Epineural repair device and method of use thereof

Abstract

The subject invention generally relates to repairing severed or damaged nerves and / or epineurium in patients in need of such treatment. In particular, the invention provides devices and methods for repairing severed or damaged nerves and / or epineurium. In some embodiments, a device is provided for repairing severed or damaged nerves and / or epineurium in vivo, comprising a flexible collagen membrane including a first surface and a second surface, the first surface having a plurality of protruding collagen bundles, and the second surface having a plurality of openings, the openings and the collagen bundles being configured to form a self-mating connection when the first surface and the second surface are placed together.

Description

[Technical field]

[0001] Priority claims and related applications This application claims priority to Australian Provisional Patent Application No. 2021901244, filed April 27, 2021, which is incorporated by reference in its entirety.

[0002] The subject invention relates generally to repairing severed or damaged nerves and / or epineurium in patients in need of such treatment. [Background technology]

[0003] Nerves carry the peripheral processes (or axons) of neurons. Nerve cell bodies are found in the spinal cord (motor neurons), in ganglia located along the spinal column (spinal sensory ganglia), or in ganglia found throughout the body's organs (autonomic and enteric ganglia). Nerves are composed of axons, Schwann cells, and a sheath of diverse connective tissue. The outer covering (epineurium) consists of collagenous connective tissue that cushions external pressure on the nerve bundle and surrounds the perineurium. The perineurium surrounds individual nerve bundles and, together with endothelial cells in the endoneurial microvessels, acts as a blood-nerve barrier. The endoneuriium lies within the perineurium and is composed of collagenous tissue that surrounds Schwann cells and axons. Nerve bundle groups are composed of two or more nerve bundles, each surrounded by a perineurium and an epineurium. The topography of nerves is consistent distally, with groups of nerve bundles that are either sensory or motor. A neuron consists of a soma (cell body) and an axon, which can reach several feet in length.

[0004] Damage to nerves can be caused by physical injury or swelling (e.g., carpal tunnel syndrome), autoimmune diseases (e.g., Guillain-Barré syndrome), infections (neuritis), diabetes, or failure of the blood vessels surrounding the nerve. Nerve injury is a major cause of chronic disability. Poorly managed nerve injury is associated with muscle atrophy and can lead to painful neuromas when severed axons are unable to reestablish continuity with the distal nerve. Although nerves have the potential to regenerate after injury, this ability is strictly dependent on regenerative nerve fibers providing adequate contact with the severed nerve segment.

[0005] Repair of injured nerves is hindered by several factors, including damage to the protective sheath surrounding the nerve (epineurium), gaps between the proximal and distal ends of the severed nerve, and the limited ability of injured nerves to regenerate.

[0006] Various sutureless nerve repair methods have been explored, including the use of biological adhesives. Advantages of adhesive techniques include ease of application and potentially rapid repair times. Unfortunately, biological adhesives such as fibrin glue lack the adhesive properties and tensile strength to ensure reliable nerve fusion in most nerve repairs without the aid of devices such as tubes or conduits.

[0007] Tubes or conduits for inducing peripheral nerve regeneration are commonly made of materials such as polylactide, polylactide / polyglycolide copolymers, acrylic copolymers, active mesothelial tubes, or various other synthetic polyesters. Disadvantages of using tubes or conduits made of these materials include, for example, immune response, induction of scar tissue, and difficulty in application. For example, many of these conduits are too rigid and not easily adapted for in vivo use.

[0008] One of the main problems in nerve repair is epineurium repair. When using currently available devices and techniques, epineurium healing is often found to be insufficient. Although such devices have been shown to have the ability to fill voids or gaps in nerves or even achieve fusion of severed nerves, repaired nerves often have problems such as scar tissue; fibrous adhesions to surrounding soft tissues in the patient; inflammation, lack of neurofilament proliferation, and failure to regenerate axons across the repair site.

[0009] Thus, a need remains for materials and techniques that provide a safe and effective means of repairing severed or damaged nerves and / or epineurium in patients. Summary of the Invention

[0010] In a first aspect, the present invention provides a device for repairing a severed or damaged epineurium in vivo, comprising a flexible collagen membrane including a first surface and a second surface, said first surface having a plurality of protruding collagen bundles and said second surface comprising a plurality of openings, said openings and said collagen bundles configured to form a self-interlocking interlocking when said first surface and said second surface are placed together.

[0011] In a second aspect, the invention provides an in vivo method of repairing a severed or damaged epineurium, comprising: (i) providing a device for repairing a severed or damaged epineurium in vivo, comprising a flexible collagen membrane including a first surface and a second surface, said first surface having a plurality of protruding collagen bundles and said second surface comprising a plurality of openings, said openings and said collagen bundles configured to form a self-interlocking interlocking when said first surface and said second surface are placed together; (ii) wrapping the flexible membrane in situ around at least a longitudinal portion of a severed or damaged nerve, wherein the severed or damaged nerve includes a severed or damaged epineurium; (iii) allowing the device to remain in place for a period of time until the severed or damaged epineurium is repaired; The present invention provides a method comprising:

[0012] In a third aspect, the present invention provides a device for repairing severed or damaged nerves in vivo comprising a flexible collagen membrane including a first surface and a second surface, said first surface having a plurality of protruding collagen bundles and said second surface comprising a plurality of openings, said openings and said collagen bundles configured to form a self-mating interlocking connection when said first surface and said second surface are placed together.

[0013] In a fourth aspect, the invention provides an in vivo method of repairing a severed or damaged nerve, comprising: (i) providing a device for repairing a severed or damaged nerve in vivo comprising a flexible collagen membrane including a first surface and a second surface, said first surface having a plurality of protruding collagen bundles and said second surface comprising a plurality of openings, said openings and said collagen bundles configured to form a self-mating interlocking connection when said first surface and said second surface are placed together; (ii) wrapping the flexible membrane in situ around at least a longitudinal portion of the severed or damaged nerve; (iii) allowing the device to remain in place for a period of time until the severed or damaged nerve is repaired; The present invention provides a method comprising:

[0014] In a fifth aspect, the present invention provides a method for producing a composition comprising: (i) providing a donor nerve including an epineurium; (ii) providing a device for repairing a severed or damaged nerve in vivo, comprising a flexible collagen membrane including a first surface and a second surface, said first surface having a plurality of protruding collagen bundles and said second surface comprising a plurality of openings, said openings and said collagen bundles configured to form a self-mating interlocking connection when said first surface and said second surface are placed together; (iii) suturing the donor nerve in place and then wrapping the flexible collagen membrane around at least a longitudinal portion of the donor and recipient nerves to contain the epineurium; (iv) allowing the device to remain in place until nerve fusion occurs; The present invention provides an in vivo nerve transplantation method, comprising:

[0015] In some embodiments, the openings and the collagen bundles are configured to interlock with one another when the first surface and the second surface are placed together.

[0016] In some embodiments, the membrane is an elongated profile membrane.

[0017] In some embodiments, the flexible collagen membrane is configured to be capable of being wrapped in situ around at least a longitudinal portion of a mammalian nerve in a manner such that the first surface and the second surface are positionable together to self-interlock with one another.

[0018] In some embodiments, the mammalian nerve comprises a severed or damaged epineurium, wherein the flexible collagen membrane is less than 200 microns thick, and wherein the device, when wrapped around the severed or damaged nerve, has the ability to repair the severed or damaged epineurium in less than four weeks.

[0019] In some aspects, the period of time is sufficient to allow the device to successfully integrate with the nerve or epineurium being repaired. Successful integration can be assessed visually or histologically.

[0020] In some embodiments, a damaged nerve or damaged epineurium causes the defect site.

[0021] In a sixth aspect, the present invention provides a repair kit for in vivo repair of a nerve and / or epineurium, comprising: (i) a device for in vivo repair of a severed or damaged nerve and / or epineurium, comprising a flexible collagen membrane including a first surface and a second surface, said first surface having a plurality of protruding collagen bundles and said second surface comprising a plurality of openings, said openings and said collagen bundles configured to form a self-interlocking interlocking when said first surface and said second surface are placed together; (ii) a sterile container for holding the device; (iii) instructions for coupling the device to a nerve and / or epineurium in need of repair; A kit comprising:

[0022] Optionally, the repair kit further comprises one or more of the following: sutures; forceps, a suture needle, fibrin glue, and accessory tools for accessing tissue (eg, clips, reference weights, an aspiration device, and a compression gauge).

