Engineered nerve grafts, methods of making and methods of treatment using same

Abstract

The present disclosure provides an engineered nerve graft, a method of making an engineered nerve graft, and a method of repairing a nerve using an engineered nerve graft. An engineered nerve graft of the present disclosure may include a body extending from a first end to a second end, the body formed from a biocompatible hydrogel; and a plurality of microchannels extending continuously through the body from the first end to the second end, wherein each of the plurality of microchannels may have an effective diameter of about 1 microns to about 200 microns.

Description

Cross - Reference to Related Applications

[0001] This patent application claims the benefit of U.S. Non - Provisional Patent Application 18 / 531,802, filed on December 7, 2023, which claims the benefit of U.S. Provisional Patent Application 63 / 594,656, filed on October 31, 2023, and U.S. Provisional Patent Application No. 63 / 477,486, filed on December 28, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present disclosure generally relates to the fields of neurobiology and medicine. More specifically, the present disclosure relates to engineered tissues, such as engineered nerve grafts, methods of manufacturing such nerve grafts, and methods of using such engineered nerve grafts to treat nerve defects. Background Art

[0003] Regardless of its cause, nerve injury can lead to severe, in some cases profound, disability and discomfort in an object. Neuropathic injury can in particular cause chronic pain, loss of sensation, loss of partial or total muscle control, or other adverse effects. Addressing the deleterious effects of peripheral nerve injury remains a significant challenge, especially when nerve repair is delayed or when axons need to re - establish connections with surrounding targets over large nerve defects or long distances. In such cases, the regenerating axons may not have the required chemical and physiological signals to effectively regenerate and re - innervate their terminal target organs. For example, relatively long nerve defects may experience depletion of neurotrophic factors at the proximal nerve stump, while the concentration of neurotrophic factors may decline in the growth - supporting environment of the distal nerve stump.

[0004] One potential treatment for nerve injury is surgical intervention via autologous tissue replacement, where nerve tissue from an uninjured area is transplanted into the injured area of the nerve. However, autologous nerve transplantation has significant associated drawbacks, such as donor - site trauma and complications, increased complexity of the transplantation surgery, increased surgery time, scarring, and loss of sensation at the donor site. In addition, hollow conduits have been used to replace grafts to provide guidance for nerve regeneration, but these conduits lack the structural support for optimal nerve regeneration and may lead to disorganized or collapsed growth of nerve fiber cables, resulting in poor clinical outcomes.

[0005] Embodiments of the present disclosure can overcome at least one or more of the above - mentioned problems by providing engineered nerve grafts that mimic the human nerve microarchitecture to promote nerve regeneration. Summary of the Invention

[0006] According to the present disclosure, an engineered nerve graft may include: a body extending from a first end to a second end, the body being formed of a biocompatible hydrogel; and a plurality of microchannels extending continuously from the first end through the body to the second end. Each of the plurality of microchannels may have an effective diameter of from about 1 micron to about 200 microns.

[0007] In one aspect, a method of manufacturing an engineered nerve graft may include: assembling a plurality of microfibers within a liquid hydrogel such that the plurality of microfibers are generally aligned with each other; curing the liquid hydrogel to form the body of the engineered nerve graft; and removing the plurality of microfibers from the body to form a plurality of microchannels extending from a first end of the body to a second end of the body.

[0008] In another aspect, an engineered nerve graft may be used in a method of repairing a nerve. The method of repairing a nerve may include implanting the engineered nerve graft into a nerve repair site of a recipient.

[0009] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and the embodiments are presented by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. Note that just because a particular compound belongs to one general formula does not mean that it cannot also belong to another.

[0010] The singular forms "a", "an", and "the" include plural referents unless the context otherwise dictates. The terms "substantially", "about", and "approximately" mean nearly the same as the reference number or value. As used herein, the terms "substantially", "about", and "approximately" should generally be understood to encompass ±10% of the specified quantity or value. The term "or" as used in the claims and the specification is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the present disclosure supports both meanings referring to alternatives only and "and / or". As used herein, "another" may mean at least a second or more.

[0011] The foregoing general description and the following detailed description are merely exemplary and explanatory and are not restrictive of the claimed features. As used herein, the terms "comprising," "including," "containing," "having," or any other variation thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Additionally, the term "exemplary" as used herein means "an example" and not "an ideal." Further, the term "between" used to describe a numerical range is intended to include the minimum and maximum values described herein.

[0012] The terms and expressions used are for description and not for limitation, and in using such terms and expressions, it is not intended to exclude any equivalents of the features shown and described or portions thereof, but it is understood that various modifications may be made within the scope of the claimed disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The following drawings form a part of this specification and are included to further illustrate certain aspects of the present disclosure. The present disclosure can be better understood by referring to one or more of these drawings in conjunction with the detailed description of the exemplary embodiments presented herein.

[0014] According to one or more aspects of the present disclosure, Figure 1 schematically shows an engineered nerve graft.

[0015] According to one or more aspects of the present disclosure, Figure 2 is an image of an enlarged cross-section of an exemplary engineered nerve graft compared to a traditional hydrogel.

[0016] According to one or more aspects of the present disclosure, Figure 3 is a flowchart of an exemplary method for fabricating an engineered nerve graft.

[0017] According to one or more aspects of the present disclosure, Figure 4 is an image of microfibers assembled within a hydrogel.

[0018] According to one or more aspects of the present disclosure, Figure 5 is an image of microfibers assembled within a hydrogel immersed in a solvent Figure 4 .

[0019] Figure 6 , Figure 7 and Figure 8 are images from a confocal microscope showing cells within the segmented microchannels.

