Nerve graft systems, devices, and methods

Abstract

Systems, devices, and methods for treating a nerve injury in a patient are provided. Systems include a nerve cap graft including tissue harvested from a tissue source. Methods include deploying a nerve cap graft at an implant site in a patient to provide a therapeutic benefit. The devices include a matrix designed to fit on one or more nerve stumps of a patient and the devices are designed to provide a patient with a therapeutic effect.

Description

[0001] Attorney Docket No. REN-008-PCT INTERNATIONAL PCT PATENT APPLICATION for NERVE GRAFT SYSTEMS, DEVICES, AND METHODS Inventors: Lorenzo Soletti, a citizen of the USA, residing at: 217 Vine Street, Pittsburgh, PA 15218 Sydney E. Borcherding a citizen of the USA, residing at: 84 South 14" Street, Pittsburgh, PA 15203 Anne E. Faust, a citizen of the USA, residing at: 5700 Centre Avenue, Apt. 916, Pittsburgh, PA 15206 Assignee: Renerva, LLC 217 Vine Street Pittsburgh, PA 15218 USA Entity: Small

[0002] Attorney Docket No. REN-008-PCT NERVE GRAFT SYSTEMS, DEVICES, AND METHODS RELATED APPLICATIONS [1] This application is related to: United States Patent No. 8,361,503, Issued January 29, 2013; United States Patent No. 8,691,276, Issued April 8, 2014; United States Patent No. 9,737,635, Issued August 22, 2017; United States Patent No. 10,004827, Issued June 26, 2018; United States Patent No. 10,179,192, Issued January 15, 2019; United States Patent No. 10,213,526, Issued February 26. 2019; United States Patent No. 10,729,813, Issued August 4, 2020; United States Patent No. 10,772,989, Issued September 15, 2020; United States Patent No. 11,338,058, Issued May 24, 2022; and United States Patent No. 11,413,375 Issued August 16, 2022 the content of each of which is incorporated herein by reference in its entirety for all purposes. [2] This application is related to: United States Patent Application Serial Number 15 / 996,916 entitled “Extracellular Matrix-Derived Gels and Related Methods”, filed June 4, 2018, published as US2019 / 0038803; United States Patent Application Serial Number 16 / 288,831 entitled “Extracellular Matrix-Derived Gels and Related Methods”, filed February 28, 2019, published as US2019 / 201581; International PCT Patent Application Serial Number PCT / US2020 / 053570 entitled “Extracellular Matrix Devices and Methods of Manufacture”, filed September 30, 2020, published as WO2021 / 067456; United States Patent Application Serial Number 17 / 762,858 entitled “Extracellular Matrix Devices and Methods of Manufacture” filed March 23, 2022, published as US20220323648; International PCT Patent Application Serial Number PCT / US2020 / 067431 entitled “Extracellular Matrix Systems, Devices, and Methods of Deployment”, filed December 30, 2020, published as WO2021 / 0138399; United States Patent Application Serial Number 17 / 788450 Attorney Docket No. REN-008-PCT entitled “Extracellular Matrix Systems, Devices, and Methods of Deployment”, filed June 23, 2022, published as US2023 / 0034585; International PCT Patent Application Serial Number PCT / US2022 / 045365 entitled “Nerve Graft Systems, Devices and Methods”, filed September 30, 2022, published as WO2023 / 056008; and International PCT Patent Application Serial Number PCT / US2022 / 054351 entitled “Neurorrhaphy Systems, Devices, and Methods”, filed December 30, 2022, the content of each of which is incorporated herein by reference in its entirety for all purposes. TECHNICAL FIELD [3] The present inventive concepts relate generally to systems, devices, and methods for improved nerve injury treatment and prevention of neuroma. BACKGROUND [4] Peripheral nerve damage caused by trauma, iatrogenic injury, or underlying disease, can have wide-ranging impacts on the quality of life, productivity, and interpersonal relationships of those affected. In cases where nerve damage is too severe to attempt surgical repair to restore function, where no surgical option or expertise is available, or where such restoration of function can be forgone, one or more nerve stumps are often left untreated. In these cases, maladaptive peripheral nerve remodeling of the nerve stump(s) following injury can cause chronic pain and related symptoms, including residual limb pain and neuroma pain. These conditions are either caused or exacerbated by the formation of neuroma, and depend on the sensitization of the nociceptive signal transduction and transmission along the pathway from the transected nerve stump and its surrounding tissue (including the significant tissue injury generated in the case of amputation) to the areas of the brain that process pain signals. In neuromas, the regenerating nerve fibers cannot reach their target tissue, and instead form a painful tangled bulbous mass. This is likely due to pathological interactions between the regenerating nerve fibers and the surrounding environment. When this happens, patients can experience severe limitations to their daily activities and psychosocial symptoms, including reduced mobility and depression. [5] While pharmacologic pain management can provide temporary pain relief, surgical treatment is the only option to remove the neuroma once formed. Surgical techniques Attorney Docket No. REN-008-PCT to correct neuroma formation involve burying the distal nerve ending in either bone or muscle, but the outcomes are highly variable with reports of reoperations ranging from between 40% and 81% for burial in muscle and between 33% and 91% for burial in bone with 2.8 re-interventions required on average. Targeted muscle reinnervation (TMR) is another surgical technique used to prevent neuroma formation. This procedure involves connection of each amputated nerve to an existing nerve-muscle pedicle, thereby creating a physiologic alternative to neuroma formation. While this technique has been shown to decrease neuropathic pain compared to the standard of care and to reduce the recurrence rate of neuroma, it requires specialized training and a dedicated surgical procedure lasting an hour or more. Moreover, in a portion of the cases, TMR is not feasible, resulting in only a fraction of affected patients receiving this procedure. [6] Commercially available nerve cap devices made of synthetic or biologically-derived materials are intended to provide protection from the tissue injury inflammatory environment. While these devices can provide a macrostructural barrier to contain axonal regeneration, they can also create nerve ‘compartment syndrome’ as these devices can become a restrictive ‘barrier / trap’ for growing / sprouting axons. Furthermore, when synthetic degradable materials are used, their degradation products can elicit a fibrotic inflammatory response, which can further increase the risk of neuroma formation. These devices have not been found to be more effective than nerve burial approaches, and their clinical adoption has been limited due to insufficient / inconclusive clinical data. [7] Prior studies in rat showed that long, decellularized nerve allografts applied to a free nerve ending arrested the formation of neuroma by exhausting the nerve regenerative potential within a length of between 2.5 and 5 cm over the course of 5 weeks and 5 months. While this promising approach benefits from both a protective environment and the longitudinal guidance for sprouting axons at the proximal nerve stump following nerve transection, the use of allografts in the clinic is not ideal due to the shortage of high-quality tissue sources, high variability, and costs associated with cadaveric procurement. Furthermore, these approaches require an extensive length of graft to complete the exhaustion of the native nerve regenerative potential as the grafts are specifically tailored and generally intended for nerve regeneration applications. Such distance requires the creation of an additional surgical pocket, which can increase the complexity and duration of the surgical procedure. Attorney Docket No. REN-008-PCT [8] The limitations of the aforementioned existing approaches suggest that there is a need for improved neuroma prevention treatment systems, devices, and methods in cases where one or more nerve stumps is present. BRIEF SUMMARY [9] According to an aspect of the present inventive concepts, a system for treating a patient comprising a nerve cap device comprising tissue derived from a tissue source. The system can be configured to provide a therapeutic benefit to the patient.