[0023] The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0024] [Figure 1]FIGURE 2 shows a surgical procedure in a pre-clinical study of the nerve repair device (10) of the present invention. The sciatic nerve was completely severed and then repaired (16) using epineural microsutures. Non-absorbable sutures (18) were used to retract the repair site (20) with the nerve repair device (10) of the present invention attached to the nerve stump (12) on either side of the repair site (20). [Diagram 2] Illustrated is the placement of A) a nerve repair device of the present invention, and B) an FDA approved collagen nerve device (NEURAWRAP®) in a rat model of peripheral nerve injury. White arrow - implanted device. [Diagram 3] Figure showing a rat model of peripheral nerve injury (4 weeks after treatment) - A) and B) nerve repair using a nerve repair device of the present invention; C) control (unoperated contralateral side) of the nerve repair device of the present invention; D) and E) FDA approved collagen nerve device (NEURAWRAP®); F) control (unoperated contralateral side) of the FDA approved device (NEURAWRAP®). White arrow - nerve device; green arrow - adhesion of nerve device to surrounding soft tissue. [Figure 4] Figure 1 shows H&E staining of nerve tissue from a rat peripheral nerve injury study. A) Nerve repair device of the present invention; B) FDA approved collagen nerve device (NEURAWRAP®). Black scale bar = 500 μm. Blue arrow - device; green arrow - nerve fibers; yellow star - inflammatory cells. [Diagram 5] FIG. 1 shows motor recovery after nerve transfer to improve arm and hand function in people with tetraplegia. Recovery of M3 or better is classified as functional motor recovery (FMR). [Figure 6] FIG. 1 shows recovery of triceps function in CG-006 compared to a published study of nerve transplantation in people with tetraplegia. [Figure 7]1 shows the transverse structure of the nerve repair device of the present invention. It can be seen that the flexible collagen membrane in the nerve repair device of the present invention comprises a bilayer including a first surface that is smooth and a second surface that is rough. The first surface and the second surface have the ability to interlock together in a locking manner. [Figure 8] 14 shows that the collagen bundles on the rough side of the nerve repair device of the present invention can collapse into the pores of the smooth side of the device to create a mating adhesive surface to encase the nerve and hold it in its shape (similar to the action of VELCRO®) when overlapping under humid conditions similar to those found in vivo. This allows the nerve repair device of the present invention to protect the nerve from soft tissue compression and act as an epineurium-like barrier structure for nerve protection. [Figure 9] FIG. 13 shows the measurement of myelinated axons at the distal site. It can be seen that after 4 weeks, the nerve repair device of the present invention induced 97% axonal preservation compared to 68% for the NEURAWRAP® product. [Figure 10] Panel A shows an SEM of a flexible collagen membrane folded with the rough (first surface) inside and the smooth (second surface) outside. Panel B shows the first (rough) surface in profile; Panel C shows the alignment of the smooth (second surface); Panel D shows the spacing of collagen fibrils; Panel E shows the frequency distribution of collagen fiber diameters within the flexible collagen membrane. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The subject invention provides devices and methods for repairing severed or damaged nerves and / or epineurium in patients in need of such treatment. In the following description, several terms related to medical devices and nerve repair are utilized. In order to provide clarity and consistency in the understanding of the specification and claims, the following definitions are provided, including the scope to be given to such terms.

[0026] In this specification, the terms "one embodiment," "one embodiment," "example embodiment," "further embodiment," "alternative embodiment," and the like are used for linguistic convenience. It is intended that any particular feature, structure, or characteristic described in connection with such an embodiment is included in at least one embodiment of the invention. The appearance of such phrases in various places in this specification does not necessarily refer to the same embodiment. In addition, any element or limitation of any invention or embodiment disclosed herein can be combined (individually or in any combination) with any and / or all other elements or limitations or with any other invention or embodiment disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.

[0027] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Terms, such as those defined in commonly used dictionaries, should be interpreted to have a meaning consistent with their meaning in the context of the present specification and the related technical field, and should not be interpreted in an idealized or overly formal sense unless expressly defined herein. Well-known functions or constructs may not be described in detail for brevity and / or clarity.

[0028] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms "comprises" and / or "comprising," as used herein, are further understood to specify the presence of the stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. As used herein, idiomatic phrases such as "X to Y" and "about X to Y" should be interpreted as including X and Y. As used herein, idiomatic phrases such as "about X to Y" mean "about X to about Y." As used herein, idiomatic phrases such as "about X to Y" mean "about X to about Y."

[0029] The term "about" as used herein refers to a deviation of 10% above or below the numerical value following the term.For example, when describing about 70% ethanol, it includes 63% to 77%, i.e., a range of 10% above or below the numerical value 70%.This includes 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, and 77% ethanol.

[0030] The term "patient" as used herein describes any mammal to which the devices and methods of the present invention are applied. Mammalian species that can benefit from the disclosed devices and methods include, but are not limited to, humans, apes, chimpanzees, orangutans, monkeys; domesticated animals (e.g., pets), such as dogs, cats, guinea pigs, hamsters, etc.; veterinary use for large animals, such as cows, horses, goats, sheep, etc.; and any wild animals for veterinary purposes. The age of the human or non-human animal patient can range from young to elderly.

[0031] The term "surgeon," as used herein, is merely for linguistic convenience. It should not be construed as limiting in any way. The devices, apparatus, methods, techniques, and / or procedures of the subject invention may be utilized by any person who desires or needs to do so and who has the requisite skill and understanding of the invention.

[0032] The terms "nerve" and "nerve tissue" as used herein interchangeably refer to the entire structure of a mammalian nerve. It is well understood by those skilled in the art that nerves are composed of axons, Schwann cells, and an extensive sheath of connective tissue. The outer covering (epineurium) consists of collagenous connective tissue that cushions external pressure on the nerve bundle and surrounds the perineurium. The perineurium surrounds the individual nerve bundles and, together with endothelial cells in the endoneurial microvessels, functions as a blood-nerve barrier. The endoneurium lies within the perineurium and is composed of collagenous tissue that surrounds Schwann cells and axons. Thus, when describing "repair" of a damaged or severed nerve, it refers equally to the damaged or severed structures, such as the endoneurium, perineurium, and epineurium.

[0033] The term "repair" or "repairing" or its grammatical equivalents are used herein to describe the in vivo effect of the device and method of the present invention on the nerve, particularly the epineurium, in a patient. "Repair" refers to the in vivo formation of new nerve and / or epineurium tissue sufficient to at least partially fill the void or structural discontinuity at the defect site. The term "repair" further encompasses successful integration of the device of the present invention into the nerve and / or epineurium without causing excessive scar tissue formation or fibrous adhesion of the repaired nerve to the surrounding tissues of the patient. However, repair does not imply or otherwise require a complete healing or treatment process that corresponds to 100% effectiveness in restoring the nerve or its epineurium to its pre-defect physiological / structural / mechanical state.

[0034] The terms "defect", "defect site", "neural defect", "epineural defect", or "neural defect site", or "epineural defect site" refer to a disruption in the nerve and / or epineurium. The defect results in a less than optimal level of performance or condition of the nerve and / or epineurium. A nerve or epineurium defect may exhibit a three-dimensional defect, such as a "void" configuration, which is understood to mean a gap, cavity, hole, or other substantial disruption in the structural integrity of the nerve or epineurium. In certain embodiments, the defect is, for example, a defect that is incapable of intrinsic or spontaneous repair. A nerve or epineurium defect may be the result of an accident, disease, and / or surgical procedure.

[0035] The phrase "severed or damaged epineurium" refers to a nerve that contains an epineurial defect requiring repair as defined herein. The epineurial defect may be complete in that the entire nerve, including the entire epineurium, is severed.

[0036] The term "device" is used interchangeably herein with the phrases "device for in vivo repair of a severed or damaged nerve" or "device for in vivo repair of a severed or damaged epineurium" and refers to a physical object that includes a "flexible collagen membrane" that can be used to aid in the in vivo repair of a severed or damaged nerve and / or epineurium within a patient.

[0037] The term "successful integration," as used herein, refers to the ability of the device of the present invention to become incorporated into a neural or epineural defect after a "period of time." Those skilled in the art will appreciate how "successful integration" may be assessed, however, in brief, one or more of the following are examples of successful integration: (i) the repair device does not protrude out of alignment with the epineurium and / or nerve to which it is connected after a suitable period of time has elapsed after ligation; (ii) there is little or no scar tissue formation at the site of ligation after a suitable period of time has elapsed after ligation; (iii) there is little or no fibrous adhesion of the repaired nerve to the surrounding soft tissues within the patient; (iv) histological examination reveals new vascularized epineurium-like tissue at the nerve repair site; (v) there is no evidence of an inflammatory response after a suitable period of time has elapsed after ligation; (vi) abundant neurofilaments are detected adjacent to new epineurium tissue by immunohistochemistry; (vii) well-organized nerve fibers are observed distal to the nerve repair site; and (viii) evidence of successful axonal regeneration across the repair site.

[0038] The term "flexible collagen membrane" as used herein refers to the collagen membrane used in the device of the present invention. The collagen membrane is not only flexible, but also pliable. There are many devices for repairing nerves and other tissues described in the literature as flexible, such as porcine small intestinal submucosa (SIS); however, the collagen membrane used in the present invention is not only flexible. The collagen membrane not only has the ability to bend (flexible) without breaking, but is also flexible enough to be easily or readily folded. The flexibility of the collagen membrane in the device of the present invention allows it to be easily wrapped around damaged but still surviving nerves in situ without destroying or causing damage to the nerve.