[0020] Figure 9 is an image of a control graft without microchannels and having DRG.

[0021] Figure 10 Image of an engineered nerve graft with microchannels.

[0022] Figure 11 Image of a control graft.

[0023] Figures 12 to 14 Image of a portion of a sample of an engineered nerve graft with microchannels.

[0024] Figures 15 to 17 Image of a portion of another sample of an engineered nerve graft with microchannels.

[0025] Figures 18 to 20 Image of a portion of another sample of an engineered nerve graft. DETAILED DESCRIPTION

[0026] Embodiments of the present disclosure relate to engineered nerve grafts and related methods of making and using engineered nerve grafts. As described above, current nerve grafts are obtained from a patient (in the case of an autograft), from a cadaver or other source (in the case of an allograft), or from another species (in the case of a xenograft). Each of these graft types (collectively referred to as harvested nerve grafts) has its own drawbacks. The engineered nerve grafts described herein provide an alternative to harvested nerve grafts and are designed to mimic the structure of endoneurial tubes in harvested nerve grafts. This is achieved by introducing microchannels into a biocompatible hydrogel matrix to mimic the microstructure of nerves such as human nerves.

[0027] Embodiments of the present disclosure include a body formed from a hydrogel matrix through which microchannels extend. The microchannels can extend from a first end of the body to a second end of the body, providing a scaffold along which nerves can regenerate when implanted into a tissue repair site. The engineered nerve grafts of the present disclosure can guide the regrowth of nerves along the conduit for repair assisted by connectors. In certain aspects, the engineered nerve grafts can also incorporate bioactive molecules and can act as a local drug delivery vehicle. Thus, the engineered nerve grafts of the present disclosure can promote nerve regeneration, which in certain aspects can in turn improve the outcomes for the recipient. Exemplary engineered nerve grafts, related methods of their preparation, and related methods of treating using nerve grafts are described in detail below.

[0028] Figure 1An engineered nerve graft 100 is schematically shown. The engineered nerve graft 100 may include a body 102 that contains microchannels 104 extending therethrough. As shown, the body 102 may extend from a first end 106 to a second end 108 along a direction L. In some embodiments, the body 102 may be substantially cylindrical, but any suitable shape may be used. The body 102 may be formed of a biocompatible material suitable for implantation in the body such that nerve axons may grow into and through the body 102 via the microchannels 104. The body 102 may have a length 110 between the first end 106 and the second end 108 that is from about 1 mm to about 200 mm. For example, the body may have a length 110 of about 1 mm to about 50 mm, about 10 mm to about 50 mm, about 15 mm to about 30 mm, about 1 mm to about 25 mm, about 5 mm to about 20 mm, about 50 mm to about 150 mm, about 50 mm to about 70 mm, about 100 mm to about 150 mm, or about 100 mm to about 120 mm. The body 102 may have a width (e.g., diameter) perpendicular to the direction L of about 0.5 mm to about 10 mm, such as about 0.5 mm to about 8 mm, about 1 mm to about 5 mm, such as about 1 mm, about 2 mm, about 3 mm, about 4 mm, or about 5 mm. The engineered nerve graft 100 may have different lengths or different diameters to facilitate use in repairing nerve injuries of different sizes. In some aspects, the engineered nerve graft 100 may be cut to a desired length prior to use by a clinician to modify the size according to the ongoing nerve repair.

[0029] As described above, the body 102 may be formed of a suitable biocompatible hydrogel material. The material forming the body 102 may be biologically inert. For example, the body 102 may include a hydrogel such as polyethyleneglycoldiacrylate (PEGDA), hyaluronic acid (HLA), an HLA-based hydrogel, a modified HLA-based hydrogel, a collagen-based hydrogel, a gelatin-based hydrogel, a photo-crosslinkable material such as lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) or a series member (a highly efficient UV-curing additive for initiating the photopolymerization of reactive prepolymers), or any other suitable biocompatible material or combination of materials. The material forming the body 102 may be UV-crosslinkable.

[0030] In some aspects, the properties of the material forming the body 102 or the curing process experienced by the hydrogel can be modified to mimic the physical properties of a nerve allograft or autograft, regulate the degradation rate of the graft, tailor the physical properties of the engineered graft to the intended use of the graft, and so on. For example, a longer graft may need to be present in the body for a longer time to allow the nerve to regenerate the graft along its entire length, while a shorter graft may require a shorter presence time. In some aspects, a longer or wider graft may be needed to have higher structural stability compared to a shorter or narrower graft. Adjusting the properties of the graft can be achieved, for example, by varying the hydrogel concentration, crosslink density, crosslinker type, curing time (e.g., UV light curing time), UV radiation dose, or other aspects of the composition or curing process. In some aspects, the physical properties of the material forming the body 102 or the curing process experienced by the hydrogel (e.g., as previously described) can be adjusted to control one or more aspects of drug delivery, such as the release of bioactive substances from the hydrogel, as will be further described below. In one example, the HLA has certain thermogel properties, so methacrylate groups can be added to the functional side chains and bonded with ultraviolet light. In some aspects, changing the concentration of the methacrylate groups can allow the properties of the body 102 to be modified.

[0031] The microchannel 104 can extend from a first end 106 of the body 102 to a second end 108 of the body 102, as Figure 1 shown. That is, the microchannel 104 can generally extend end-to-end within the body 102. Specifically, the microchannel 104 can extend continuously from the first end 106 to the second end 108 along the body 102, although there may also be manufacturing limitations that represent the possibility that some of the microchannels 104 may not extend completely from the first end 106 to the second end 108. In an embodiment, most, if not all, of the microchannels 104 can be separated from each other. In other words, each of the microchannels 104 can be separated from each other microchannel in the microchannel 104, but it is contemplated that manufacturing limitations may mean that some of the microchannels 104 may at least partially overlap each other. As described above, the microchannels 104 can be generally longitudinally aligned and proceed end-to-end to mimic the natural nerve microarchitecture.

