10.5%, or up to 50.0% or more to ensure that the multiple nanofiber yarns fit inside the tube lumen.

[0067] In some embodiments, the interstitial spaces between the nanotube yarn bundles can include one or more polymer matrices to support nerve fiber growth. In some examples, the polymer matrix can be one or more of collagen, gelatin, fibrin, neural extracellular matrix (ECM) molecules, ECM proteins, and analogs thereof. In some other examples, the polymer matrix can include one or more of glycosaminoglycans.

[0068] In some embodiments, the nanotube yarn bundles and / or the interstitial spaces of the nanotube yarn bundles may further comprise one or more proteins or growth factors to improve growth rate and reduce the time required for a cell division cycle. The one or more proteins or growth factors may promote the growth or division of one or more specific cell types. In some examples, the proteins or growth factors may be at least one of vascular endothelial growth factor (VEGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), neuregulin 1, glial-derived neurotrophic factor, pleiotrophin, or brain-derived neurotrophic factor (BDGF). In one particular example, the proteins and / or growth factors in the interstitial spaces have a sustained-release formulation.

[0069] In one embodiment, the nanotube yarn bundles or the interstitial spaces of the nanotube yarn bundles may be further impregnated with at least one inhibitor to prevent less desirable or undesirable adverse reactions or outcomes, including, but not limited to, foreign body type multinucleated giant cell inhibitor (FBGC-I).

[0070] In another embodiment, the FBGC-I may be selected from at least a diacylglycerol kinase inhibitor, a protein kinase C (PKC) inhibitor, or a combination thereof. Exemplary diacylglycerol kinase inhibitors include, but are not limited to, R59022 and R59949 (3-{2-(4-[bis-(4-fluorophenyl)methylene]-1-piperidinyl)ethyl}-2,3-dihydro-2-thioxo-4(1H)quinazolinone) or analogs or derivatives thereof. Exemplary PKC inhibitors include, but are not limited to, (±)-1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride and calphostin C.

[0071] In yet another embodiment, the interstitial spaces of the nanotube yarn bundles can include one or more polymer matrices in combination with one or more proteins. In a particular example, the interstitial spaces include collagen in combination with nerve growth factor.

[0072] In some instances, the polymer matrix and / or protein growth factors may have a concentration gradient from the proximal end of the nerve regeneration scaffold to the distal end of the scaffold, or vice versa.

[0073] In other examples, the appropriate spacing between adjacent nanotube yarn bundles of the plurality of nanotube yarn bundles 104 described above and the number of channels can be indirectly measured as the percentage of nanotube yarn volume relative to the total volume of the nerve scaffold lumen. This percentage can be greater than 0.1%, greater than 2%, less than 10%, or less than 30%. In some other examples, nerve fiber regrowth does not occur when the percentage of nanotube yarn volume relative to the scaffold lumen volume is less than 0.1% or greater than 30%. Nerve fiber regrowth can occur when the nanotube yarn volume percentage falls within the range of 0.1% to 30%, with sufficient functional recovery detected by electrophysiological measurements at volume percentages of 2% to 10%.

[0074] In some instances, it was experimentally found that false-twisted nanofiber yarns with a volume percentage range of 2% to 10% were found to have interfiber spacing distances of 8 μm to 10 μm.

[0075] The nerve regeneration scaffold can contain any number of nanotube yarns ranging from 10 to 3,000 or 10 to 8,000.

[0076] Exemplary embodiments of the present disclosure incorporate modified nanotube yarn bundles in place of pristine nanotube yarn bundles, including oxidation with an oxidizing gas, an oxidizing solution, or a combination thereof, which exhibit improved wettability, as measured by water absorption, as previously described.

[0077] Animal model test results Five animal groups (rats) received implantation of nerve regeneration scaffolds containing different nanotube yarn bundles. Each animal group in this study included at least five rats in the sciatic nerve translocation model. The five groups were autograft (AG), pristine CNT yarn (P-CNT), oxidized CNT yarn (O-CNT), strong acid-treated CNT yarn (SA-CNT), and silicone tube alone (ST). To demonstrate the effects of different nanotube yarn bundles in the nerve regeneration scaffolds, the rats were examined at 8 and 16 weeks after surgical implantation.