[0039] In some embodiments, the flexible collagen membrane is less than 200 microns thick. In further embodiments, the flexible collagen membrane is between 100 microns and 200 microns thick. In yet further embodiments, the flexible collagen membrane is less than 100 microns thick. In yet further embodiments, the flexible collagen membrane is less than 75 microns thick.

[0040] The flexible collagen membrane of the present invention comprises type I collagen. Type I collagen is composed of two α1 chains and one α2 chain. In some embodiments, the flexible collagen membrane of the present invention comprises greater than 80% type I collagen. In other embodiments, the collagen membrane comprises at least 85% type I collagen. In yet other embodiments, the collagen membrane comprises greater than 90% type I collagen. The flexible collagen membrane of the present invention further comprises type III collagen.

[0041] The flexible collagen membrane of the present invention comprises two surfaces (bilayer). The first surface comprises a plurality of collagen bundles protruding from the surface of the membrane. These collagen bundles extend from the first surface as shown in FIG. 7, panel A (cross section). When the flexible collagen membrane is viewed from above (front), it appears rough, which is caused by the presence of the plurality of collagen bundles. In contrast, when viewed from below (from the bottom), the second surface of the flexible collagen membrane appears smooth. No collagen bundles are present on the second surface as shown in FIG. 7, panel A (smooth).

[0042] The term "collagen bundles", as used herein, refers to masses of collagen fibers composed of three polypeptide chains intertwined to form a right-handed triple helix. The collagen bundles are generally arranged along a first surface direction. These collagen bundles are found primarily in dense connective tissues found in mammalian tendons, ligaments, and the dermis. Dense connective tissues are distinct from "loose connective tissues". Loose connective tissues are characterized by loosely arranged fibers and abundant cells, and are found, for example, under the epithelium that covers the body surface and lines internal organs.

[0043] The second surface of the flexible collagen membrane contains primarily loose connective tissue containing collagen types I and III fibers and provides multiple openings. These openings are essentially "voids" or "gaps" between the collagen "microfibrils," "fibrils," and "fibers" found in the loose connective tissue. Microfibrils are approximately 3.5-50 nm in diameter. Fibrils are approximately 50 nm-50 μm in diameter. Fibers are greater than 50 μm in diameter.

[0044] Although the flexible collagen membrane of the present invention is comprised primarily of types I and III collagen, it is recognized that other materials may also be included in the flexible collagen membrane as long as they do not adversely affect the repair benefits of the flexible collagen membrane, as described below.

[0045] Other possible materials include, but are not limited to, hydroxyapatite, or drugs that promote tissue growth; growth factors such as basic fibroblast growth factor, tumor growth factor beta, bone morphogenetic proteins, platelet-derived growth factor, and insulin-like growth factors; chemotactic factors such as fibronectin and hyaluronan; and extracellular matrix molecules such as aggrecan, biglycan, and decorin.

[0046] "Dense connective tissue" and "loose connective tissue" are readily obtained from "collagen-containing tissues," such as skin, muscle, etc., which are isolatable from mammalian bodies that contain collagen. In some embodiments, tissues containing dense and loose connective tissue are isolated from tendons.

[0047] Generally, the device of the present invention is connected to the defect site and fixed in place. Factors such as the size of the nerve or epineurium defect will determine how much of the nerve or epineurium needs to be covered by the device of the present invention. The surgeon or other skilled artisan can determine which portion of the nerve or epineurium is covered. In some embodiments, at least a longitudinal portion (along the nerve or epineurium) is covered by the repair device. General knowledge of the placement of the repair device is required. For example, the entire nerve or epineurium does not need to be contained with the repair device. Similarly, covering the defect site alone is not sufficient to protect the repair site.

[0048] The term "wrapping" as used herein refers to the preferred process of connecting a flexible collagen membrane to a cut or damaged nerve or epineurium. Wrapping is particularly useful when the nerve containing the epineurium defect site is still viable, i.e., in an uncut state. In this situation, wrapping the device around the nerve to contain the defect site is the best option for connection. Wrapping can also be used when the nerve or epineurium is cut, such as during nerve grafting. In this case, other connection means can be used to supplement the connection, as previously described.

[0049] The flexible collagen membrane can be wrapped around the nerve or epineural defect site so that the first and second surfaces are positioned together, i.e., overlapping. The flexible collagen membrane is typically wrapped so that the first surface (rough) faces the outside of the nerve while the second surface (smooth) faces inwards towards the nerve surface.

[0050] The amount of overlap depends on several factors known to surgeons and others skilled in the art, but a sufficient amount of overlap is required to allow at least a portion of the plurality of protruding collagen bundles to mate with at least a portion of the plurality of openings on the second surface. This arrangement allows the collagen bundles to mate with the openings with little or no (at a minimum) physical pressure. That is, the first and second surfaces of the flexible collagen membrane are held together (self-interlocking) by the mating of the protruding collagen bundles with the openings. In some embodiments, the overlap is at least 10%, while in other embodiments, the overlap is at least 20%. In some embodiments, the degree of overlap is selected from the group consisting of 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60%.

[0051] In some embodiments, the first and second surfaces of the flexible collagen membrane interlock to provide a stronger fit. Thus, as described herein, the device of the present invention, once placed on the nerve and / or epineurium, can form a self-fitting fit without the need for sutures or other attachment means. This is especially true when the flexible collagen membrane is applied in vivo, which is generally a "wet" environment. However, if required by the surgeon, the flexible collagen membrane is also adaptable to be used in combination with sutures to hold it in place when placed on the nerve.

[0052] The term "suture," as used herein, is for linguistic convenience only. This term should not be construed as limiting in any way. Embodiments of the subject invention may be utilized with any of a variety of devices, materials, and techniques useful for fastening and / or binding neural tissue. This may include, but is not limited to, sutures, staples, vascular clips, hydrogels, fibrin, urethane-based adhesives, and other medical adhesives, or combinations thereof.

[0053] The phrase "allowing the device to remain in place" refers to coupling of the device of the present invention to the nerve or epineurium requiring repair, including the period of time that the device is coupled to the nerve and / or epineurium.

[0054] The term "period of time" as used herein refers to the time it takes for the device of the invention to become successfully integrated with the nerve or epineurium to which it is connected, as defined herein. In some embodiments, the period of time is less than 4 weeks. In some embodiments, the period of time is between 2 weeks and 4 weeks.

[0055] The length of the device may vary depending on various factors understood by those skilled in the art, including, but not limited to, the length of the damaged or severed nerve, the diameter of the damaged or severed nerve (or donor nerve diameter, if a donor nerve is used), the locations and number of sutures available, and the location of the severed or damaged nerve in vivo, as well as other factors. In one embodiment, the length of the device is about 0.5 cm to about 3.0 cm. In a more specific embodiment, the length of the device is about 0.75 cm to about 2.0 cm. In a particular embodiment, the device has a length of about 1.0 cm.

[0056] In some aspects of the invention, the methods and devices are used in in vivo nerve grafting. This may require the removal of all or part of a damaged nerve in a recipient patient. Once the damaged nerve is removed, a graft can be used on the nerve to restore function. Various types of grafts, such as autografts, syngrafts, allografts, and xenografts, are encompassed within the subject invention. The size (e.g., length and diameter) of the graft is not critical to the subject invention. For example, the length of the nerve graft can be about 1 cm to about 10 cm, or more than about 10 cm. The diameter of the nerve graft can match the diameter of any injured nerve or portion of a nerve, as desired. The nerve graft can be a structurally intact segment of a nerve to bridge a gap along the length of the recipient nerve or to replace the distal end, i.e., end-to-end grafting. Alternatively, the nerve graft can be a partial nerve segment, or an eccentric shape (e.g., a nerve valve), and can be intended to reconstruct a lacerated nerve with some structural disruption but retaining physical continuity.

[0057] Once the donor nerve is placed end-to-end with the recipient nerve stump at the defect site and properly positioned, one or more sutures can be used to hold them in place until the repair device of the present invention is wrapped around the defect site. Alternatively, the procedure can be accomplished without the use of sutures, i.e., by simply wrapping the device of the present invention around the donor and recipient nerves.

[0058] Optionally, a tissue adhesive material, such as a biological adhesive, is applied to the donor and recipient nerve stumps. Preferably, the biological adhesive is a fibrin-containing adhesive material, such as fibrin glue, fibrin sealant, or platelet gel. Biological adhesives are well known in surgical techniques (Suri A et al.

[2002] Neurol. India 50:23-26; Alibai E et al.

[1999] Irn J. Med. Sci. 24(3&4):92-97; Sames M et al.

[1997] Physiol. Res. 46(4):303-306; Jackson M et al.

[1996] Blood Coag. Fibrinolysis 7:737-746; Fasol R et al.

[1994] J. Thorac. Cardiovasc. Surg. 107:1432-1439). As used herein, the terms "fibrin glue," "fibrin sealant," and "fibrin tissue adhesive" are used interchangeably to refer to a group of preparations containing fibrinogen and thrombin that cause the formation of a fibrin clot at the site of application.