[0032] In some embodiments, the microchannels 104 can be substantially cylindrical. The microchannels 104 can be narrower than the body 102. Specifically, the microchannels 104 can have an effective width of from about 1 micron to about 200 microns, such as from about 1 micron to about 100 microns, from about 10 microns to about 150 microns, from about 10 microns to about 70 microns, from about 40 microns to about 70 microns, from about 30 microns to about 60 microns, from about 40 microns to about 60 microns, from about 20 microns to about 30 microns, from about 10 microns to about 40 microns, or from about 10 microns to about 30 microns, e.g., diameter 112. Thus, in some embodiments, the dimensions of the microchannels 104 can be similar to those of endoneurial tubes found in tissue allografts and autografts, which can have an effective width of, for example, from about 20 microns to about 30 microns. The microchannels 104 can be arranged and distributed throughout the body 102 at a density of from about 1000 to about 30000 microchannels per square millimeter of the body, as measured in a plane perpendicular to the transverse direction L. For example, such density can be from about 5000 to about 20000 microchannels per square millimeter of the body, from about 8000 to about 12000 microchannels per square millimeter of the body, or from about 10000 to about 30000 microchannels per square millimeter of the body. As further described below Figure 2 An enlarged cross-sectional image showing an exemplary microchannel 104 within the engineered nerve graft 100.

[0033] In certain aspects, the microchannels 104 can be aligned with each other, where each microchannel 104 extends substantially parallel to the other microchannels 104. Alternatively, the microchannels 104 can extend end-to-end but can be non-parallel to each other. In some embodiments, the microchannels 104 can be spaced apart from each other at a predetermined spacing, while in other aspects, the microchannels 104 can be spaced apart randomly from each other. If desired, the alignment and spacing of the microchannels 104 can be confirmed experimentally. The microchannels 104 can be formed within the engineered nerve graft 100 such that they constitute from about 50% to about 90% of the cross-sectional area of the engineered nerve graft 100.

[0034] Still referring to Figure 1, the engineered nerve graft 100 may include a membrane 114. The membrane 114 may extend around at least a portion of the body 102 such that the membrane 114 completely or at least partially surrounds the body 102. The membrane 114 may extend completely or partially along the length 110 of the body 102, or may extend completely or partially along the outer periphery (e.g., circumference) of the body 102. The membrane 114 may be made of a biocompatible material. For example, the membrane 114 may be a natural material such as amnion-based tissue (e.g., amnion / chorion), recombinant denatured collagen, dermis, fascia, pericardium, or small intestinal submucosa (SIS). In certain aspects, the membrane 114 may be formed from a synthetic material such as a non-woven or woven structure, which may include one or more of the following monomers: glycolide, lactide, caprolactone, dioxanone, trimethylene carbonate, monomers of cellulose derivatives, and homopolymers, copolymers, and / or polymeric blends of monomers that polymerize to form polyesters. Additional synthetic materials that may be included therein to replace natural materials or that may be included in addition to natural materials include: silicone membranes, expanded-polytetrafluoroethylene (ePTFE), polyethylene terephthalate (Dacron), polyurethane aliphatic polyesters, poly(amino acids), poly(propylene fumarate), copoly(ether-ester), polyalkylenes oxalates, polyamides, tyrosine-derived polycarbonates, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyaminoesters, amino-containing polyoxaesters, poly(anhydrides), polyphosphazenes, and mixtures thereof. Natural polymers may include collagen, elastin, thrombin, fibrillin, starch, poly(amino acids), gelatin, alginate, pectin, fibrin, oxidized cellulose, chitin, chitosan, tropoelastin, hyaluronic acid, fibrin-based materials, collagen-based materials, hyaluronic acid-based materials, glycoprotein-based materials, cellulose-based materials, silk, polyglycolide (PGA), or other biocompatible materials or combinations of materials. In certain aspects, the membrane 114 may be a mesh. The membrane 114 may provide a structure through which the engineered nerve graft 100 may be sutured in place during nerve repair. The membrane 114 may provide the ability to physically support and hold the engineered nerve graft 100 in place, e.g., suturing the engineered nerve graft 100 in place such that it is configured to be implanted in the vicinity of proximal and distal nerve endings.In some embodiments, the membrane 114 may have one or more prefabricated suture holes or may have a preset suture, such as a suture (not shown), to facilitate implantation at the nerve repair site. In other aspects, the membrane 114 may allow the nerve graft 100 to more closely mimic the natural environment in vivo, or may increase the time required for the degradation of the nerve graft 100, for example, by protecting the degrading nerve graft 100 from enzymes in the environment that may degrade the nerve graft 100.

[0035] After the formation of the body 102 and / or the microchannels 104, the membrane 114 may be wrapped around the engineered nerve graft 100. The membrane 114 may then be fixed in place, for example, by joining the membrane 114 to the body 102. In certain aspects, the same or another suitable hydrogel material used to form the body 102 may be used to join the membrane 114 in place. In other aspects, an adhesive may be used to hold the membrane 114 in place, or the inner surface of the membrane 114 may be textured or may include one or more barbs, for example, to hold the membrane 114 in place on the body 102. In other aspects, the membrane 114 may be formed as a tube, and the hydrogel forming the body 102 may be poured into the membrane 114, and the body 102 may gel within the membrane 114, fixing the membrane 114 to the body 102 during the formation of the body 102.