[0010] According to another aspect of the present inventive concepts, a method for treating a patient comprising the deployment of a system comprising a nerve cap device at an implant site in the patient. The system can be configured to provide a therapeutic benefit at the implant site.

[0011] According to another aspect of the present inventive concepts, a method for manufacturing a system for treating a patient comprising a nerve cap device configured to provide a therapeutic benefit to patients.

[0012] The technology described herein, along with the attributes and attendant advantages thereof, will best be appreciated and understood in view of the following detailed description taken in conjunction with the accompanying drawings in which representative embodiments are described by way of example. INCORPORATION BY REFERENCE

[0013] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figs. 1A-C illustrate a schematic view of a System comprising one or more nerve cap graft devices, additional methods of using, and methods of manufacturing and storage.

[0015] Fig. 2 illustrates a schematic view of a nerve-cap graft consistent with the present inventive concepts. Attorney Docket No. REN-008-PCT

[0016] Fig 3. illustrates a schematic view of nerve-cap graft degradation and axon infiltration behavior over time, consistent with the present inventive concepts.

[0017] Figs. 4A-F illustrate a schematic view of multiple implantation configurations of a nerve cap graft device, consistent with the present inventive concepts.

[0018] Fig. 5 illustrates a schematic view of a sizing tool, consistent with the present inventive concepts.

[0019] Fig. 6 illustrates a method for producing a nerve cap graft device from tissue, consistent with the present inventive concepts.

[0020] Fig. 7 illustrates a method for selecting and harvesting tissue for further processing, consistent with the present inventive concepts.

[0021] Fig. 8 illustrates a method for nerve tissue cleaning and segmentation for further processing, consistent with the present inventive concepts.

[0022] Fig. 9 illustrates a method for chemically treating a nerve segment to remove cells and other immunogenic components, consistent with the present inventive concepts.

[0023] Fig. 10 illustrates a method for inserting a nerve segment into a correctly sized support assembly, consistent with the present inventive concepts.

[0024] Fig. 11 illustrates a method for inserting support assemblies containing nerve segments into a holding rack prior to further processing, consistent with the present inventive concepts.

[0025] Fig. 12 illustrates a method for creating desired features in a nerve segment, consistent with the present inventive concepts.

[0026] Fig. 13 illustrates a method for kitting and packaging a nerve cap graft device, consistent with the present inventive concepts.

[0027] Fig. 14 illustrates a method for sterilizing a nerve cap graft device, consistent with the present inventive concepts.

[0028] Fig. 15 illustrates a method for storing a nerve cap graft device, consistent with the present inventive concepts.

[0029] Fig. 16 illustrates a method for implanting a nerve cap graft device, consistent with the present inventive concepts.

[0030] Figs. 17A-C illustrate a schematic view of raw and cleaned nerve tissue, consistent with the present inventive concepts.

[0031] Figs. 18A-C illustrate a schematic view of a method to clean and segment nerve tissue branches, consistent with the present inventive concepts. Attorney Docket No. REN-008-PCT

[0032] Fig. 19 illustrates a schematic view of a cassette configured to secure nerve segments during processing, consistent with the present inventive concepts.

[0033] Fig. 20 illustrates a schematic view of a bioreactor shaft assembly configured to secure cassettes comprising nerve segments during processing, consistent with the present inventive concepts.

[0034] Figs. 21A-B illustrate a schematic view of a mixing device configured to hold bioreactor shaft assembly comprising nerve segments during processing, consistent with the present inventive concepts.

[0035] Fig. 22 illustrates a schematic and graphical view of a method for chemical treatment verification analysis, consistent with the present inventive concepts.

[0036] Figs. 23A-B illustrate schematic views of a nerve size discrimination tool and methods, consistent with the present inventive concepts.

[0037] Fig. 24 illustrates a schematic view of a support assembly to hold a nerve segment, consistent with the present inventive concepts.

[0038] Fig. 25 illustrates a schematic view of a tool configured to insert a nerve segment into a support assembly, consistent with the present inventive concepts.

[0039] Figs. 26A-F illustrate schematic views of different configurations and methods to contain nerve segments within support assemblies, consistent with the present inventive concepts.

[0040] Figs. 27A-F illustrate schematic views of a holding rack for use in manufacturing a nerve cap graft device, consistent with the present inventive concepts.

[0041] Fig. 28 illustrates a schematic view of a nerve segment holder for use in MicroCT scanning and assessing nerve cap graft device porosity, consistent with the present inventive concepts.

[0042] Figs. 29A-B illustrate schematic views of a deflaring tool and methods to create desired features in a nerve cap graft device, consistent with the present inventive concepts.

[0043] Figs. 30A-B illustrate schematic views of an assembly to align the feature creation of a nerve cap graft device, consistent with the present inventive concepts.

[0044] Fig. 31 illustrates a schematic view of a method to create a tab feature in a nerve cap graft device, consistent with the present inventive concepts.

[0045] Fig. 32 illustrates a schematic view of the materials and methods for the packaging of a nerve cap graft device, consistent with the present inventive concepts. Attorney Docket No. REN-008-PCT

[0046] Fig. 33 illustrates a table of nerve cap graft device sizes and features, consistent with the present inventive concepts.

[0047] Fig. 34 illustrates a method for obtaining ideal decellularization in order to obtain desired structure and function in the nerve cap graft device, consistent with the present inventive concepts. DETAILED DESCRIPTION OF THE DRAWINGS

[0048] Reference will now be made in detail to the present embodiments of the technology, examples of which are illustrated in the accompanying drawings. Similar reference numbers may be used to refer to similar components. However, the description is not intended to limit the present disclosure to particular embodiments, and it should be construed as including various modifications, equivalents, and / or alternatives of the embodiments described herein.

[0049] It will be understood that the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") when used herein, specify the presence of 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.

[0050] It will be further understood that, although the terms first, second, third, etc. may be used herein to describe various limitations, elements, components, regions, layers and / or sections, these limitations, elements, components, regions, layers and / or sections should not be limited by these terms. These terms are only used to distinguish one limitation, element, component, region, layer or section from another limitation, element, component, region, layer, or section. Thus, a first limitation, element, component, region, layer, or section discussed below could be termed a second limitation, element, component, region, layer, or section without departing from the teachings of the present application.