[0078] Muscle weight loss is a common clinical symptom caused by nerve injury. An increase in muscle weight indicates effective functional recovery. Muscle ratio, which compares muscle weight from the injured site with that from the healthy contralateral site, is often an excellent indicator for demonstrating differences between various treatments. The effects of different nanotube yarn bundles on muscle ratios of the tibialis anterior (TA) and gastrocnemius (GA) muscles are presented in Table 2 below. [Table 2] At week 8, the TA and GA ratios were significantly higher in the AG group compared with all CNT groups. The O-CNT group gained more weight than the P-CNT group, while the SA-CNT group showed a clear advantage over the P-CNT group. At week 16, both the TA and GA ratios were more than twice the corresponding TA and GA ratios in the P-CNT group.

[0079] Electrophysiological measurements were performed and the results are presented below in Table 3. At 8 and 16 weeks, standard compound muscle action potentials (CMAPs) were measured and recorded after stimulation using a VikingQuest system (Nicolet Biomedical, Madison, WI, USA). The CMAP is an electromyography test involving the summation of a group of simultaneous action potentials from several muscles targeted by the test. It reflects the status of the motor units. The onset latency and peak-to-peak amplitude of the CMAP on the experimental side of the animal are recorded. [Table 3] At 8 weeks, CMAP was detected in 5 of 7 rats (71.4%) in the P-CNT group, 6 of 8 rats (75.0%) in the O-CNT group, 9 of 9 rats (100.0%) in the SA-CNT group, and 8 of 8 rats (100.0%) in the AG group. When the same study was repeated at 16 weeks, results showed no improvement in the P-CNT group (71.4%) and further improvement in the O-CNT group (100.0%). No CMAP was observed in the ST group. This data set provides strong evidence to demonstrate the superiority of modified and oxidized nanofiber yarn in functional recovery.

[0080] The mean latency and mean amplitude of nerve conduction velocity (NCV) with standard error (SE) at 8 weeks were 4.61 ± 0.23 ms and 4,791.1 ± 1,518.2 μV in the AG group, 4.80 ± 0.40 ms and 451.5 ± 168 μV in the P-CNT group, 4.15 ± 0.19 ms and 702.7 ± 400.4 μV in the O-CNT group, and 3.97 ± 0.08 ms and 728.3 ± 157.9 μV in the SA-CNT group.

[0081] At 16 weeks, the NCV plus SE results were 5.14 ± 0.3 ms and 1,841.2 ± 1,038.3 μV in the P-CNT group, 3.28 ± 0.21 ms and 5,453.4 ± 2,195.2 μV in the O-CNT group, and 4.01 ± 0.2 ms and 8,215.5 ± 2,813.5 μV in the SA-CNT group.

[0082] NCV and amplitude test data from both modified nanofiber yarn groups demonstrate continued recovery significantly greater than that of the P-CNT group. NCV and amplitude test data from both modified nanofiber yarn groups demonstrate continued recovery significantly greater than that of the P-CNT group. At 8 weeks, NCV data for the O-CNT and SA-CNT groups demonstrated initial restoration of electrochemical impulse propagation comparable to that of the P-CNT group, but at lower levels than the AG group in terms of signal amplitude. NCV amplitude data collected at 16 weeks strongly suggests an advantage of O-CNT, and particularly SA-CNT, over P-CNT. Because CMAP and NCV often indicate motor function status, embodiments of the present disclosure further suggest that motor function recovery takes precedence over sensory function recovery.

[0083] The sciatic functional index (SFI) is a widely used index for quantitative testing of neurological pathology and potential treatments. This index correlates with the ratio of nerve fiber to axon diameter and the ratio of myelin thickness to axon diameter, with a reduction indicating functional recovery. Measurements were performed at 4-week increments, and the results are shown in Figure 3.