[0036] Still referring to Figure 1 In some embodiments, the engineered nerve graft 100 may include one or more bioactive molecules (bioactive substances) 116. The engineered nerve graft 100 may be functionally modified with one or more growth factors or bioactive molecules (collectively referred to as bioactive substances 116) to further promote nerve regeneration. In this regard, the engineered nerve graft 100 may also be used as a local drug delivery system to introduce pro-regenerative cues to promote axonal regeneration into the network of microchannels 104. The bioactive substances 116 may include, for example, laminin, collagen, collagen with HLA, PLGA, or one or more other suitable bioactive substances. In certain aspects, human or animal peripheral nerve tissue may be dissolved and incorporated into the engineered nerve graft 100 as the bioactive substance 116. In other aspects, human or animal peripheral nerve tissue may be micronized into particles or powder and incorporated into the engineered nerve graft 100 as the bioactive substance 116. In certain aspects, the homogenized nerve tissue or other bioactive substances 116 may be methacrolated to make them UV curable. One or more bioactive substances 116 may promote nerve growth through the microchannels 104.

[0037] The bioactive agent 116 may be distributed throughout the body 102, may be disposed on the surface of the body 102, or may be disposed within the microchannel 104, as will be further discussed below. In some embodiments, the bioactive agent 116 may be coated or lined within the microchannel 104. The location of the bioactive agent 116 within the engineered nerve graft 100 may be selected and / or focused such that its release is controlled. For example, the proximal region of the microchannel 104 may be loaded with a bioactive agent configured to be released at a first time to promote early growth of regenerating axons into the microchannel 104, and the distal region of the microchannel 104 may be loaded with a bioactive agent configured to be released at a second time later than the first time to promote growth as the axons begin to further extend into the microchannel 104. In other examples, the distal region of the microchannel 104 may have a higher concentration of the bioactive agent as compared to the proximal region such that the bioactive agent is still released as the axons further extend into the microchannel 104. By controlling the properties of the hydrogel, the timing, rate, and / or dose of one or more bioactive agents 116 may be controlled and adjusted. Additionally, the concentration of one or more bioactive agents 116 in different regions of the body 102 may be controlled to achieve the desired release characteristics.

[0038] In embodiments where one or more bioactive agents 116 are incorporated throughout the body 102, a pre-gel or liquid hydrogel solution may be mixed with one or more bioactive agents 116. When the hydrogel solution gels, one or more bioactive agents 116 may be incorporated throughout the body 102. In other aspects, chemical crosslinking may be used to facilitate the incorporation of the bioactive agent 116. In other aspects, surface modification may be used to incorporate the bioactive agent 116. For example, at least some of the microchannels 104 may be coated with a negatively charged sulfate-based solution, and a positively charged bioactive agent 116 may then be coated onto the microchannels 104 by, for example, immersing the body 102 in a solution having one or more positively charged bioactive agents 116. In other aspects, a positively charged solution may be used to incorporate the bioactive agent 116. For example, a positively charged amino acid polymer such as a polylysine solution may be used to coat the microchannels 104. Subsequently, a negatively charged bioactive agent 116 such as laminin may be coated onto the microchannels 104. As described above, the body 102 may be immersed in a solution containing one or more charged substrate coatings and / or one or more bioactive agents 116 in order to coat the outer surface of the body 102 and / or the microchannels 104. In other aspects, the bioactive agent 116 may be entrapped on or within the body 102 and / or the microchannels 104 in a chemical or physical manner.

[0039] As will be appreciated, the engineered nerve graft 100 can provide a nerve repair product that is engineered to support and promote nerve regeneration. Embodiments of the present disclosure can provide clinicians with a hydrogel-based device for nerve repair applications and obviate the need for autologous, allogeneic, or xenogeneic grafts. Thus, a method of using the engineered nerve graft 100 can include implanting the engineered nerve graft 100 into a nerve gap of a recipient, such as between two damaged nerve ends of the recipient. The length of the nerve gap can be up to 200 mm. The length of the nerve gap can be in the range of about 1 mm to about 200 mm, in the range of about 5 mm to about 100 mm, in the range of about 10 mm to about 50 mm, in the range of about 10 mm to about 30 mm, in the range of about 15 mm to about 30 mm, in the range of about 1 mm to about 25 mm, in the range of about 5 mm to about 20 mm, in the range of about 5 mm to about 30 mm, in the range of about 50 mm to about 150 mm, in the range of about 50 mm to about 70 mm, in the range of about 100 mm to about 150 mm, or in the range of about 100 mm to about 120 mm. The engineered nerve graft 100 can be sutured in place relative to the damaged nerve ends. This can facilitate axonal regeneration into and through the microchannels 104 of the engineered nerve graft 100.

[0040] Now referring to Figure 3 and Figure 4 in combination, there is shown a method 200 of fabricating an exemplary engineered nerve graft 100. In method 200, a hydrogel matrix can be cast around aligned microfibers 120, and then the microfibers 120 can be removed, leaving a hydrogel matrix with aligned microchannels 104 to form the engineered nerve graft 100.

[0041] Method 200 can include a step 202 of assembling the microfibers 120 within a liquid or pre-gel hydrogel 122. In some embodiments, the hydrogel 122 and the microfibers 120 can be assembled within a mold 124 so as to maintain the shape of the hydrogel 122. The mold 124 can completely or partially surround the hydrogel 122 and the microfibers 120. In some embodiments, the mold 124 can be formed of a transparent material that permits UV light to pass through the mold 124. The mold 124 can be cylindrical in some aspects to give the hydrogel a shape similar to that of a nerve. In some embodiments, the hydrogel 122 can be cast around the microfibers 120.