[0051] It will be further understood that when an element is referred to as being "on", "attached", "connected" or "coupled" to another element, it can be directly on or above, or connected or coupled to, the other element, or one or more intervening elements can be present. In contrast, when an element is referred to as being "directly on", "directly attached", "directly connected" or "directly coupled" to another element, there are no intervening Attorney Docket No. REN-008-PCT elements present. Other words used to describe the relationship between elements should be interpreted in a like fashion (e.g., "between" versus "directly between," "adjacent" versus "directly adjacent," etc.).

[0052] It will be further understood that when a first element is referred to as being "in", "on" and / or "within" a second element, the first element can be positioned: within an internal space of the second element, within a portion of the second element (e.g., within a wall of the second element); positioned on an external and / or internal surface of the second element; and combinations of one or more of these.

[0053] As used herein, the term “proximate”, when used to describe proximity of a first component or location to a second component or location, is to be taken to include one or more locations near to the second component or location, as well as locations in, on and / or within the second component or location. For example, a component positioned proximate an anatomical site (e.g., a target tissue location), shall include components positioned near to the anatomical site, as well as components positioned in, on and / or within the anatomical site.

[0054] Spatially relative terms, such as "beneath," "below," "lower," "above," "upper" and the like may be used to describe an element and / or feature's relationship to another element(s) and / or feature(s) as, for example, illustrated in the figures. It will be further understood that the spatially relative terms are intended to encompass different orientations of the device in use and / or operation in addition to the orientation depicted in the figures. For example, if the device in a figure is turned over, elements described as "below" and / or "beneath" other elements or features would then be oriented "above" the other elements or features. The device can be otherwise oriented (e.g., rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.

[0055] The terms “reduce”, “reducing”, “reduction” and the like, where used herein, are to include a reduction in a quantity, including a reduction to zero. Reducing the likelihood of an occurrence shall include prevention of the occurrence. Correspondingly, the terms “prevent”, “preventing”, and “prevention” shall include the acts of “reduce”, “reducing”, and “reduction”, respectively.

[0056] The term "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Attorney Docket No. REN-008-PCT

[0057] The term “one or more”, where used herein can mean one, two, three, four, five, six, seven, eight, nine, ten, or more, up to any number.

[0058] The terms “and combinations thereof” and “and combinations of these” can each be used herein after a list of items that are to be included singly or collectively. For example, a component, process, and / or other item selected from the group consisting of: A; B; C; and combinations thereof, shall include a set of one or more components that comprise: one, two, three or more of item A; one, two, three or more of item B; and / or one, two, three, or more of item C.

[0059] In this specification, unless explicitly stated otherwise, “and” can mean “or”, and “or” can mean “and”. For example, if a feature is described as having A, B, or C, the feature can have A, B, and C, or any combination of A, B, and C. Similarly, if a feature is described as having A, B, and C, the feature can have only one or two of A, B, or C.

[0060] As used herein, when a quantifiable parameter is described as having a value “between” a first value X and a second value Y, it shall include the parameter having a value of: at least X, no more than Y, and / or at least X and no more than Y. For example, a length of between 1 and 10 shall include a length of at least 1 (including values greater than 10), a length of less than 10 (including values less than 1), and / or values greater than 1 and less than 10.

[0061] The expression “configured (or set) to” used in the present disclosure may be used interchangeably with, for example, the expressions “suitable for”, “having the capacity to”, “designed to”, “adapted to”, “made to” and “capable of” according to a situation. The expression “configured (or set) to” does not mean only “specifically designed to” in hardware. Alternatively, in some situations, the expression “‘a device configured to” may mean that the device “can” operate together with another device or component.

[0062] As used herein, the term “threshold” refers to a maximum level, a minimum level, and / or range of values correlating to a desired or undesired state. In some embodiments, a system parameter is maintained above a minimum threshold, below a maximum threshold, within a threshold range of values, and / or outside a threshold range of values, such as to cause a desired effect (e.g., efficacious therapy) and / or to prevent or otherwise reduce (hereinafter “prevent”’) an undesired event (e.g., a device and / or clinical adverse event). In some embodiments, a system parameter is maintained above a first threshold (e.g., above a first temperature threshold to cause a desired therapeutic effect to tissue) and below a second threshold (e.g., below a second temperature threshold to prevent Attorney Docket No. REN-008-PCT undesired tissue damage). In some embodiments, a threshold value is determined to include a safety margin, such as to account for patient variability, system variability, tolerances, and the like. As used herein, “exceeding a threshold” relates to a parameter going above a maximum threshold, below a minimum threshold, within a range of threshold values and / or outside of a range of threshold values.

[0063] The term “diameter” where used herein to describe a non-circular geometry is to be taken as the diameter of a hypothetical circle approximating the geometry being described. For example, when describing a cross section, such as the cross section of a component, the term “diameter” shall be taken to represent the diameter of a hypothetical circle with the same cross-sectional area as the cross section of the component being described.

[0064] As used herein, the term “functional element” is to be taken to include one or more elements constructed and arranged to perform a function. A functional element can comprise a sensor and / or a transducer. In some embodiments, a functional element is configured to generate and / or deliver energy and / or otherwise treat tissue (e.g., a functional element configured as a treatment element). Alternatively, or additionally, a functional element (e.g., a functional element comprising a sensor) can be configured to record one or more parameters, such as a patient physiologic parameter; a patient anatomical parameter (e.g., a tissue geometry parameter); a patient environment parameter; and / or a system parameter. In some embodiments, a sensor or other functional element is configured to perform a diagnostic function (e.g., to gather data used to perform a diagnosis). In some embodiments, a functional element is configured to perform a therapeutic function (e.g., to deliver therapeutic energy and / or a therapeutic agent). In some embodiments, a functional element comprises one or more elements constructed and arranged to perform a function selected from the group consisting of: deliver energy; extract energy (e.g., to cool a component); deliver a drug or other agent; manipulate a system component or patient tissue; record or otherwise sense a parameter such as a patient physiologic parameter or a system parameter; and combinations of one or more of these. A functional element can comprise a fluid and / or a fluid delivery system. A functional element can comprise a reservoir, such as an expandable balloon or other fluid-maintaining reservoir. A “functional assembly” can comprise an assembly constructed and arranged to perform a function, such as a diagnostic and / or therapeutic function. A functional assembly can comprise an expandable assembly. A functional assembly can comprise one or more functional elements. -1ll- Attorney Docket No. REN-008-PCT

[0065] As used herein, the term “fluid” can refer to a liquid, gas, gel, or any flowable material, such as a material which can be propelled through a lumen and / or opening.