[0059] The present invention also includes kits for use in repairing severed or damaged nerves or epineurium. Such kits can be used for laboratory or clinical purposes. Such kits include a device as described herein and instructions for applying said device to repair the epineurium of a patient. The kits can include a storage container, e.g., a light-proof and / or cool-insulated container for storing the device. Optionally, the kits can include additional agents used in epineurium repair, e.g., antibiotics, suture material, etc.

[0060] The kits described herein may also include means for applying the device to the epineurium, such as forceps, suture needles, fibrin glue, etc. The kits may further include accessory tools for accessing tissue, such as clips, reference weights, an aspiration device, and a compression gauge.

[0061] The kit may include instructions for use.

[0062] All patents, patent applications, provisional patent applications, and publications referenced or cited herein, including all figures and tables, are incorporated by reference in their entirety to the extent they do not conflict with the explicit teachings of this specification.

[0063] Below are examples that illustrate the procedures for practicing the present invention.These examples are intended to be limited for illustrative purposes, i.e., are not intended to be limiting, since numerous modifications and variations thereof will be obvious to those skilled in the art.Unless otherwise stated, all percentages are by weight, and all solvent mixture percentages are by volume.

[0064] Example 1: Flexible collagen membrane preparation Collagen segments derived from the lining of porcine internal organs were carefully isolated, placed in a solution containing approximately 70% ethanol, and incubated briefly at room temperature. The collagen-containing tissue was then stretched, fat side up, against a working surface, and as much fat tissue and blood as possible was removed.

[0065] To visualize the adipose tissue present, the collagen-containing tissue was coated with glycerol for about 10 minutes. At this point, the collagen was transparent, but the adipose tissue was white. Using forceps, the white adipose tissue was separated from the collagen under a dissecting microscope.

[0066] Once complete, the collagen-containing tissue was carefully transferred to a sealed container and incubated in a solution containing approximately 1% (v / v) sodium dodecyl sulfate (SDS) and 0.2% (v / v) lithium chloride (LiCl) to denature non-collagenous proteins. Incubation was carried out at 4°C overnight.

[0067] The collagen-containing tissue was then carefully washed twice in 100% acetone to remove denatured non-collagenous proteins, and then centrifuged at 100 RPM in a 200 ml container to briefly spin down residual solution, non-collagenous proteins, and nucleic acids from the collagen-containing tissue.

[0068] The collagen-containing tissue was carefully removed and washed again three times in Steripure® water. Optionally, the collagen-containing tissue was washed in a solution containing NaOH:NaCl, which was then centrifuged at 100 RPM for 90 minutes.

[0069] The collagen-containing tissue was then immersed in 0.5% (v / v) HCl and placed on a shaker for 30 min to denature the collagen. The concentration of HCl and incubation time were found to be important to avoid damage to the mechanical structure of the resulting flexible collagen membrane.

[0070] The collagen-containing tissue was then removed and washed again three times in Steripure® water.

[0071] The collagen-containing tissue was then neutralized using 0.5% (v / v) NaOH. At this stage, preliminary tests may be performed on the mechanical properties of the resulting flexible collagen membrane.

[0072] The flexible collagen membrane was then manipulated using mechanical forces (compression and tension) using a stainless steel frame. Once the flexible collagen membrane was stretched to the correct size, thickness (e.g., less than 200 microns, preferably less than 100 microns, even more preferably less than 70 microns), etc., the tissue was denatured in situ, i.e., immersed in a solution containing 1% (v / v) HCl, within the frame. Typically, the tissue was incubated for 22-25 hours with shaking at 100 RPM until the collagen fiber bundles were aligned.

[0073] The flexible collagen membrane was then washed with water and rinsed with a mixture of 1% (v / v) SDS and 0.2% (v / v) LiCl.

[0074] Finally, the flexible collagen membrane was treated with acetone and air-dried while still stretched in the frame so that the protruding collagen bundles on the rough surface of the collagen membrane were immobilized. The flexible collagen membrane was then stretched, compressed, and / or rolled to create a smooth surface (second surface) on the other side of the membrane. The final flexible collagen membrane was then inspected and cut to size using a laser cutter.

[0075] Example 2: Nerve transplantation Nerve grafting has recently transformed standard medical care, which was based on accommodating physical and mental disabilities, into functional enhancement. Several studies have demonstrated that nerve grafting alone or in conjunction with tendon grafting allows for subtle enhancement in function of basic daily activities. This has been a game changer for patients with SCI, especially quadriplegia.

[0076] In patients with quadriplegia, nerve grafts are used as a "bypass" for spinal cord injury. When spinal cord injury occurs, there are three distinct regions of injury. Above the level of injury, both upper and lower motor neurons remain intact and continue to function. At the level of injury, both upper and lower motor neurons are damaged. The involved nerve axons undergo Wallerian degeneration and pathological changes occur at the neuromuscular junction. However, below the level of injury, there is no injury to the lower motor neurons, but there is loss of function due to the loss of upper motor neurons. Due to the fact that there is no injury to the peripheral nerves (lower motor neurons), Wallerian degeneration on the axons does not occur in the peripheral nerves. These peripheral nerves are not damaged and the neuromuscular junctions often remain intact at the motor nerve endplates. Nerve grafts utilize functional nerves supplied above the level of spinal cord injury to innervate muscles supplied by nerves originating at or below the level of injury. During a nerve transplant procedure, an injured peripheral nerve (recipient) reconnects with the spinal cord by redirection of a functional peripheral nerve (donor). The donor nerve is severed and the "viable" stump (i.e., the end still connected to the spinal cord) is attached to the distal stump of the recipient nerve. Regenerating axons from the donor nerve grow into the endoneurial tube of the recipient nerve and merge with receptors on the recipient nerve's target organs, restoring connectivity to the spinal cord and brain.

[0077] Nerve transplant procedures can restore function to elbows, wrists, fingers, and thumbs. Compared to muscles, there is more redundancy in peripheral nerves, so there is a larger pool of donors for nerve transplants compared to tendon transplants. This allows surgeons to select donors that can be attached to the recipient in close proximity to the recipient's target organ, thereby reducing the distance required for axon regeneration and thereby increasing the probability of successful treatment.

[0078] For people living with quadriplegia, the advantage of nerve grafts over tendon grafts is the ability to restore sensory function, and functional muscle loss below the elbow does not preclude treatment. Nerve grafts avoid the mechanical challenges of tendon grafts, such as tensioning the tendon during surgery, and post-treatment failures due to tendon rupture, adhesion, or overstretching. Nerve grafts are an elegant and effective solution to restore motor function, as the resulting movement is powered by muscles anatomically designed for the function, preserving natural biomechanics and range of motion, and providing superior mobility and control compared to tendon grafts. Clinical trial participants who received both types of grafts reported that the hand treated with the tendon graft felt stronger, but the hand that received the nerve graft had a natural appearance, was more adaptable to social interactions, had greater mobility, and was better at holding large objects and using electronic devices.

[0079] A search of the PubMed database (November 2020) yielded 12 clinical trials of nerve grafts for tetraplegia that included data on motor recovery using the MRC rating scale for muscle function (Table 1). There were no controlled trials, and eight studies were case reports describing outcomes in a single patient. MRC data were extracted for nerve grafts performed to restore function to the elbow (triceps), hand grip / release (flexion / extension of fingers), and thumb pinch / release (flexion / extension of thumb). Participants were considered to have achieved functional motor recovery (FMR) if muscle function was restored to Roganovic MRC grade ≥3.

[0080] [Table 1]

[0081] A total of 56 study participants underwent 132 nerve transfers, of which 70 transfers were performed to restore 202 muscle functions, including two muscle functions (finger + thumb extension or finger + thumb flexion). The meta-analysis revealed that at a mean assessment time of 22.0 months (SD 6.0, range 6-24.9) after treatment, the pooled functional motor recovery rate was 61.9% (95% CI: 55.2-68.6). Finger extension was the most successful reconstructive procedure, with 81.8% of transfers achieving FMR (95% CI: 68.6-59.0) at 21.6 months after surgery, and thumb flexion was the least successful, with 46.2% of transfers achieving FMR (95% CI: 30.5-61.8) at 22.8 months after surgery (Table 2). These data suggest that nerve grafting is a feasible approach for the surgical reconstruction of arm and hand function and that the development of novel techniques is required to improve the consistency and predictability of treatment outcomes.

[0082] [Table 2]

[0083] Restoration of arm and hand function is the most desirable treatment goal for people with quadriplegia, yet reconstructive surgery is underutilized. It is estimated that up to 75% of people with quadriplegia would benefit from reconstructive surgery to improve arm and hand function. In a recent US survey, less than 50% of patients were informed of the existence of reconstructive surgery, yet only 9% of surveyed patients underwent surgical treatment.