[0042] The microfibers 120 can be generally aligned with each other such that they extend in a similar direction within the hydrogel 122. The microfibers 120 can be aligned with each other, and each microfiber 120 extends substantially parallel to the other microfibers 120 or, if not parallel, end-to-end adjacent to each other. In some embodiments, the microfibers 120 can be placed at a predetermined spacing from each other, while in other respects, the spacing between the microfibers 120 can be random. The microfibers 120 can have an effective width that matches the effective diameter of the microchannels 104 described above, such as the diameter 112. For example, the diameter 112 of the microfibers 120 can be from about 1 micron to about 200 microns, from about 1 micron to about 100 microns, from about 10 microns to about 150 microns, from about 10 microns to about 70 microns, from about 40 microns to about 70 microns, from about 30 microns to about 60 microns, from about 40 microns to about 60 microns, from about 20 microns to about 30 microns, from about 10 microns to about 40 microns, or from about 10 microns to about 30 microns. The microfibers 120 can be generally aligned with each other within the hydrogel 122, as Figure 4 shown. The microfibers 120 are used as a mold to form the microchannels 104 within the engineered nerve graft 100. In some aspects, the microfibers 120 can be made of a dissolvable material such as cellulose acetate, such that once cured, the microfibers 120 can be dissolved out of the hydrogel 122. That is, the microfibers 120 can be composed of a material that can be selectively dissolved without affecting the surrounding hydrogel 122. In other embodiments, the microfibers 120 can be multiple sutures arranged generally parallel to each other, such as 8-0 to 10-0 sutures. In other embodiments, the microfibers 120 can be formed as part of a mold made of, for example, resin, polymer, wire, or other suitable materials. In some aspects, the mold used to form the body 102 and / or the microchannels 104 can be additively manufactured or 3D printed. As described above, the hydrogel 122 can be a suitable biocompatible hydrogel material, such as PEGDA, HLA, collagen-based hydrogels, gelatin-based hydrogels, fibrin-based hydrogels, photo-crosslinkable materials such as LAP, or one or more of any other biocompatible materials or combinations of materials. In some embodiments, the hydrogel 122 can also contain one or more bioactive substances 116, such as laminin, collagen, collagen with HLA, micronized nerve tissue, or one or more other suitable bioactive substances. The number of microfibers 120 assembled within the liquid hydrogel can be approximately equal to the number of microchannels 140 incorporated into the ultimately produced engineered nerve graft 100.

[0043] Method 200 may also include a step 204 of curing the hydrogel 122. In an embodiment, this may include curing the hydrogel 122 with ultraviolet (UV) light (e.g., photo-crosslinking) or heat so that the hydrogel 122 is substantially solid. As used herein, "substantially solid" means capable of maintaining a shape and is not intended to imply rigidity or stiffness. The hydrogel 122, when cured, may form the body 102 of the engineered nerve graft 100 or a precursor of the body 102 (as Figure 1 shown). The step 204 of curing the hydrogel 122 with UV light can be performed for a predetermined amount of time with a predetermined UV radiation dose or UV light intensity. The predetermined amount of time and the predetermined dose for curing the hydrogel 122 can be selected based on the intensity of the UV light, the concentration of the crosslinker, or the desired physical properties or bioactivity release profile of the engineered nerve graft 100 obtained, such as hardness or degradation rate after implantation in the body. The predetermined amount of time can be, for example, from about 5 seconds to about 10 minutes, from about 10 seconds to about 10 minutes, or about 5 minutes. The UV light intensity can be, for example, from about 0.5 mW / cm2 to about 40 mW / cm2, or about 4 mW / cm2. Also, before the step 204 of curing the hydrogel 122, method 200 may include a step of adding one or more crosslinkers to the hydrogel 122. The number or type of crosslinkers added can also be selected according to the desired physical properties of the engineered nerve graft 100 obtained. The one or more crosslinkers may include Irgacure or lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). As a specific example, LAP at a concentration of 2.5 mg / ml can be used. The LAP concentration can be varied to obtain the desired physical properties and curing time. In the case of adding one or more crosslinkers to the hydrogel 122, the step 204 of curing the hydrogel 122 may also be referred to as crosslinking. The predetermined time depends at least in part on the concentration of the one or more crosslinkers added. After the step 204 of curing the hydrogel 122, the microfibers 120 can be distributed in the solid hydrogel 122. In the solid hydrogel 122, the distribution of the microfibers 120 corresponds to the position of the microchannels 104 within the body 102. In an embodiment where the hydrogel 122 contains one or more bioactive substances 116, the bioactive substances 116 can be dispersed throughout the body 102 or concentrated in one or more regions of the body 102. After curing the hydrogel 122, the body 102 can be removed from the mold 124.

[0044] Referring to Figure 3 and 5In the combination of, method 200 may include step 206 of removing microfibers 120 from the self-curing hydrogel 122 to form microchannels 104. In some aspects, for example, if cellulose acetate fibers are used as microfibers 120, removing microfibers 120 may include dissolving microfibers 120. In other words, in step 206, microfibers can be chemically removed from the cured hydrogel 122, leaving the cured hydrogel 122 containing microchannels 104. If microfibers 120 are formed from, for example, sutures, resins, polymers, wires, or other materials, or using molds or 3D printing, removing such microfibers can be performed by physically pulling the microfibers out of the cured hydrogel 122, obtaining a hydrogel 122 with substantially aligned microchannels 104. In some embodiments, step 206 may optionally include trimming the ends of the body 102 to expose microfibers 120. This can ensure that the microchannels 104 created by microfibers 120 will extend from the first end 106 of the engineered nerve graft 100 to the second end 108 of the engineered nerve graft 100. Additionally, trimming the ends of the body 102 can increase the exposure of microfibers 120 to the liquid or other materials used to dissolve microfibers 120. If microfibers 120 are dissolved in the cured hydrogel 122, trimming to expose microfibers 120 also allows the solution to contact microfibers 120 to dissolve them.