[0066] It is appreciated that certain features of the present inventive concepts, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present inventive concepts which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. For example, it will be appreciated that all features set out in any of the claims (whether independent or dependent) can be combined in any given way.

[0067] It is to be understood that at least some of the figures and descriptions of the present inventive concepts have been simplified to focus on elements that are relevant for a clear understanding of the present inventive concepts, while eliminating, for purposes of clarity, other elements that those of ordinary skill in the art will appreciate may also comprise a portion of the present inventive concepts. However, because such elements are well known in the art, and because they do not necessarily facilitate a better understanding of the present inventive concepts, a description of such elements is not provided herein.

[0068] Terms defined in the present disclosure are only used for describing specific embodiments of the present disclosure and are not intended to limit the scope of the present disclosure. Terms provided in singular forms are intended to include plural forms as well, unless the context clearly indicates otherwise. All of the terms used herein, including technical or scientific terms, have the same meanings as those generally understood by an ordinary person skilled in the related art, unless otherwise defined herein. Terms defined in a generally used dictionary should be interpreted as having meanings that are the same as or similar to the contextual meanings of the relevant technology and should not be interpreted as having ideal or exaggerated meanings, unless expressly so defined herein. In some cases, terms defined in the present disclosure should not be interpreted to exclude the embodiments of the present disclosure.

[0069] Provided herein are improved nerve injury treatment and prevention of neuroma systems, devices, and methods.

[0070] Referring now to Fig, 1A-C, a schematic view of a System 1000 comprising one or more nerve cap graft devices System 1000A, additional methods of using System 1000B, and methods of manufacturing and storage System 1000C. Attorney Docket No. REN-008-PCT

[0071] In Fig. 1A, Device 10 comprises one or more nerve cap graft devices, Nerve Cap 100 shown, as well as various components used to package, deploy, or aid in the deployment of Nerve Cap 100. Device 10 can be configured as a terminally sterilized, biocompatible, resorbable medical device available off-the shelf in multiple sizes, with handling characteristics suitable for surgical use in the intended application. One or more Nerve Cap 100 are configured to be deployed (e.g., implanted and the like) at one or more “implant sites” (one Nerve Cap 100 per implant site), such as to provide a therapeutic benefit at each implant site. Each implant site can comprise a peripheral nerve stump or stumps for which functional recovery is not desirable or possible, produced by trauma, iatrogenic injury, or underlying disease. A Nerve Cap 100 can be implanted at each implant site to prevent, inhibit, and / or reduce nerve tissue growth, neuroma formation, or other neuropathic conditions from the treated nerve stump. In some embodiments, Nerve Cap 100 is remodeled over time into native tissue of the patient.

[0072] In some embodiments, the implant site requires an additional surgical incision to reach a more proximal location of the nerve(s) affected by a downstream amputation. In this embodiment, the tissue nociceptive innervation surrounding the implant site would not experience the significant nociceptive sensitization activated by the chemical milieu released in the diffuse tissue damage at the site of extremity amputation. In this embodiment, an effective isolation of the nociceptive innervation between the implant site and the downstream nerve stump at the amputation site is achieved, leading to an elimination / reduction of pain.

[0073] Nerve Cap 100 comprises an acellular nerve graft obtained from a treated nerve segment comprising decellularized extracellular matrix, Matrix 110 shown. Matrix 110 can comprise structural and non-structural endogenous (i.¢e., naturally present in Matrix 110) biomolecules, including, but not limited to collagens, elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and / or matrix bound vesicles. In some non-limiting embodiments, Matrix 110 can comprise modified amounts and combinations of the aforementioned endogenous structural and non-structural components. In some additional non-limiting embodiments, Nerve Cap 100 can include exogenous (.e., artificially added to those endogenously present) molecules such as Small Molecules / Drugs (anti-inflammatory, etc.) 140, Large Molecules / Biologics (growth factors, immunomodulators, etc.) 150, Anti-Microbial Agents 160, or other Molecules 170 and combinations thereof. Matrix 110 can be configured to inhibit the growth of nerve tissue Attorney Docket No. REN-008-PCT and / or associated processes leading to the formation of neuroma or other neuropathic conditions causing pain.

[0074] Matrix 110 can be derived, or otherwise produced, from one or more Raw Material 1010 as described herein. In some embodiments, Matrix 110 is derived from a raw material as described in applicant’s co-pending United States Patent Application Serial Number 17 / 762,858 entitled “Extracellular Matrix Devices and Methods of Manufacture” filed March 23, 2022, published as US20220323648. For overall understanding and in no way meant to be limiting to the inventive concepts disclosed in this application, Raw Material 1010 generally refers to sourced nerve tissue prior to any treatments including chemical treatments, decellularization, or other treatments disclosed. Acellular nerve graft generally refers to a segment of nerve tissue treated to obtain decellularization and that can make up the Matrix 110. Matrix 110 generally refers to nerve tissue that has been decellularized and possibly further treated, and that comprises the Nerve Cap 100.

[0075] Raw Material 1010 can comprise sensory, motor, and / or mixed nerve tissue. In some embodiments, Raw Material 1010 comprises autonomic nerve tissue. In some embodiments, Raw Material 1010 comprises spinal cord nerve tissue. In some embodiments, Raw Material 1010 comprises ventral and / or dorsal root ganglion. In some embodiments, Raw Material 1010 comprises somatic nerve tissue. In some embodiments, Raw Material 1010 comprises sciatic nerve tissue, such as bilateral sciatic nerves and their distal branches (i.e., sural branch, tibial branch, peroneal branch, etc.) and sub-branches. In some embodiments, Raw Material 1010 comprises brachial plexus tissue. In some embodiments, Raw Material 1010 is selected and harvested from a specific animal species of a specific age, sex, and / or weight, and from a specific anatomic location within the peripheral nervous system in order to produce one or more desired features in Nerve Cap 100. In some embodiments, this specific anatomic location includes a specific nerve branch or sub-branch, as well as a specific position within them (e.g., a position at a defined distance from a prior and / or neighboring nerve branch or sub-branch). In some embodiments, the anatomic location includes the nerve branching points. In some embodiments, this specific anatomic location excludes segments of nerve tissue that contain branches and sub-branches. In some embodiments, specific nerve branches are indicated for specific sizes / diameters of nerves to be treated. Desired features of Nerve Cap 100 can include, but are not limited to: structural and functional qualities relating to the overall nerve area (e.g., external diameter); structural and functional qualities of the area occupied by nerve fascicles (e.g., number of fascicles, Attorney Docket No. REN-008-PCT and / or fascicular density, such as can be measured by the area of fascicles divided by the total nerve area); structural and functional qualities of the area occupied by axons (e.g., average axonal cross-sectional area, average myelin sheath thickness, and / or axonal density, such as can be as measured by the total axonal area divided by the total nerve cross-sectional area); and combinations of these.