[0084] There are many factors that contribute to the underutilization of reconstructive surgery. The main barrier to wider patient uptake appears to be lack of referral to specialty surgeons, especially given the limited window to restore motor function after nerve injury, as well as lack of knowledge and coordination of reconstructive procedures in the U.S. health care system. An additional problem is the lack of U.S. surgeons willing and able to perform nerve transfer procedures. Only three of the 12 published trials of nerve transfer for quadriplegia were performed in the United States, and all were conducted by the Washington University School of Medicine. The majority of reconstructions performed in the United States were tendon transfers (a simpler procedure compared to nerve transfers, but with a more limited scope of repair), which require patients to have fully functional arm muscles to use as donors and are less attractive to patients because they require prolonged immobilization and physical rehabilitation of the operated limb.

[0085] Nerve grafting is a promising reconstructive procedure intended to restore arm and hand function in patients with quadriplegia. However, published trials of nerve grafting in people with quadriplegia have highlighted the need for further investigation into improving the predictability of outcome, outcomes at the upper and lower end of patient age, the optimal time after injury, and the utility of nerve grafting in patients older than 24 months after injury.

[0086] The use of nerve grafts for patients with quadriplegia is promising in the treatment of upper limb paralysis, but the assessment, selection, and timing of suitable nerve grafts are always complicated. One of the important effects of successful nerve grafts is that the surgical procedure must be able to reconnect functional nerves originating from different regions of the spinal cord to dysfunctional nerves originating from dead neurons in the spine. To do so, the surgical procedure must be able to provide sufficient protection and microenvironment for myelination, as well as the extension and reconnection of nerve axons, quickly after surgery.

[0087] The epineurium, an outer layer of dense irregular connective tissue that surrounds peripheral nerves, is an extension of the cerebral dura mater. Under normal physiological conditions, the epineurium acts as an anatomical barrier for peripheral nerves, protecting them from nerve outgrowth injury, and providing nutrients for nerve growth. The epineurium surrounds multiple nerve bundles and also contains blood vessels that supply the nerves. There are two distinct epineurium layers. The outermost layer consists of connective tissue with vascular components. The inner sheath is composed of collagen fibrils and collagen fibers. The epineurium also contains fibroblasts, variable amounts of fat, and hyaluronic acid. The human epineurium accounts for up to 70% of the cross-section of the peripheral nerve and also contains types I and III collagen.

[0088] The ideal situation for a successful nerve grafting procedure would be to reconstruct a barrier structure similar to the epineurium for nerve grafting at the site. However, the current state-of-the-art surgical technique for nerve grafting in treating upper limb paralysis only uses sutures on the epineurium to connect the nerve end-to-end. Some surgeons may also apply fibrin sealant locally to provide a hemostatic barrier for nerve grafting. In most cases, the ideal situation of introducing an epineurium barrier structure is still far from being met. Although there have been some attempts to develop an autologous epineurium conduit to repair nerves, it is almost impossible for patients with paralysis to obtain autologous epineurium tissue for nerve grafting.

[0089] An alternative approach is to use nerve repair devices that have been developed to assist in nerve repair surgery. Current nerve repair devices (either hollow tubes or sheaths) are composed of either polymers or reconstituted collagen (Table 3).

[0090] [Table 3]

[0091] Although these nerve repair devices have been shown to be effective for common peripheral nerve injuries, they are hardly suitable for use in nerve grafting surgery to treat upper limb paralysis due to the inability to introduce an epineural barrier structure. According to the literature, most nerve repair devices suffer from poor biocompatibility problems (leading to adhesions, scar tissue formation, and neuromas), are too rigid for easy manipulation, and cannot be adjusted to fit different sized nerves when the size of the donor nerve is different from that of the recipient nerve. A literature review revealed that there are no published studies using nerve repair devices to repair peripheral nerves in patients with quadriplegia.

[0092] To address the unmet medical need for nerve grafts for patients with paralysis, applicants have developed a repair device comprising a flexible collagen membrane as discussed in Example 1 that mimics the native structural, physiological, and biological performance of the epineurium. Preclinical performance testing has revealed that the device of the present invention has the right mechanical properties, semi-permeability, and suitable degradation kinetics structure to allow Schwann cell migration and axonal outgrowth.

[0093] Proof-of-concept evidence was obtained from preclinical studies of peripheral nerve repair using a rat model. The sciatic nerve was completely cut and then repaired using three epineural microsutures, similar to normal surgical practice in human procedures. The suture repairs graded as excellent or good were housed in the repair device of the present invention or a commonly used FDA-approved collagen nerve device (NEURAWRAP®). To connect the device to the epineurium, the location of the device was marked using non-absorbable "stay" sutures at each end of the device (see, for example, Figures 1 and 2).

[0094] After 4 weeks of healing, the repair sites of two animals treated with the nerve repair device of the present invention (FIGS. 3A and 3B) were similar in appearance to the untreated nerve on the contralateral side of the animal (FIG. 3C). The nerve repair device of the present invention appeared well integrated into the epineurium and did not protrude. There was no fibrous adhesion of the nerve to scar tissue or surrounding soft tissue. In contrast, the repair sites of animals treated with an FDA-approved collagen device (NEURAWRAP®) showed extensive hypertrophic, dense connective tissue overlying the surface of the nerve device and causing adhesion of such connective tissue to the surrounding soft tissue (FIGS. 3D and 3E). NEURAWRAP® did not appear integrated into the epineurium and remained bulkier in appearance compared to the untreated nerve on the contralateral side (FIG. 3F).

[0095] Histological examination was consistent with the macroscopic examination of the nerve repair device of the present invention. Histologically, new vascularized epineurium-like tissue was found to have formed from the tissue at the nerve repair site (Figure 4A). No evidence of an inflammatory response was observed. Collagen fibers from the nerve repair device of the present invention were well integrated into the epineurium-like tissue and were similar in appearance to normal connective tissue. Abundant neurofilaments were detected by immunohistochemistry adjacent to the new epineurium tissue, and well-organized nerve fibers were found distal to the nerve repair site (evidence of successful axonal regeneration across the repair site). In contrast, the repair site of the FDA-approved collagen device (NEURAWRAP®) was encapsulated with fibrous tissue, causing axonal entrapment and nerve compression (Figure 4B). Infiltration of lymphocytes, plasma cells, macrophages, and foreign body reactive multinucleated giant cells was observed at the repair site (evidence of an inflammatory response). Neurofilaments and axonal growth were observed distal to the repair, but the newly regenerated nerve tissue was less organized compared to the repair sites of our product.

[0096] Results from this study demonstrated that treatment with the nerve repair device of the present invention caused the formation of new epineurium-like tissue at the repair site, no inflammation, nerve compression, or axonal entrapment was observed, and the use of our product provides a suitable environment for regenerating axons across the repair site.

[0097] Example 3: Clinical trial: Use of the nerve repair device of the present invention to improve the outcome of nerve graft surgery to restore arm and hand function in patients with quadriplegia. Based on preclinical observations, applicants conducted a prospective case series, clinical trial CG-006, to investigate the use of the nerve repair device of the present invention to improve nerve graft outcomes in patients with tetraplegia. Fourteen nerve grafts were performed to restore arm and hand function in four patients with tetraplegia. Recovery of motor function was assessed using manual muscle testing and graded using the UK Medical Research Council (MRC) standardized criteria for motor function for up to two years after treatment (Table 1 below).

[0098] Functional motor recovery of previously paralyzed muscles was observed in 71.4% of grafts at 12 months post-treatment, increasing to 88.9% at 24 months post-treatment (Figure 5). In comparison, a meta-analysis of nerve graft surgery (using suture repair only) in people with tetraplegia achieved an FMR of 61.9% from 202 nerve grafts at 22 months post-treatment (Table 4).

[0099] [Table 4]

[0100] Loss of elbow extension affects approximately 75% of people with tetraplegia. Restoring elbow stability and function is essential to the ability to independently transfer (e.g., from a bed to a wheelchair), perform pressure-relieving activities, maintain balance while sitting, reach for overhead objects, and operate a manual wheelchair.

[0101] Elbow extension is provided by the triceps muscle. At 12 months after nerve grafting, functional recovery of the triceps muscle was observed in 100% of patients treated with the nerve repair device of the present invention, compared with a published study reporting a 73.9% recovery at 21 months (Figure 6, Table 5). Participants in the clinical trial were able to perform a range of daily activities that were not possible before receiving the study treatment, such as moving around and turning in bed, using a manual wheelchair, and transferring from bed to a wheelchair using a slide board.

[0102] The nerve repair device of the present invention demonstrated faster and better results in restoring triceps function compared to data obtained from trials of conventional nerve graft surgery, a level of recovery not possible using standard nursing care treatment (physical rehabilitation) for quadriplegia.

[0103] [Table 5]

[0104] Overall, these data support the positioning of the nerve repair device of the present invention as a viable alternative epineural substitute and to enable peripheral nerve repair.

[0105] Example 4: Evaluation of nerve repair device in reconstruction of upper limb peripheral nerve injury Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and enhanced micro-computed tomography (micro-CT) were used to examine the ultrastructural features of the nerve repair device of the present invention. A prospective case series with 2-year follow-up was performed and reported. Nerve reconstruction was performed on patients who required nerve transfer or grafting. Participants underwent single or multiple nerve reconstruction in one or both upper limbs.