[0045] To dissolve microfibers 120, step 206 may include immersing the body 102 and microfibers 120 in a solvent 128. Specifically, step 206 may include immersing the body 102 and microfibers 120 in a solvent 128 for at least about 2 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours, at least 36 hours, or at least 48 hours. In some aspects, the body 102 may be immersed for about 2 hours to about 48 hours, about 2 hours to about 36 hours, or about 8 hours to about 24 hours to dissolve microfibers 120 from the body 102. The amount of time immersed may depend at least in part on the type of microfibers 120 used, the type of solvent 128 used, the size of the microfibers 120 used, the number of microfibers 120 used, the type of hydrogel 122 used, the size of the hydrogel 122 used, etc. The solvent 128 can be selected such that it can selectively dissolve microfibers 120 without dissolving the body 102 or having a negative impact on the body 102. For example, in some embodiments, microfibers 120 may be cellulose acetate fibers, and the solvent 128 may include acetone. In such embodiments, the body 102 may be formed from, for example, PEGDA. In such embodiments, the acetone of the solvent 128 can dissolve or substantially dissolve the cellulose acetate of microfibers 120 without dissolving the body 102 or otherwise having a negative impact on the body 102.

[0046] In some embodiments, when the body 102 is immersed in the solvent 128, stirring or agitation may be performed to facilitate the selective dissolution of the microfibers 120. For example, agitation at about 20 rotations per minute (RPM) to about 80 RPM may be used, such as about 50 RPM, about 60 RPM, or about 70 RPM.

[0047] In embodiments where sutures are used as the microfibers 120, if dissolvable sutures are used, the sutures themselves may be dissolved. In other cases, the sutures may be pulled out from the body 102. Additionally, if a mold formed of, for example, resin, polymer, wire, or other suitable materials is used, the female mold may be removed (e.g., physically pulled out from the body 102), leaving microchannels 104 within the body 102.

[0048] As will now be understood, after removing the microfibers 120, the body 102 may remain and may contain the microchannels 104 described above. Thus, after removing the microfibers 120, the remaining body 102 may form an engineered nerve graft 100 (as Figure 1 shown).

[0049] Referring Figure 1 and Figure 3 in combination, the method 200 may include an optional step 208 of coating the microchannels 104 with one or more bioactive substances 116. Step 208 may include first coating the microchannels 104 with a negatively charged solution such as a sulfate solution. In other embodiments, step 208 may include first coating the microchannels 104 with a positively charged solution such as a polylysine solution. For example, coating the microchannels 104 with a charged solution may be related to immersing the body 102 in the charged solution. Step 208 may include immersing the body 102 in one or more bioactive substances 116 such that after immersion, the bioactive substances 116 coat the microchannels 104. If a negatively charged solution is applied before coating with one or more bioactive substances 116, then a positively charged bioactive substance 116 may then be used to coat the microchannels 104. If a positively charged solution is applied before coating with one or more bioactive substances 116, then a negatively charged bioactive substance 116 such as laminin may then be used to coat the microchannels 104.

[0050] Still referring Figure 1 and Figure 3For the combination, method 200 may include an optional step 210 of applying membrane 114. Step 210 may include wrapping the body 102 in membrane 114. As described above, membrane 114 may at least partially or completely cover the perimeter or length of body 102. After membrane 114 is disposed on the outer surface of body 102, the membrane may be fixed in place via an adhesive, via curing or joining the membrane 114 in place, or via another suitable process. As described above, in some aspects, membrane 114 may be joined in place using the same or another suitable hydrogel material as that used for body 102. In other aspects, an adhesive may be used to hold membrane 114 in place. In other aspects, the outer surface of body 102 may be rough, or the inner surface of membrane 114 may be textured or may include one or more barbs, for example to hold membrane 114 in place on body 102. In other aspects, step 210 may be performed in combination with step 202 and / or step 204. For example, membrane 114 may be formed as a tube and used as a mold, and the hydrogel 122 forming body 102 may be poured into membrane 114 during step 202, and body 102 may form a cured hydrogel within membrane 114, fixing membrane 114 to body 102. Thus, membrane 114 can be fixed to body 102 in any suitable manner. Although reference is made to one layer of membrane 114, more than one layer of membrane 114 may also be applied to body 102, or multiple layers of membrane 114 may be applied to body 102.

[0051] Example 1

[0052] Cellulose acetate fibers with a diameter of 40 to 70 microns are used in combination with PEGDA hydrogel to form an engineered nerve graft. The cellulose acetate fibers are aligned with each other in the liquid PEGDA hydrogel, and then the hydrogel is crosslinked and cured using UV light. After crosslinking, the ends of the cured hydrogel are trimmed to increase the exposure of the cellulose acetate fibers. Then the cured hydrogel with cellulose acetate fibers is immersed in an acetone solution at a stirring setting of 60 RPM for about 24 hours to dissolve the cellulose acetate fibers. Figure 2 The six images on the far right in [figure number] show an enlarged cross-sectional view of the engineered nerve graft after dissolution of the cellulose acetate fibers. Figure 2 The six images on the far right in [figure number] show microchannels with increasing magnification from left to right. These can be compared with Figure 2 the images of the control hydrogel shown in the two leftmost images in [figure number]. Visual comparison shows that more defined and larger microchannels are formed in the engineered nerve graft compared to the control hydrogel.