[0076] In some embodiments, Nerve Cap 100 is designed and / or manufactured with one, two, or more structural and functional qualities intended to match, mismatch, or adapt to the structural and functional qualities of the nerve stump intended to be treated. For example, Nerve Cap 100 may be intentionally designed and / or manufactured to mismatch fascicle number and / or fascicle area to inhibit nerve regeneration by creating a convergence (e.g., funneling excessive axonal growth into an insufficient distal space) and / or a divergence (e.g., excessive splicing of axonal growth into multiple distal recipients) in the regenerating axons.

[0077] In some embodiments, Nerve Cap 100 comprises nerve tissue segments that have different degrees of decellularization depending on the radial depth. During decellularization, nerve segments are submerged in a detergent solution (in some embodiments combined with mechanical, chemical, and / or physical treatment). The radial distance from the edge of the nerve segment can affect the degree mass transport of detergent solution into each specific area and the resulting treatment. As a function of this difference in mass transport, the outer portions of the nerve segment treated can be exposed to a higher degree of treatment and are subsequently more decellularized than the inner portions of the nerve segment, resulting in a gradient of treatment (e.g., residual cellular and myelin content) along the radial direction of the nerve segment, with the highest level of treatment (and lowest level of residual content) proximate to the outer portion of the nerve segment and the lowest level of treatment (and highest level of residual content) proximate to the innermost portion of the nerve segment.

[0078] Raw Material 1010 can comprise tissue harvested from a Tissue Source 1020 selected from the group consisting of: a mammal, such as pig, human, cow, horse, and the like; an amphibian, such as salamander, axolotl, frog, and the like; a chondrichthyan, such as shark and the like; reptile, such as chelonians, crocodiles, snakes, and the like; a cephalopod, such as squid and the like; marine invertebrate animals, such as starfish, tunicate, geoduck, and the like; and combinations of these. In some embodiments, Raw Material 1010 comprises tissue harvested from a Landrace, Landrace X, or Yorkshire pig. For example, Raw Material 1010 can comprise sciatic nerve tissue, such as sciatic nerve tissue harvested from a Tissue source 1020 comprising a pig (e.g., a male pig), such as a pig with a weight between Ilbs and Attorney Docket No. REN-008-PCT 400lbs, such as a weight between S50lbs and 300lbs, such as a weight of approximately 250 Ibs.

[0079] Raw Material 1010 can comprise tissue harvested from one, two, or more similar and / or dissimilar Tissue Sources 1020. Tissue harvested from multiple (e.g., two or more) Tissue Sources 1020 can be pooled to provide a larger quantity of homogeneous Raw Material 1010. Raw Material 1010 can comprise tissue harvested from a uniform sex, such as tissue harvested from all male Tissue Sources 1020 or all female Tissue Sources 1020, wherein tissue harvested from a uniform sex can increase tissue consistency. Raw Material 1010 can comprise tissue harvested from both male and female Tissue Sources 1020. Raw Material 1010 can comprise tissue harvested from an adult and / or juvenile (1.e., less than 3 months old) Tissue source 1020, such as tissue harvested from all adult tissue sources, all juvenile tissue sources, or both adult and juvenile tissue sources. Raw Material 1010 can comprise tissue harvested from a genetically uniform Tissue source 1020 (e.g., tissue from a single genetic strain of animals). Alternatively, or additionally, Raw Material 1010 can comprise tissue harvested from a genetically modified Tissue source 1020. For example, Raw Material 1010 can comprise tissue harvested from an a1 ,3-galactosyltransferase knockout pig.

[0080] Raw Material 1010 can comprise tissue harvested from one or more Tissue Sources 1020 that provide an increased potency and / or altered mechanical, physical, and / or chemical characteristics of Raw Material 1010. Raw Material 1010 can comprise an increased potency and / or altered characteristic of an element selected from the group consisting of: nerve tissue type; adult tissue; juvenile tissue; tissue from genetically-modified animals or tissue transfected with genetic material; tissue from mechanically conditioned animals or mechanically conditioned tissue; tissue from chemically conditioned animals or chemically conditioned tissue; tissue from pharmacologically conditioned animals or pharmacologically conditioned tissue; tissue from physically conditioned animals or physically conditioned tissue; tissue from psychologically conditioned animals; and combinations of these.

[0081] Raw Material 1010 can comprise tissue harvested from one or more Tissue Sources 1020 that is subsequently subjected to a conditioning and / or other tissue regimen. The tissue regimen can be configured to modify the mechanical, physical, and / or chemical characteristics of Raw Material 1010. In some embodiments, Raw Material 1010 is cross- linked to alter its degradation rate, mechanical properties, and / or orient its microstructure. Attorney Docket No. REN-008-PCT

[0082] Raw Material 1010 can comprise tissue harvested from one, two, or more Tissue Sources 1020 comprising an animal that adhered to a pre-determined diet, exercise, chemical, pharmacological, physical, psychological stimulation, and / or other regimens. The pre- determined regimen can be configured to modify the anatomical and / or physiological characteristics of Tissue source 1020. In some embodiments, the animal adhered to a physical stimuli regimen, such as exercise, electrical stimulation, mechanical conditioning (e.g., stretch, compression), physical conditioning (e.g., thermal, light exposure), and radiation. In some embodiments, the applicable animal adhered to a psychological conditioning regimen, such as different levels of daily stress or lack thereof, amount of space per animal, level of socialization, different sleep / light cycles, and level of induced sexual or reproductive activity.

[0083] Nerve Cap 100 can be constructed and arranged to be used for applications involving neuropathic conditions causing pain following nerve injury for which nerve functional recovery is not attainable or desirable (e.g., nerve / limb amputations).

[0084] Nerve Cap 100 can be constructed and arranged for therapeutic and / or clinical applications, including veterinary applications for small and large animals.

[0085] Nerve Cap 100 can comprise a mechanical strength and / or degradation rate / durability that is modified via at least one of chemical cross-linking or other chemical treatments, physical cross-linking or other physical or mechanical treatments, selection of Raw Material 1010, and / or combinations.

[0086] Nerve Cap 100 can comprise a degradation rate in vivo of between 2 weeks and 1 year, such as a degradation rate in vivo of between 8 and 16 weeks.

[0087] In some embodiments, Nerve Cap 100 is constructed and arranged as a scaffold configured to provide structural support for cell attachment, cell migration, cell alignment, cell proliferation, cell differentiation, cell dedifferentiation, cell phenotype, cell selection, cell development, gene expression, protein expression, protein secretion, tissue alignment, tissue development and / or tissue remodeling at a treatment site.