[0106] method Structural characterization All analyses were performed at the Centre for Microscopy, Characterization and Analysis at The University of Western Australia. For SEM assessment, 1 cm x 1 cm square pieces of the nerve repair device of the present invention were mounted on stubs and sputter coated with a layer of platinum (Pt). Samples were analyzed using a Zeiss 1555 VP-FESEM (high resolution field emission variable pressure scanning electron microscope). For micro-CT assessment, to visualize soft tissues under micro-CT, the nerve repair device of the present invention was first stained with 0.3% potassium iodine solution as a contrast agent, and 3D image reconstruction was performed using XM Reconstructor software, v10.7.3679.13921, Zeiss. Such staining techniques provided high resolution images without causing damage to the samples.

[0107] Clinical Trial Design and Participants A prospective case series of 19 patients with 2-year follow-up was performed and reported according to the PROCESS guidelines. The study was approved by the Human Research Ethics Committee of St John of God Health Care and registered with the Australian and New Zealand Clinical Trials Registry (ACTRN12616001157460). Patients with nerve truncation (losing of connection of the injured nerve with resultant complete loss of function) according to the Seddon classification of peripheral nerve injury were selected. Inclusion criteria were men and women aged 8-50 years with traumatic injury to one or more peripheral nerves occurring in the upper limb or hand within the past 18 months.

[0108] surgical intervention Nerve reconstructions were performed in patients who required nerve transfer or grafting. Participants underwent single or multiple nerve reconstructions in one or both upper limbs. Primary reconstructions included triceps nerve transferred to axillary nerve in the shoulder; posterior axillary nerve to biceps or triceps nerve, or gracilis nerve to biceps nerve in the elbow; supinator or brachialis nerve to anterior, middle, or posterior interosseous nerve, or extensor carpi radialis nerve to posterior interosseous nerve in the hand. All surgical procedures were performed by a single surgeon (AOB). Operations were performed under general anesthesia with skeletal muscle relaxants and thromboprophylaxis, with the patient positioned as required for optimal surgical approach.

[0109] The donor and recipient nerves were identified using a nerve stimulator and locator device and dissected from the surrounding fascia. The nerve stumps were coapted with two tethered sutures, using 8-0 nylon for larger nerves or 10-0 nylon for smaller nerve stumps. The nerve repair device of the present invention was placed below the coaptation site with the rough side (first surface) facing out. The nerve repair device was then wrapped around the coaptation site to cover at least 1.5 cm across the proximal and distal nerve stumps, and with a minimum of 30% overlap to create an interlocking adhesive contact between the rough (first surface) and smooth (second surface) surfaces of the nerve repair device. This ensured that proper contact occurred between the nerve repair device and the nerve, regardless of the size mismatch between the donor and recipient nerves, as well as mechanically protecting the nerve from soft tissue compression. Fibrin sealant (TISSEEL, Baxter, Deerfield, IL, USA) was placed around the nerve repair device to stabilize the position of the device surrounding the nerve.

[0110] The operated limb(s) was placed in a sling for 1-2 weeks after surgery to protect the attachment site. All postoperative therapy was performed on an outpatient basis.

[0111] Outcome assessment Physical examination of the affected limb was performed by a duly qualified investigator at baseline and at each postoperative clinic visit. Efficacy endpoints, including functional assessments and patient-reported outcomes, were described using the following:

[0112] motor function The UK Medical Research Council (MRC) rating system is used to rate muscle strength as an assessment of motor function involving manual muscle testing. The MRC scale has been demonstrated to have substantial inter- and intra-rater reliability for the upper limbs and is suitable for use even when severe muscle weakness is present.

[0113] Sensory function Discrimination is the ability to recognize the properties of an object through touch (e.g., texture) and is an important indicator of sensory function. To determine sensory function, the static two-point discrimination test (s2PD) and the dynamic two-point discrimination test (m2PD) were used. s2PD and m2PD are assessment tools that test the patient's ability to discriminate the shortest distance between two points that can be perceived as separate. Normal values ​​for the fingers were less than 6 mm for static 2PD and 2-3 mm for dynamic 2PD. The static test measures the innervation density of slow adapting neuroreceptors, while the dynamic test measures fast adapting neuroreceptors.

[0114] QuickDASH The QuickDASH (Quick Disabilities of the Arm, Shoulder and Hand) questionnaire is a validated outcome measure of symptoms and disability in patients with musculoskeletal disorders of the upper limb. Eleven items are scored on a 5-point scale ranging from 1 (no difficulty) to 5 (unable to perform the task). Raw scores are scaled to a final score of 100, with higher scores representing greater disability. An optional task-specific module consisting of four additional questions was also administered in this study.

[0115] Visual Analog Pain Scale (VAS) Pain is a common outcome after peripheral nerve injury and contributes significantly to disability. Paradoxically, both decreased and increased pain may be associated with nerve regeneration after surgical repair. Participants' experience of overall pain, nighttime pain, and activity-related pain was rated using a standardized numeric visual analog scale ranging from 0 (no pain) to 10 (worst pain ever).

[0116] Quality of Life Assessment (AQoL-6D) The AQoL-6D is a validated health-related multi-attribute utility quality of life measure. Participants complete a questionnaire consisting of 20 items arranged across six dimensions, each of which is assigned a score ranging from 0 (dead) to 100 (perfect health).

[0117] Adverse events All adverse events and serious adverse events were monitored and recorded.

[0118] Data Management and Analysis The study was conducted according to an approved protocol (GLP) and all applicable regulatory requirements. Assessments were recorded on individual case report forms and data were managed using the Research Electronic Data Capture tool provided by St John of God Hospital, Subiaco, Western Australia. Monitoring queries were generated and, if necessary, inquiries were answered by the responsible investigator or his / her designee at each site.

[0119] Data from patient-reported outcomes were imported into SPSS (v27) for statistical analysis. One-way repeated measures ANOVA was performed with timepoint as a within-subject factor. Greenhouse-Geisser correction was applied to the data set (returning significant results for Mauchly's test of sphericity). Post-hoc tests were performed using Bonferroni correction (where applicable). MRC scores were reported as median and interquartile range (IQR).

[0120] result Structural Characterization of the Nerve Repair Device of the Present Invention To examine whether the nerve repair device of the present invention mimics the bilayer structure of the epineurium, applicants have examined the morphological properties of the flexible collagen membrane using SEM, enhanced micro-CT, and TEM. Enhanced micro-CT revealed that the nerve repair device is composed of two distinct layers, with the outer layer (first surface) containing densely parallel collagen bundles and the inner layer (second surface) containing loose bundles and fibers, some of which are vertically distributed along the flexible collagen membrane. D-spacing of collagen fibrils was observed in the nerve repair device. The thickness of the nerve repair device was approximately 100 μm, and the average diameter of the collagen fibrils was 90 nm ± 20 nm.

[0121] Patient demographics A total of 19 participants (17 men and 2 women) were eligible for inclusion in the study. Five participants had cervical spinal cord injuries and were candidates for nerve transfer surgery to restore upper limb function. Eight patients had traumatic injuries to the brachial plexus and required reconstructive surgery to primarily restore shoulder or elbow function. Six patients had upper limb peripheral nerve injuries and required surgery to restore shoulder, elbow, wrist, or hand function. The most common cause of injury was motor vehicle accidents (60%), but other causes included sports and recreation-related injuries, falls, and iatrogenic causes. All patients were followed up for 24 months after surgery, except for one participant who died 12 months after surgery (due to lung infection) and two patients who were lost to follow-up after 6 months.

[0122] Outcome assessment A total of 36 peripheral nerve reconstructions were performed (35 for motor function and 1 for sensory function). Of the 36 peripheral nerve transfers, 4 involved the shoulder, 11 involved the elbow, 2 involved the wrist, and 19 involved the hand.

[0123] At baseline, the median overall MRC score based on nerve graft type was 0 (IQR-0; n=60). After surgery, at 6 months, the median MRC score improved to 1 (IQR2; n=60), with further improvements observed at 12 months (median MRC score=3; IQR2; n=59) and 24 months (median MRC score=4; IQR1.25; n=48). Reinnervation of adjacent target muscles was observed in all nerve repairs evaluated.

[0124] Improvements were also observed in upper extremity function, as assessed by QuickDASH scores, at screening (baseline), 12 months, and 24 months visits. The mean QuickDASH score was 52.9 (SD 20.8) at baseline and improved to 46.8 (SD 30.2) at 24 months. Although the mean improvement in QuickDASH score was not statistically significant, 62% of participants reported an improvement in QuickDASH score at 12 months compared to baseline.