[0053] Example 2

[0054] The migration of cells into the microchannels of an engineered nerve graft was evaluated. Specifically, an engineered nerve graft was formed having a hydrogel body formed of 10% PEGDA gel in which microchannels were formed. The engineered nerve graft was cut into fragments approximately 3 mm in length. The diameter of the fibers used to form the microchannels was between approximately 40 μm and 70 μm. The graft fragments were transferred into a 6-well plate inside a silicone insert, and a cell matrix was added. Then the graft fragments were incubated at 37 °C for 30 minutes. Then the graft fragments were transferred to a 24-well plate, and neuroblastoma stem cells - 34 (NSC-34) and additional cell matrix were added. NSC-34 is a hybrid cell line produced by fusing spinal cord cells rich in mouse embryonic motor neurons with mouse neuroblastoma. Then the graft fragments were incubated at 37 °C for 24 hours, followed by imaging using phase contrast and confocal microscopy. Figure 6 , Figure 7 and Figure 8 are images from a confocal microscope showing cells within the microchannels of the segments. In the images of these figures, the cultured NSC-34 has a diameter of less than 10 μm. Specifically, Figure 6 shows the top edge of the graft fragment, with green indicating phalloidin-conjugated F-actin (FITC). Figure 7 and Figure 8 are images with different focal planes compared to Figure 6 and show cells within multiple microchannels at different depths within the graft fragment. The evaluation showed that a cell matrix with a diameter of approximately 10 μm could migrate within the microchannels of the engineered nerve graft.

[0055] Example 3

[0056] The neurite growth in the aligned microchannels of an engineered nerve graft formed of 10% PEGDA gel was evaluated. Specifically, according to the present invention, rat embryos (E18) dorsal root ganglions (DRG) were obtained and placed on an engineered nerve graft having microchannels, and the main body of the graft was formed of 10% PEGDA gel. The DRG and the graft were incubated at 37 °C, 5% CO2 for 7 days. After incubation, the graft was fixed in 10% formalin, stained with βIII tubulin antibody, and imaged using phase contrast and confocal microscopy. Figure 9 is an image of a control graft without microchannels with DRG placed thereon, while Figure 10 is an image of an engineered nerve graft having microchannels as described above. As Figure 9As shown, no neurite extension through the hydrogel matrix of the control graft occurred, while as Figure 10 shown, neurite extension through the microchannels within the engineered nerve graft occurred. Thus Figure 9 and Figure 10 show that the microchannels allow neurites to extend therein and promote neurite growth into the engineered nerve graft.

[0057] Figures 11 to 20 Images of a control graft without microchannels and three sample engineered nerve grafts with microchannels based on video screenshots, respectively. Figure 11 Image of the control graft, showing no neurite extension within the hydrogel matrix in the absence of microchannels. Figures 12 to 14 Image of a portion of one sample (Sample 1) of the engineered nerve graft with microchannels, showing neurite extension through its microchannels stained in red. Figures 15 to 17 Image of a portion of another sample (Sample 2) of the engineered nerve graft with microchannels, showing neurite extension through its microchannels stained in red. Figures 18 to 20 Image of a portion of another sample (Sample 3) of the engineered nerve graft, showing neurite extension through its microchannels stained in red. The assessment shows that neurite extension from the DRG occurred within the microchannels of the engineered nerve graft, while no neurite extension occurred within the hydrogel of the control graft lacking microchannels. Thus, it can be inferred that neurite extension occurred within the microchannels rather than into the hydrogel, and thus the hydrogel was able to separate the individual microchannels from each other to prevent or avoid cross-talk between the microchannels. The assessment also shows that no neurite extension occurred on the outer surface of the engineered nerve graft.

[0058] With the engineered nerve graft 100 and related methods 200 described herein, a biocompatible matrix with a microarchitecture similar to that of human nerves is provided to permit nerve regeneration. More specifically, uniaxially aligned three-dimensional microchannels formed by physically extracting or chemically and / or enzymatically dissolving microfibers in a hydrogel matrix that promotes regeneration are used to form a biocompatible matrix that mimics the human nerve microarchitecture. Such nerve grafts can be used, for example, in the field of nerve repair, particularly for repairing or filling short to medium-sized gaps within a nerve. Additionally, compared to autograft and allograft, the engineered nerve grafts according to the present disclosure can be relatively longer and / or thicker (i.e., can have a relatively larger diameter). Furthermore, the engineered nerve grafts formed from biocompatible hydrogels as described herein can be used as alternatives to autograft and allograft to assist in the regeneration of damaged tissues including, for example, nerve tissue. Still further, the engineered nerve grafts as described herein can provide improved clinical outcomes compared to grafts using hollow conduits because the aligned microchannels mimic the microarchitecture of human nerves and provide a supportive structure for nerve regeneration along the entire length of each graft. In other words, the engineered nerve grafts according to the present disclosure provide end-to-end support for nerve regeneration throughout the graft.

[0059] In some aspects, the engineered nerve grafts can be designed to provide properties not obtainable in naturally occurring autograft, allograft, or xenograft. For example, the grafts according to the present disclosure can be designed to have a wider diameter or longer length than what occurs naturally. The engineered grafts can have shapes not found in nature, such as a branched configuration or a curved profile. They can be filled with one or more bioactive substances, or can have controlled degradation properties. The physical properties of the engineered grafts can be affected such that, compared to natural nerves, the endoneurial microchannels intended to mimic those in nerve grafts can be distributed over a wider cross-section of the engineered graft. Thus, while only a portion of a natural nerve graft can be used to provide a scaffold for nerve regeneration, a larger portion or nearly all of the engineered nerve graft can be used to provide a scaffold for nerve regeneration.