[0084] The indices provided in Figure 3 show a general improvement across all transplant groups, as shown by the ST group versus the transplant group. Among the transplant groups, the SA-CNT group showed the best recovery, with the O-CNT group (shown as SO-CNT in Figure 3) recovering better compared to the P-CNT group at the end of weeks 8, 12, and 16. There is a significant difference in the SA-CNT group (-63.2 ± 4.3) compared to the P-CNT group (-88.9 ± 5.7).

[0085] Further histological and immunochemical examinations were performed on longitudinal sections of nerve tissue harvested from the scaffolds using anti-neurofilament and Schwann cell-specific S100 protein antibodies. Staining revealed the presence of axons and myelin sheaths 8 weeks after implantation. Regrowth extended from the proximal junction of the severed sciatic nerve and scaffold to the distal terminal of the scaffold in all CNT groups. In contrast, axonal regeneration was not observed in the silicon group (data not included). Furthermore, among all CNT groups, the SA-CNT group showed a significant increase in axonal regeneration compared to the P-CNT group, as evidenced by immunochemical staining for neurofilament and Schwann cells at the distal terminal. There was no statistically significant difference in the number of cell nuclei (DAPI) in the proximal and distal regenerating nerve terminals between groups (see Table 4 below). [Table 4]

[0086] The results of the analysis of CD68-positive cells and foreign body multinucleated giant cells (FBGCs) further support one of the embodiments and significance of the present disclosure.

[0087] CD68 is a marker of the macrophage lineage in the circulation and tissues. Staining in proximal longitudinal sections of regenerated tissue from different treatment groups is presented here (see Figure 4). In the P-CNT group, there was significant positive CD68 staining (CD68 + ) cell staining, while CD68 in the O-CNT and SA-CNT groups + Although staining was dramatically reduced, the O-CNT and SA-CNT groups still showed CD68 staining. + Shows slightly higher levels of staining.

[0088] FBGCs are fused forms of macrophages. They are a prominent cell type in implanted biomaterials and have long been considered a hallmark of chronic inflammation.

[0089] In Figure 4, DRAQ5, a known nuclear stain, inhibits CD68 +Compared with the staining images, and among several CNT groups, the formation, increase, or decrease of FBGCs was observed (DRAQ5 plus CD68 + (See clusters of strong signals from stained images.) In proximal longitudinal sections of the regenerated tissue, FBGCs are observed around the CNT fibers in all CNT groups. The number of FBGCs was significantly reduced in the O-CNT and SA-CNT groups compared to the P-CNT group, with p values ​​less than 0.01 (O-CNT compared to P-CNT) or less than 0.001 (SA-CNT compared to P-CNT) (see Table 5 below). [Table 5]

[0090] CD68 around the graft after hydrophilization of nanotube yarn bundles + The reduction of both FBGCs and foreign body giant cells can be interpreted as a new strategy for resolving transplant rejection. Small molecule inhibitors of FBGCs have been suggested, developed, and reported to reduce or eliminate FBGC formation. Incorporating an FBGC inhibitor into a scaffold, particularly in a sustained-release form, may constitute another embodiment of the present disclosure for further reduction of FBGCs. The FBGC inhibitor can be selected from one or more of a diacylglycerol kinase inhibitor, a protein kinase C inhibitor, and their analogs or derivatives.

[0091] The present disclosure may further include one or more anti-inflammatory agents in the scaffold.The present disclosure may include methods for repair and regeneration of neural tissue by applying any of the embodiments disclosed herein.

[0092] Ex vivo neuronal cell culture and uses Various nanofibers and nanotubes with different properties have been explored and investigated for ex vivo use and testing, either for academic research purposes or for pharmacological and toxicological testing services that rely on the expansion of neuronal cell populations. Functionalized nanotubes and / or nanotube bundles with increased hydrophilicity and reduced undesired reactions can modulate neuronal growth, such as neurite outgrowth of hippocampal neurons in culture. Ex vivo expansion of neuronal stem cells and glial cells can also rely on exemplary embodiments of functionalized nanotubes presented herein. The resulting cell populations are ideal for scientific research, drug safety testing, and cell transplantation for injury or neurodegenerative disease.