[0125] The mean pain score assessments for worst pain, pain at rest, pain while performing repetitive tasks, and night pain remained stable up to 12 months. However, 77%, 46%, and 54% of participants reported a decrease in pain at this time point compared to baseline for worst pain, pain at rest, and pain at night, respectively. To investigate whether pain medication had any impact on the outcome of this assessment, applicants analyzed patients' medication use during the study. This analysis revealed that participants were receiving a median of two pain medications at baseline (mean 1.8; SD 1.56, range 0-4). These included neuroleptics, antidepressants, opioids, and nonopioid analgesics. Pain medication use decreased to a median of one at 6 months (mean 1.4; SD 1.40; range 0-4), and zero at 12 months (mean 1.1; SD 1.55; range 0-4). Five participants reported not taking any pain medications during the 12-month period, and seven participants significantly reduced or discontinued their pain medication use at 12 months compared with baseline.

[0126] Participants completed the AQoL-6D questionnaire at screening (baseline), 12 months, and 24 months study visits. Results demonstrated improvement in AQoL-6D scores after surgery. Mean AQoL-6D scores remained stable up to 24 months after treatment. However, individual improvements in AQoL-6D scores were reported for 60% of participants at 6 months and 54% of participants at 12 months.

[0127] Adverse events Nine adverse events were reported in four participants during the study period, all deemed unrelated to the nerve repair device. At 12 months, one nerve repair was classified as a treatment failure (due to wound infection after surgical correction of a concomitant fracture injury). One participant died due to pneumonia 12 months after surgery.

[0128] result Morphological characterization demonstrated that the nerve repair device mimicked the structural and biological properties of the epineurium. The thickness of the nerve repair device (100 μm) is within the range of epineurium found in most human peripheral nerves (Figure 10). A total of 36 peripheral nerve reconstructions were performed in 19 patients with upper limb nerve injuries. Results demonstrated that nerve reconstructions using the nerve repair device restored peripheral nerve function compared to baseline, and restored motor function was maintained in 90% of reconstructions at 6 months. Improvements in motor function were observed in all repairs at 24 months, with 96% of reconstructions restoring active motion to the target muscle.

[0129] Consideration Patients who lose the function of their upper limbs are severely disabled. Using the nerve repair device as an adjunct to nerve reconstruction surgery, the applicant successfully performed 36 nerve reconstructions in 19 participants with upper limb paralysis due to peripheral nerve injury. Clinical assessment revealed that 90% of the reconstructed nerves showed improved motor function as early as 6 months to the target muscles adjacent to the reconstruction site. At 12 months, improved motor function was seen in all reconstructed nerves, and 97% of the reconstructed nerves restored active motion to the target muscles. Reductions in pain and disability were also observed at 12 months, with improvements continuing through 24 months. QuickDASH scores improved in 62% of participants, and 77% of participants reported a decrease in their worst pain experience. Use of pain medication was reduced or stopped in 77% of participants who required medication for their nerve pain at baseline.

[0130] Notably, of the 19 patients who underwent peripheral nerve reconstruction, 5 had C5–C8 spinal cord injuries that caused tetraplegia. Nerve grafts performed to restore upper limb function in these patients were promising, with all nerve grafts aimed at restoring triceps function achieving functional motor recovery (MRC 3 or 4) after 12 months. Improved triceps function enabled participants to perform daily activities that were not possible before surgery (e.g., turning in bed, using a manual wheelchair, and transferring from bed to a wheelchair independently using a slide board). These results compare positively with those reported by van Zyl et al. (2019), who reported a median MRC score of 3 at 24 months after surgery.

[0131] Applicants found that all participants had significantly improved functional outcomes based on MRC scores after 12 months, whereas pain and disability scores were not significantly different from baseline.

[0132] There are multiple factors that influence the outcome of nerve reconstruction. These include age, sex, injured nerve, time between injury and surgery, type of graft, and repair material used. However, when comparing outcomes between repair materials, it was revealed that surgery using the nerve repair device of the present invention achieved relatively better outcomes than those reported in the literature. In this study, more than 97% of the nerve constructs resulted in functional motor recovery of the target muscle at 12 months. This compares positively with other studies showing FMR of 86% using polyglycolic acid, 76.5% using NEUROTUBE®, 75%-88% using NEURAGEN®, and 65% using human acellular nerve grafts.

[0133] conclusion Quadriplegia is a devastating, irreversible condition that disproportionately affects young, healthy males. People with quadriplegia have little or no voluntary control of their bowels or bladder. Their legs and torso are paralyzed, and although they may have some movement in their shoulder muscles, sensation and movement of their arms and hands are limited or nonexistent. People with quadriplegia have a shortened lifespan and experience significant morbidity.

[0134] The standard of care in the United States for people with quadriplegia is rehabilitation. Patients undergo therapy focused on enhancing remaining muscle function, employing compensatory techniques, using prosthetic devices, and environmental modifications. However, the ability of patients with quadriplegia to regain motor and sensory function through rehabilitation alone is inherently limited by the extent of their spinal cord injury. Once the connection between the brain and peripheral nerves is lost, it cannot be regenerated by physical or drug therapy.

[0135] Surveys of people with quadriplegia and their caregivers have revealed that regaining arm and hand function takes the highest priority in functional recovery over bowel / bladder function, walking, pain management, or sexual function. For chronic quadriplegics (i.e., after reaching maximum neurological recovery), surgical reconstruction is the most common alternative to rehabilitation therapy and provides a means by which additional arm and hand function can be restored by strategically repositioning remaining function.

[0136] Nerve transfers have recently transformed standard medical care, such as nursing assistance, for patients with SCI, especially quadriplegia, by restoring basic daily activities and enhancing function. Nerve transfers re-establish basic arm and hand function for people with quadriplegia. Nerve transfers are used to "bypass" spinal cord injuries. Multiple nerve transfers can be performed in one surgical procedure, and one nerve transfer may restore function to multiple muscle targets.

[0137] The ideal situation for a successful nerve grafting procedure would be to reconstruct a barrier structure similar to the epineurium for nerve grafting at the site, but the current state-of-the-art surgical technique for nerve grafting in treating upper limb paralysis only uses sutures in the epineurium to tie the nerve end-to-end. In most cases, the ideal situation of introducing an epineurium barrier structure is still far from being met.

[0138] To address the unmet medical need for nerve grafting for patients with paralysis, the applicant has developed a repair device that includes a flexible collagen membrane that mimics the natural structural, physiological, and biological performance of epineurium when successfully integrated. Preclinical performance testing has revealed that the repair device of the present invention has the right mechanical properties, semi-permeability, and suitable degradation kinetics structure to allow Schwann cell migration and axonal extension. Clinical trial CG-006 has demonstrated that nerve grafting using the repair device of the present invention in people with quadriplegia induces faster and better recovery of triceps function compared to standard nerve grafting (suture only) tests cited in the literature.

[0139] The nerve repair device of the present invention is a technology that can simplify and increase the reproducibility of nerve graft surgical techniques, reduce intraoperative handling of delicate nerve tissue, and increase the consistency and predictability of treatment outcomes. Use of the nerve repair device of the present invention increases access to treatments that address the highest functional priorities for Americans living with quadriplegia.

[0140] Reference materials Van Zyl, N. et al. (2019), Lancet, 394(10198):565-575. Fox, IK et al. (2015), Plastic and Reconstructive Surgery, 136(4):780-792. Khalifeh, JM et al. (2019), JNS, 31(5):629-640. Bertelli, J et al. (2011), J Neurosurg, 114:1457-1460. Bertelli, J et al. (2012), J. Hand Surgery, 37(10): 1990-1993. Van Zyl, N. et al. (2014), J. Hand Surgery, 39(9): 1779-1783. Sananpanich, K. et al. (2018), J. Hand Surgery, 43(10): 920-926.

Claims

1. 1. A device for repairing severed or damaged nerves in vivo, comprising a flexible collagen membrane including a first surface and a second surface, said first surface having a plurality of protruding collagen bundles and said second surface comprising a plurality of openings, said openings and said collagen bundles configured to form a self-interlocking interlock when said first surface and said second surface are positioned together.

2. The device of claim 1 , wherein the openings and the collagen bundles are configured to interlock with one another when the first surface and the second surface are placed together.

3. The device of claim 1 or 2, wherein the flexible collagen membrane is an elongated profile membrane.

4. 2. The device of claim 1, wherein the flexible collagen membrane is configured to be wrapped in situ around at least a lengthwise portion of a mammalian nerve in a manner such that the first surface and the second surface are positionable together to self-connect to one another.

5. The device of claim 4 , wherein the flexible collagen membrane is less than 200 microns thick.

6. The device of claim 4, wherein the flexible collagen membrane is 100 to 200 microns thick.

7. The device of claim 5 , wherein the flexible collagen membrane is less than 100 microns thick.

8. The device of claim 7 , wherein the flexible collagen membrane is less than 75 microns thick.

9. 9. The device of any one of claims 4 to 8, wherein the mammalian nerve comprises a cut or damaged epineurium and the device, when wrapped around the cut or damaged nerve, has the ability to repair the cut or damaged epineurium in less than four weeks.