[0060] Moreover, with the engineered nerve graft 100 according to the present disclosure, and by using a biocompatible material to form the membrane 114, for example, if the membrane degrades over time, it can prevent interference with nerves (or neurites) growing inside and around the engineered nerve graft after implantation into a recipient.

[0061] It should be understood that although the present disclosure is obtained with reference to preferred embodiments, exemplary embodiments, and optional features, those of ordinary skill in the art may make modifications and variations to the concepts disclosed herein, and such modifications and variations are considered to fall within the scope of the present disclosure as defined by the appended claims. The specific embodiments and examples provided herein are examples of useful embodiments of the present disclosure and are non-limiting and are for illustrative purposes only. It will be apparent to those of ordinary skill in the art that numerous variations of the apparatus, apparatus components, methods, and steps described in this specification may be used to practice the present disclosure. As will be understood by those of ordinary skill in the art, the means and apparatus that may be used in the present method may include a large variety of alternative compositions and processing components and steps. Aspects of the present invention will now be described in detail, examples of which are illustrated in the accompanying drawings. Wherever possible, the same or similar reference numerals will be used throughout the drawings to refer to the same or like parts. The term "distal" refers to the part that is furthest from the user when the device is introduced into a subject (e.g., a patient) body. Conversely, the term "proximal" refers to the part that is closest to the user when the device is placed in the subject body.

Claims

1. An engineered nerve graft, comprising: A body extending from a first end to a second end, the body being formed of a biocompatible hydrogel; And A plurality of microchannels extending continuously from the first end through the body to the second end, wherein each of the plurality of microchannels has an effective diameter of about 1 micron to about 200 microns.

2. The engineered nerve graft according to claim 1, wherein each of the plurality of microchannels has an effective diameter of about 1 micron to about 100 microns.

3. The engineered nerve graft according to claim 1, wherein the plurality of microchannels are arranged in the body at a density of about 1000 to about 30000 microchannels per square millimeter of the body.

4. The engineered nerve graft according to claim 1, further comprising a membrane extending at least partially around an outer surface of the body.

5. The engineered nerve graft according to claim 1, further comprising one or more bioactive molecules distributed throughout the body.

6. The engineered nerve graft according to claim 1, further comprising one or more bioactive molecules disposed in at least some of the plurality of microchannels.

7. The engineered nerve graft according to claim 1, further comprising one or more bioactive molecules, wherein the one or more bioactive molecules include one or more of laminin, collagen, or collagen having hyaluronic acid.

8. The engineered nerve graft according to claim 1, wherein the body comprises one or more of polyethylene glycol diacrylate, hyaluronic acid, a collagen-based hydrogel, or a gelatin-based hydrogel.

9. The engineered nerve graft according to claim 1, wherein the body comprises a photo-crosslinked hydrogel.

10. A method of manufacturing an engineered nerve graft, the method comprising: Assembling a plurality of microfibers within a liquid hydrogel such that the plurality of microfibers are generally aligned with each other; Curing the liquid hydrogel to form the body of the engineered nerve graft; And Removing the plurality of microfibers from the body to form a plurality of microchannels extending from a first end of the body to a second end of the body.

11. The method according to claim 10, wherein each of the plurality of microfibers has an effective diameter of about 1 micron to about 200 microns.

12. The method according to claim 10, wherein the plurality of microchannels are formed in the body at a density of about 1000 to about 30000 microchannels per square millimeter of the body.

13. The method according to claim 10, wherein the plurality of microfibers are formed of one or more of: dissolvable fibers, sutures, resins, polymers, or wires.

14. The method according to claim 10, wherein the plurality of microfibers are cellulose acetate fibers.

15. The method according to claim 10, wherein removing the plurality of microfibers comprises dissolving the plurality of microfibers.

16. The method according to claim 15, wherein dissolving the plurality of microfibers comprises immersing the body in a solvent comprising acetone.

17. The method according to claim 10, wherein removing the plurality of microfibers comprises physically pulling the plurality of microfibers out of the body.

18. The method according to claim 10, wherein curing the hydrogel comprises photo-crosslinking the hydrogel.

19. The method according to claim 10, further comprising coating at least some of the plurality of microchannels with one or more bioactive molecules.

20. The method according to claim 19, wherein coating at least some of the plurality of microchannels comprises immersing the body in a solution comprising the one or more bioactive molecules.

21. The method according to claim 10, further comprising applying a membrane to an outer surface of the body.

22. A method of repairing a nerve using an engineered nerve graft, the method comprising: implanting the engineered nerve graft into a nerve repair site of a recipient, the engineered nerve graft comprising: a body extending from a first end to a second end, the body formed of a biocompatible hydrogel; and a plurality of microchannels extending from the first end through the body to the second end, wherein each of the plurality of microchannels has an effective diameter of from about 1 micron to about 200 microns.

23. The method according to claim 22, wherein the plurality of microchannels are arranged at a density of from about 1000 to about 30000 microchannels per square millimeter of the body.

24. The method according to claim 22, further comprising suturing the engineered nerve graft in place at the nerve repair site.

25. The method according to claim 24, wherein the engineered nerve graft further comprises a membrane extending at least partially around an outer surface of the body, and wherein suturing the engineered nerve graft in place comprises suturing through the membrane.

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