[0093] In one embodiment of the present disclosure, modified nanofiber yarns, yarn bundles, or modified nanofiber sheets can be used as substrates for the growth of harvested primary neuronal tissue and cells, purified primary neuronal cells, or established neuronal cell lines, resulting in the growth of larger cell populations compared to unmodified nanotube materials. They can also, optionally, function as substrates for stem cell differentiation into neuronal cells in the presence of one or more selected growth factors. Modified nanofiber yarn bundles can be configured as scaffolds or randomly oriented in an ex vivo culture environment. Modified nanofiber sheets can have nanofibers aligned or randomly dispersed in the plane of the sheet.

[0094] A further embodiment of the present disclosure is the application of the nerve regeneration scaffold to artificial systems to grow nerve tissue, or the application of the modified nanotubes, nanotube yarns, or bundles to the research and production of prosthetic organs.

[0095] Further Considerations The foregoing description of embodiments of the present disclosure has been presented for purposes of illustration and is not intended to be exhaustive or to limit the scope of the claims to the precise form disclosed. Those skilled in the art will recognize that many modifications and variations are possible in light of the above disclosure.

[0096] The language used herein has been selected primarily for purposes of readability and instruction, and the language may not have been selected to describe or limit the subject matter of the present disclosure. Accordingly, it is intended that the scope of the present disclosure be limited not by this Detailed Description, but rather by any claims that issue in an application based thereon. Accordingly, the disclosure of the embodiments is intended to illustrate, but not limit, the scope of the invention, which is set forth in the following Claims.

[0097] While the present invention has been described with reference to certain exemplary embodiments, it is understood that the words that have been used are words of description and illustration, rather than words of limitation. Changes may be made within the purview of the appended claims, as presently stated and as amended, without departing from the scope and spirit of the disclosure in its aspects. While the invention has been described with reference to particular means, materials, and embodiments, the invention is not intended to be limited to the disclosed details. Rather, the invention extends to all functionally equivalent structures, methods, and uses that are within the scope of the appended claims.

[0098] The illustrations of the embodiments described herein are intended to provide a general understanding of various embodiments. The figures are not intended to serve as a complete description of all elements and features of apparatus and systems that utilize the structures or methods described herein. Many other embodiments may be apparent to those skilled in the art upon reviewing the present disclosure. Since other embodiments may be utilized and derived from the present disclosure, structural and logical substitutions and changes may be made without departing from the scope of the present disclosure. Moreover, the illustrations are merely representative and may not be drawn to scale. Certain proportions within the illustrations may be exaggerated, while other proportions may be minimized. Therefore, the present disclosure and figures should be considered illustrative and not limiting.

[0099] One or more embodiments of the present disclosure may be referred to herein, individually and / or collectively, by the term "invention," merely for convenience and without any intention to intentionally limit the scope of the present application to any particular invention or inventive concept. Furthermore, although specific embodiments have been illustrated and described herein, it should be recognized that any subsequent arrangement designed to achieve the same or similar purpose may be substituted for the specific embodiment shown. The present disclosure is intended to cover any and all subsequent adaptations or modifications of the various embodiments. Combinations of the above embodiments, as well as other embodiments not specifically described herein, will be apparent to those skilled in the art upon reviewing this description.

[0100] The Abstract of the Disclosure is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. Additionally, the foregoing Detailed Description may group or describe various features together in a single embodiment for the purpose of streamlining the disclosure. The disclosure should not be interpreted as reflecting an intention that the claimed embodiments require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter may be directed to less than all features of any of the disclosed embodiments. Accordingly, the following claims are incorporated into the Detailed Description, with each claim standing on its own as defining separately claimed subject matter.