10. The device of claim 1 , wherein the flexible collagen membrane comprises greater than 80% type I collagen.

11. The device of claim 10 , wherein the flexible collagen membrane comprises greater than 85% type I collagen.

12. The device of claim 11 , wherein the flexible collagen membrane comprises greater than 90% type I collagen.

13. 13. The device of any one of claims 10 to 12, wherein the flexible collagen membrane further comprises type III collagen.

14. 3. The device of claim 1 or 2, further comprising one or more of hydroxyapatite, a growth factor, a chemotactic factor, or an extracellular matrix molecule.

15. The device of claim 14, wherein the growth factor is selected from the group consisting of basic fibroblast growth factor, tumor growth factor beta, bone morphogenetic protein, platelet-derived growth factor, and insulin-like growth factor.

16. The device of claim 14 , wherein the chemotactic factor is fibronectin or hyaluronan, or a combination thereof.

17. The device of claim 14 , wherein the extracellular matrix molecule is selected from the group consisting of aggrecan, biglycan, and decorin.

18. 1. An in vivo method for repairing a severed or damaged nerve, comprising: (i) a device for repairing a severed or damaged nerve in vivo comprising a flexible collagen membrane including a first surface and a second surface, said first surface having a plurality of protruding collagen bundles and said second surface comprising a plurality of openings, said openings and said collagen bundles configured to form a self-interlocking interlock when said first surface and said second surface are placed together; (ii) wrapping said flexible membrane in situ around at least a longitudinal portion of a severed or damaged nerve at the defect site, wherein said severed or damaged nerve includes a severed or damaged epineurium; (iii) allowing the device to remain in place until the severed or damaged epineurium has repaired, for a period of time that is less than four weeks; The method includes:

19. The method of claim 18 , wherein the openings and the collagen bundles are configured to interlock with one another when the first surface and the second surface are placed together.

20. The method of claim 18, wherein the flexible collagen membrane is an elongated profile membrane.

21. 20. The method of claim 18, wherein the flexible collagen membrane is configured to be wrapped in situ around at least a longitudinal portion of a nerve in a manner such that the first surface and the second surface are positionable together to self-connect to one another.

22. 22. The method of claim 21, wherein the flexible collagen membrane is less than 200 microns thick.

23. 22. The method of claim 21, wherein the flexible collagen membrane is 100 to 200 microns thick.

24. 23. The method of claim 22, wherein the flexible collagen membrane is less than 100 microns thick.

25. 25. The method of claim 24, wherein the flexible collagen membrane is less than 75 microns thick.

26. 22. The method of claim 21, wherein the nerve comprises a cut or damaged epineurium and the device, when wrapped around the cut or damaged nerve, has the ability to repair the cut or damaged epineurium in less than four weeks.

27. 20. The method of claim 18, wherein the flexible collagen membrane comprises greater than 80% type I collagen.

28. 28. The method of claim 27, wherein the flexible collagen membrane comprises greater than 85% type I collagen.

29. 30. The method of claim 28, wherein the flexible collagen membrane comprises greater than 90% type I collagen.

30. 28. The method of claim 27, wherein the flexible collagen membrane further comprises type III collagen.

31. 20. The method of claim 18, wherein the device further comprises one or more of hydroxyapatite, a growth factor, a chemotactic factor, or an extracellular matrix molecule.

32. 32. The method of claim 31, wherein the growth factor is selected from the group consisting of basic fibroblast growth factor, tumor growth factor beta, bone morphogenetic protein, platelet-derived growth factor, and insulin-like growth factor.

33. 32. The method of claim 31 , wherein the chemotactic factor is fibronectin or hyaluronan, or a combination thereof.

34. 32. The method of claim 31 , wherein the extracellular matrix molecule is selected from the group consisting of aggrecan, biglycan, and decorin.

35. 35. The method of any one of claims 18 to 34, wherein the repair of the severed or damaged epineurium comprises successful integration of the device into the nerve and / or epineurium without scar tissue formation or fibrous adhesion of the repaired nerve to the patient's surrounding tissue.

36. The successful integration includes: (i) the device does not protrude from the repaired nerve; (ii) little or no scar tissue formation at the defect site; (iii) little or no fibrous adhesion of the repaired nerve to surrounding soft tissues within the patient; (iv) histological examination reveals new vascularized epineurium-like tissue at the site of nerve repair; (v) there is no evidence of an inflammatory response after a suitable period of time following ligation; (vi) abundant neurofilaments were detected adjacent to the new epineural tissue by immunohistochemistry; (vii) well-organized nerve fibers are observed distal to the nerve repair site; and (viii) Evidence of successful axonal regeneration across the repair site.

36. The method of claim 35, comprising one or more of:

37. (i) providing a donor nerve containing a severed or damaged epineurium; (ii) providing a device for repairing a severed or damaged nerve in vivo, comprising a flexible collagen membrane including a first surface and a second surface, said first surface having a plurality of protruding collagen bundles and said second surface comprising a plurality of openings, said openings and said collagen bundles configured to form a self-interlocking interlock when said first surface and said second surface are placed together; (iii) suturing the donor nerve in place and then wrapping the flexible collagen membrane around at least a longitudinal portion of the donor nerve to contain the severed or damaged epineurium; (iv) allowing the device to remain in place for a period of time less than four weeks until the severed or damaged epineurium has repaired; An in vivo nerve transplantation method comprising:

38. 38. The method of claim 37, wherein the openings and the collagen bundles are configured to interlock with one another when the first surface and the second surface are placed together.

39. 38. The method of claim 37, wherein the flexible collagen membrane is an elongated profile membrane.

40. 38. The method of claim 37, wherein the flexible collagen membrane is configured to be wrapped in situ around at least a longitudinal portion of a nerve in a manner such that the first surface and the second surface are positionable together to self-connect to one another.

41. 41. The method of claim 40, wherein the flexible collagen membrane is less than 200 microns thick.

42. 41. The method of claim 40, wherein the flexible collagen membrane is 100 to 200 microns thick.

43. 42. The method of claim 41, wherein the flexible collagen membrane is less than 100 microns thick.

44. 44. The method of claim 43, wherein the flexible collagen membrane is less than 75 microns thick.

45. 41. The method of claim 40, wherein the nerve includes a cut or damaged epineurium and the device, when wrapped around the cut or damaged nerve, has the ability to repair the cut or damaged epineurium in less than four weeks.

46. 38. The method of claim 37, wherein the flexible collagen membrane comprises greater than 80% type I collagen.

47. 47. The method of claim 46, wherein the flexible collagen membrane comprises greater than 85% type I collagen.

48. 47. The method of claim 46, wherein the flexible collagen membrane comprises greater than 90% type I collagen.

49. 38. The method of claim 37, wherein the flexible collagen membrane further comprises type III collagen.

50. 38. The method of claim 37, wherein the device further comprises one or more of hydroxyapatite, a growth factor, a chemotactic factor, or an extracellular matrix molecule.

51. 51. The method of claim 50, wherein the growth factor is selected from the group consisting of basic fibroblast growth factor, tumor growth factor beta, bone morphogenetic protein, platelet-derived growth factor, and insulin-like growth factor.

52. 51. The method of claim 50, wherein the chemotactic factor is fibronectin or hyaluronan, or a combination thereof.

53. 51. The method of claim 50, wherein the extracellular matrix molecule is selected from the group consisting of aggrecan, biglycan, and decorin.

54. 54. The method of any one of claims 37 to 53, wherein the repair of the severed or damaged epineurium comprises successful integration of the device into the nerve and / or epineurium without scar tissue formation or fibrous adhesion of the repaired nerve to the patient's surrounding tissue.

55. The successful integration includes: (i) the device does not protrude from the repaired nerve; (ii) little or no scar tissue formation at the defect site; (iii) little or no fibrous adhesion of the repaired nerve to surrounding soft tissues within the patient; (iv) histological examination reveals new vascularized epineurium-like tissue at the site of nerve repair; (v) there is no evidence of an inflammatory response after a suitable period of time following ligation; (vi) abundant neurofilaments were detected adjacent to the new epineural tissue by immunohistochemistry; (vii) well-organized nerve fibers are observed distal to the nerve repair site; and (viii) Evidence of successful axonal regeneration across the repair site.

55. The method of claim 54, comprising one or more of:

56. A repair kit for in vivo repair of severed or damaged nerves and / or epineurium, comprising: (i) a device for in vivo repair of a severed or damaged nerve and / or epineurium comprising a flexible collagen membrane including a first surface and a second surface, said first surface having a plurality of protruding collagen bundles and said second surface comprising a plurality of openings, said openings and said collagen bundles configured to form a self-interlocking interlocking when said first surface and said second surface are placed together; (ii) a sterile container for holding the device; (iii) instructions for coupling the device to a nerve and / or epineurium in need of repair; Includes repair kit.

57. 57. The repair kit of claim 56, further comprising one or more of the following: sutures, forceps, suture needles, fibrin glue, and accessory tools for accessing tissue.

58. 58. The repair kit of claim 57, wherein the accessory tool for accessing tissue is selected from the group consisting of a clip, a calibration weight, an aspiration device, and a compression gauge.

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