[0088] In some embodiments, Nerve Cap 100 is deployed at one or more implant sites via minimally invasive methods, including those using robotic actuators. For example, Nerve Cap 100 can be implanted minimally invasively following an amputation procedure at one or more upstream locations via small skin incisions and use of existing minimally invasive surgery tools. Attorney Docket No. REN-008-PCT

[0089] In some embodiments, the implant site comprises a nerve stump, or a portion of a nerve stump such as one or more fascicular stump(s) or fractions of fascicular stumps, within the peripheral nervous system, such as a site located outside the brain and spinal cord, including any location spanning from the dorsal and / or ventral root ganglia to motor, sensory, or autonomic endings (e.g., muscle end plate, Pacinian corpuscle, Ruffini endings).

[0090] In some embodiments, Nerve Cap 100 connected to a peripheral nerve stump as described above can be placed in any natural or surgically created anatomic location and tissue type that can host the dimensions of Nerve Cap 100. For example, Nerve Cap 100 connected to a peripheral nerve stump can be hosted in a subcutaneous or intramuscular space / pocket.

[0091] In some embodiments, the implant site comprises the location of a nerve amputation (for example, following limb amputation 2 or more nerves are left amputated and each of such nerve stumps represents a potential implant site). In some embodiments, the implant site comprises a peripheral nerve stump which is acutely damaged by trauma, such as compressed / crushed, stretched / avulsed, partially transected / torn, burned / charred, chemically or electrically injured, inflamed, infected, pharmacologically damaged, edematous, perforated, and the like. In some embodiments, the implant site comprises a nerve stump generated by iatrogenic (medical, surgical, etc.) injury to remove tissue, such as a limb amputation, tumor resection, etc. In some embodiments, the implant site comprises a nerve stump generated by iatrogenic injury to alleviate or eliminate intolerable acute or chronic neuropathic or non-neuropathic local pain, including resection of a painful neuroma. Examples include Morton’s neuroma, facial pain, migraine, burns, and other conditions. In some embodiments, the implant site comprises a nerve stump generated by chronic disease, such as peripheral vascular disease, diabetes, other conditions causing poor microvascular perfusion, chronic drug abuse, chronic compression or inflammation, pharmacological damage, and the like.

[0092] In some embodiments, Nerve Cap 100 is deployed into an implant site comprising oral tissue (e.g., oral mucosa, teeth, tooth pulp, cranial nerve, tongue). In some embodiments, Nerve Cap 100 is deployed into or below the tooth root following a root canal or pulpectomy procedure. In some embodiments, Nerve Cap 100 is implanted onto cranial nerve stumps.

[0093] In some embodiments, Nerve Cap 100 is implanted onto an implant site comprising heart nervous system including both the heart conduction system (e.g., sino-atrial node, atrio-ventricular node, Purkinje fibers, etc.) and the heart nervous system tissue from Attorney Docket No. REN-008-PCT the lower cervical to the upper thoracic ganglia (e.g., cardiac plexus, parasympathetic and sympathetic nerve fibers, vagal cardiac nerve, vagus nerve, etc.). In some embodiments, Nerve Cap 100 can be implanted in the atrial tissue to treat irregular heart rhythms.

[0094] Nerve Cap 100 can be implanted in combination with one, two, or more additional treatments provided to the patient (e.g., one or more treatments deployed at the implant site, and / or another patient location) with a common or complementary goal. In some embodiments, Nerve Cap 100 is implanted in combination with a pharmacological treatment. In some embodiments, Nerve Cap 100 is implanted in combination with a thermal treatment. In some embodiments, Nerve Cap 100 is implanted in combination with a cellular treatment. In some embodiments, Nerve Cap 100 is implanted in combination with a structural element (e.g., sutures, conduit, wrap, glue). In some embodiments, Nerve Cap 100 is implanted for treatment in combination with physical and / or occupational therapy, and / or with electrical, magnetic, and laser stimulation.

[0095] In some embodiments, Nerve Cap 100 further comprises one or more compounds and / or materials, Agent 140 (shown), with the goal of controlling the release of such compound and / or material over time. Agent 140 can be included in (e.g., integrated into) Nerve Cap 100, or it can be provided separately with or without a separate delivery system. Agent 140 can comprise an adhesive. In some embodiments, combination of Nerve Cap 100 with an adhesive is configured to reinforce a suture site or connect Nerve Cap 100 to the nerve stump. Agent 140 can comprise a drug with or without separate controlled release material. For example, Agent 140 can comprise antiproliferative drugs configured to inhibit nerve regeneration, such as rapamycin, sirolimus, or paclitaxel. Agent 140 can comprise a chemical agent configured to modify, or otherwise affect, an immune response at the implant site, such as when Agent 140 comprises an immunomodulator (e.g., immunosuppressant) or an anti-fibrotic agent. Agent 140 can comprise an antimicrobial agent configured to deactivate, or otherwise stop or slow down, the growth of microorganisms at the implant site, such as when Agent 140 comprises one, two, or more agents selected from the group consisting of: disinfectant; antiseptic; antibiotic; and combinations of these. Agent 140 can comprise a visual additive, such as a visual additive configured to provide visibility to Nerve Cap 100 during the implantation surgery. Agent 140 can comprise a visual additive, such as a dye configured to improve identification of one or more specific features of Nerve Cap 100 or configured to aid the implantation by providing geometrical landmarks. In other embodiments, Agent 140 can allow the visualization of Nerve Cap 100 via external imaging Attorney Docket No. REN-008-PCT devices. For example, iodine compounds can be used as radio-opaque agents for fluoroscopy, X-ray, or CT scans, paramagnetic substances such as gadolinium can be used for MRI. Agent 140 can comprise a lubricating substance configured to minimize friction at the implant site. Agent 140 can comprise an electrically insulating agent configured to decrease electrical conductivity at the implant site. Agent 140 can comprise one or more anti-adhesive agents configured to prevent an adhesion at the implant site, such as to prevent fibrotic adhesions. Agent 140 can comprise a thermally insulating agent configured to screen the nerve stump from external thermal excursions. Agent 140 can comprise a thermally conductive material configured to facilitate thermal transfer with the nerve stump. Agent 140 can comprise an anesthetic and / or pain reliever agent configured to induce an insensitivity to pain and / or sensation at the implant site (e.g., lidocaine, epinephrine, tetracaine, and the like), or to reduce neuropathic pain (e.g., botulinum toxin, gabapentin, and the like). Agent 140 can comprise a hemostatic agent configured to promote hemostasis at the implant site. Agent 140 can comprise an antidote configured to counteract a poison and / or toxin at the implant site, such as an antidote configured to counteract a nerve agent. Agent 140 can comprise an anti- inflammatory and / or anti-fibrotic agent configured to reduce inflammation and / or fibrosis at the implant site. Agent 140 can comprise a chemoattractant configured to attract motile cells to the implant site, such as a motile cell selected from the group consisting of: Schwann cells; macrophages; endothelial cells; progenitor cells; and combinations of these. Agent 140 can comprise an agent configured to promote the production of angiogenic factors at the implant site, such as an angiogenic factor selected from the group consisting of: angiogenin; growth factors, such as fibroblast growth factors, transforming growth factors; lipids; and combinations of these. Agent 140 can comprise an agent configured to promote cell migration, tissue development, and / or maturation at the implant site. Agent 140 can comprise a chemical agent producing an exothermic and / or endothermic reaction causing an increase and / or decrease in temperature, with the purpose of obtaining analgesia or other effects. Agent 140 can be a multi-agent comprising multiple combinations of the different aforementioned agents.