[0101] The subject matter disclosed above should be considered illustrative and not limiting, and the appended claims are intended to cover all such modifications, extensions, and other embodiments that fall within the true spirit and scope of the present disclosure. Accordingly, to the maximum extent permitted by law, the scope of the present disclosure should be determined by the broadest permissible interpretation of the following claims and their equivalents, and should not be limited or constrained by the foregoing detailed description.

Claims

1. A nerve regeneration scaffold, comprising: a tube having a first end and a second end opposite the first end, the tube defining a lumen having a diameter and openings at the first end and the second end, the tube comprising a biocompatible material; one or more modified nanotube yarn bundles disposed within the lumen and extending from the first end to the second end; an intermediate space between the modified nanotube yarn bundles and inside the lumen; the modified nanotube yarn bundle includes at least two nanotube yarns; the modified nanotube yarn bundles define a plurality of channels from the first end to the second end and a plurality of gaps between the modified nanotube yarn bundles; The nerve regeneration scaffold, wherein the modified nanotube yarn bundles are bundles of oxidized nanofiber yarns.

2. The nerve regeneration scaffold of claim 1 , wherein the modified nanotube yarn bundle has a water absorption rate of at least 18 mg per mg of the modified nanotube yarn bundle.

3. The nerve regeneration scaffold of claim 1 , wherein the nanotube yarn bundles are false-twisted nanofiber yarns.

4. The nerve regeneration scaffold of claim 1 , wherein the nanotube yarn is a single nanofiber yarn.

5. The nerve regeneration scaffold of claim 1 , wherein the nanotube yarn is a multi-stranded nanotube yarn.

6. 2. The nerve regeneration scaffold of claim 1, wherein the diagonal distance between the nanofiber yarn bundles defining one of the channels is between 5 μm and 20 μm.

7. The nerve regeneration scaffold of claim 1 , wherein the distance between adjacent nanotube yarn bundles defining one of the channels is between 2 μm and 15 μm.

8. The nerve regeneration scaffold of claim 1 , wherein the collective volume of the modified nanotube yarn bundles is 0.1% to 30% of the volume of the lumen.

9. The nerve regeneration scaffold of claim 1 , wherein at least one of the modified nanotube yarn bundles comprises 10 to 3000 nanotube yarns.

10. The nerve regeneration scaffold of claim 1, wherein the diameter of the nanotube yarn is in the range of 5 μm to 30 μm.

11. The nerve regeneration scaffold of claim 1 , wherein the tube comprises at least a first polymer.

12. The nerve regeneration scaffold of claim 11 , wherein the first polymer is a bioabsorbable polymer.

13. The nerve regeneration scaffold of claim 1, wherein the diameter of the lumen ranges from about 1.0 mm to about 21 mm.

14. 10. The nerve regeneration scaffold of claim 1, wherein the nanotube yarn comprises carbon nanotubes.

15. 10. The nerve regeneration scaffold of claim 1, wherein the nanofiber yarns comprise boron nitride nanotubes.

16. 10. The nerve regeneration scaffold of claim 1, wherein the intermediate spaces between the modified nanofiber yarn bundles comprise at least a second polymer between the modified nanotube yarn bundles.

17. The nerve regeneration scaffold of claim 16, wherein the second polymer is collagen, gelatin, or a matrix protein.

18. The nerve regeneration scaffold of claim 1 , further comprising at least a protein.

19. 19. The nerve regeneration scaffold of claim 18, wherein the protein is a growth factor selected from vascular endothelial growth factor, nerve growth factor, hepatocyte growth factor, neuregulin 1, glial-derived neurotrophic factor, pleiotrophin, fibrin matrix gel, or a combination thereof.

20. The nerve regeneration scaffold of claim 1 , further comprising at least one foreign body type multinucleated giant cell inhibitor.

21. The nerve regeneration scaffold of claim 1 , wherein the minimum length of the nerve regeneration scaffold is 10 mm.

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