[0096] In some embodiments, Nerve Cap 100 is configured to exhibit cell adhesion properties configured to interact and / or attach to neighboring cells at the implant site, such as one, two, or more cell adhesion properties associated with cell-adhesion molecules selected from the group consisting of: integrins; laminin, immunoglobulins; cadherins; selectins; and combinations of these. In some embodiments, Nerve Cap 100 is configured to exhibit cell Attorney Docket No. REN-008-PCT signaling properties configured to communicate and / or coordinate cell actions at the implant site, such as one, two, or more properties associated with cell signals selected from the group consisting of: intracrine signals; autocrine signals; juxtracrine signals; paracrine signals; endocrine signals; and combinations of these. In some embodiments, Nerve Cap 100 is configured to exhibit pharmacologic and / or biologic properties configured to support the local microenvironment at the implant site, such as to promote immunomodulatory action, revascularization, cell chemotaxis, cell development, nerve tissue deposition, tissue remodeling, nutrient transfer, and / or waste removal. In some embodiments, Nerve Cap 100 is configured to increase remodeling and / or vascularization of a nerve stump.

[0097] Nerve Cap 100 can be configured to respond to electrical, mechanical, physical, and / or chemical factors, such as factors internal and / or external to Nerve Cap 100. For example, Nerve Cap 100 can be configured to respond to temperature, sound waves, vibrations, electromagnetic waves, and / or light (e.g., coherent light, such as laser). In some embodiments, Nerve Cap 100 is configured to undergo a physical change upon the application of an ultraviolet light internally and / or externally. In some embodiments, Nerve Cap 100 is configured to release an agent (e.g., agent 140, such as a chemical, pharmaceutical, and / or other agent) upon the application of an external vibration to Nerve Cap 100.

[0098] In some embodiments, Nerve Cap 100 further comprises one or more electronic devices, peripheral nerve-computer interface (PNCI 150 shown), with the goal of acting as an intermediary between the nerve stump, the surrounding environment, and external prosthetic devices to aid in the restoration or enhancement of nerve function by communicating with the patient’s central nervous system. For example, PNCI 150 can comprise a sensing array unit (input) for mechanical, electrical, physical, chemical, and biological signals, including encoding efferent (motor) electrical signals from different locations of the nerve stump, and / or afferent (sensory) electrical signals from external devices. PNCI 150 can comprise outputs, such as mechanical, electrical, physical, chemical, and biological actions, including communicating efferent (motor) electrical signals derived from different locations of the nerve stump to external devices, such as robotic prostheses, or afferent (sensory) electrical signals to different locations of the nerve stump. PNCI 150 can comprise internal electronic components such as RFIDs, antennas, microcomputers, batteries, integrated circuitry, actuators, amplifiers, and the like. Attorney Docket No. REN-008-PCT

[0099] Nerve Cap 100 can be included into a Sealed Inner Sterile Barrier 200. Inner Barrier 200 can comprise a laminated foil pouch which provides sterility, moisture, light, and oxygen barriers. In some embodiments, Inner Sterile Barrier 200 measures 4 inches by 4 3 / 4 inches. Further included into Sealed Inner Sterile Barrier 200 are a Protective Bracket 205, Rehydrating Solution 210, a Sizing Tool 220, and Trimming Tool 230.

[0100] Device 10 can further comprise one or more Protective Brackets 205 shown, which can be configured to mechanically protect Nerve Cap 100, such as a thermoformed small plastic tray that can contain or otherwise surround Nerve Cap 100, preventing Nerve Cap 100 from being bent, compressed, or otherwise damaged or deformed by external forces during storage and shipment.

[0101] Device 10 can further comprise one or more Rehydrating Solutions 210 shown, held in a sealed container and comprising saline, Ringer’s lactate, PBS, or other water solutions, which can be configured to rehydrate Nerve Cap 100 before surgical implantation.

[0102] Device 10 can further comprise one or more Sizing Tools 220 shown, which can be configured to measure one or more parameters relevant for Nerve Cap 100 use. Sizing Tool 220 can be configured to measure one or more parameters of the implant site, such as the diameter of a nerve stump, and / or the available space in the surgical pocket, with the purpose of selecting the correct size of Nerve Cap 100. Sizing Tool 220 can be configured to measure the diameter, length, depth, and / or width of Nerve Cap 100 or one or more features of Nerve Cap 100 (e.g., Matrix 110, Socket 120, and / or Tab 130). For example, the length of Nerve Cap 100 may be trimmed to a specific length for a specific application based on measurements from Sizing Tool 220.

[0103] Device 10 can further comprise one or more Trimming Tools 230 shown, which can be configured to trim or split a Nerve Cap 100 into one or more shorter Nerve Caps 100, each of a desired smaller length. Trimming Tool 230 can comprise a scalpel, surgical scissors, mechanical punch die, hot wire cutter, and combinations of these. Trimming Tool 230 can be used in conjunction with Sizing Tool 220 to trim or split Nerve Cap 100 to specific lengths based on implant site size.

[0104] Sealed Inner Sterile Barrier 200 can be included into a Sealed Outer Sterile Barrier 300. Outer Sterile Barrier 200 can comprise a Tyvek pouch measuring 9 inches by 5 inches.

[0105] Sealed Outer Sterile Barrier 300 can be included into a Protective Packaging 400. Protective Packaging 400 can comprise a cardboard presentation box measuring 6 % Attorney Docket No. REN-008-PCT inches by 5 % inches by 1 inch. Protective Packaging 400 can further comprise Instructions for Use, IFU 410, and Item 420, such as additional product documentation.

[0106] In Fig. 1B, a continuation of the schematic view of a System 1000 comprising additional methods of using System 1000B, is illustrated consistent with the present inventive concepts.

[0107] System 1000 can further comprise one or more imaging devices, Device 30 shown, which can be configured to visualize, measure, and / or monitor an object (e.g., Nerve Cap 100, nerve stump, etc.) prior to, during, or after the implant of Nerve Cap 100. Device 30 can comprise an imaging device selected from the group consisting of: microscope, such as a surgical microscope; loupes; magnifying lens; device that provides virtual and / or augmented reality visualization; device that provides stereo visualization; device that provides infrared / near-infrared visualization; device that provides thermal imaging; Schlieren photography; medical imaging device, such as an X-ray, a fluoroscope, an MRI, a CT scanner, OCT, an ultrasound, an endoscope; device that images using UV light; device that images using polarized light; device that images using fluorescent light; and combinations of these.

[0108] System 1000 can further comprise one or more surgical devices, Device 40 shown, which can be configured to aid in the implantation of Nerve Cap 100 by either enabling the surgical positioning and placement of Nerve Cap 100 or supporting the connection of Nerve Cap 100 to the nerve stump and / or adjacent tissue. Device 40 can comprise one or more of the following: surgical scalpels, forceps, dissecting instruments, surgical shears, sutures, tissue glues, retractors, trocars, tunneling instruments, size measurement instrument, minimally invasive access, manipulation, and visualization devices, robotic surgery devices, and the like.

[0109] System 1000 can further comprise one or more surgical diagnostic devices, Device 50 shown, which can be configured to identify or monitor activity of the injured nerve stump to establish eligibility for the implant of Nerve Cap 100. Device 50 can comprise an electrophysiology system, an intrasurgical nerve stimulator, evoked potential system, superconductive quantum interference device, external computer interface, and the like.

[0110] System 1000 can further comprise one or more controllers to prosthetic robotic devices, Controller 60 shown, which can interface with PNCI 150 to respond to motor signals derived from different locations of the nerve stump to actuate a prosthesis accordingly. Controller 60 can additionally process information from sensors for temperature, Attorney Docket No. REN-008-PCT force / pressure, pain, etc. located on the external prosthetic device and communicate these signals to PNCI 150 to be relayed to the central nervous system.

[0111] System 1000 can further comprise one or more external Therapeutic Agents 70 shown, which can facilitate or improve the use and / or function of Nerve Cap 100. Therapeutic Agent 70 may comprise a pharmacologic agent, electrical waveforms, thermal waveforms, ultrasonic waveforms, light waveforms (e.g., laser stimulation) and the like. Agent 70 can be a multi-agent comprising multiple combinations of the components previously described for Agent 70 and Agent 140.

[0112] In Fig. 1C, a continuation of the schematic view of a System 1000 comprising methods of manufacturing and storage System 1000C, can further comprise one or more Segmentation Tools 1025 shown, which can be configured to cut Raw Material 1010 to a pre-defined length.

[0113] System 1000 can further comprise one or more Buffer Solutions 1030 shown, which can be configured to resist changes in pH when an acid and / or alkali is added to it (e.g., maintain a constant pH). In some embodiments, Buffer Solution 1030 comprises phosphate buffered solution or phosphate buffered saline (PBS, 1X).

[0114] System 1000 can further comprise one or more environmental chambers, Chamber 1040 shown. Chamber 1040 can comprise a temperature-controlled environmental chamber configured to chill and / or freeze an object (e.g., Raw Material 1010) through non- cyclic and / or cyclic refrigeration. In some embodiments, Chamber 1040 consists of ice or frozen synthetic ice packs within an insulated container. In some embodiments, Chamber 1040 consists of a refrigerator.

[0115] System 1000 can further comprise one or more Cooling Agents 1050 shown, which can be configured to reduce, and / or otherwise regulate, the temperature of a product (e.g., Raw Material 1010). Cooling Agent 1050 can comprise an agent selected from the group consisting of: dry ice; dry ice with ethanol; dry ice with acetone; liquid nitrogen; wet ice; frozen ice packs; and combinations of these.

[0116] System 1000 can further comprise one or more Vessels 1060 shown, which can be configured to store an object (e.g., Raw Material 1010). Vessel 1060 can comprise a vented container configured to comprise one or more openings to allow for the passage of air, gas, and / or liquid through Vessel 1060. In some embodiments, Vessel 1060 is configured to store a tissue sample during tissue processing, embedding, and / or sectioning. In some embodiments, Vessel 1060 is configured to store one or more tissue samples during Attorney Docket No. REN-008-PCT decellularization, disinfection, and / or other tissue processing (e.g., lyophilization, freezing, etc.). Vessel 1060 can be configured to store and handle one, two, or more tissue samples with minimal manipulation, such as individual nerve segments. Vessel 1060 can comprise a rigid or flexible material and can form a cassette, pouch, or cylinder in which one, two, or more tissue samples are stored. Vessel 1060 can comprise: silicone; polyolefins, such as polypropylene (PP); other biocompatible and chemically resistant materials; and combinations of these.

[0117] System 1000 can further comprise one or more Detergent Solutions, 1070 shown, which can be configured to lyse and / or permeabilize tissue, cells, and proteins, such as Raw Material 1010. In some embodiments, Detergent Solution 1070 comprises Triton X-100. In some embodiments, Detergent Solution 1070 comprises a 4% (w / v) sodium deoxycholate in water solution.

[0118] System 1000 can further comprise one or more Disinfecting Solutions, 1080 shown, which can be configured to reduce bioburden or otherwise destroy one, two, or more microorganisms (e.g., bacteria, virus, fungi). In some embodiments, Disinfecting Solution 1080 comprises a co-solution comprising 0.1% peracetic acid and 4% ethanol in water.

[0119] System 1000 can further comprise one or more Dissociation Solutions 1210 shown, which can be configured to dissociate adherent cells, cell aggregates, and / or tissues into single-cell suspensions. In some embodiments, Dissociation Solution 1210 comprises a co-solution comprising 0.02% trypsin and 0.05% ethylenediaminetetraacetic acid (EDTA). Dissociation Solution 1210 can comprise a solution that is warmed to a temperature of approximately 35°C.

[0120] System 1000 can further comprise one or more Excipient Solutions 1220 shown. In some embodiment, Excipient Solution 1220 comprises a sucrose solution, such as 1M sucrose solution.

[0121] System 1000 can further comprise one or more Digestion Solutions 1230 shown, which can be configured to break down tissue. In some embodiments, Digestion Solution 1230 comprises a 0.01 N hydrochloric acid (HCI) solution.

[0122] System 1000 can further comprise one or more Enzymes 1240 shown, which can be configured to break down polymeric macromolecules. In some embodiments, the digestive enzyme comprises pepsin comprising an activity level of between 0.5U / mg and 5000U / mg, such as an activity level of approximately 2500U / mg. Enzyme 1240 is then placed into Digestion Solution 1230 such that the final concentration results in an activity level of Attorney Docket No. REN-008-PCT between 10 U / mL and 2500 U / mL, such as activity levels of 250 U / mL. In some embodiments, Enzyme 1240 comprises chondroitinase.

[0123] System 1000 can further comprise one or more stabilizing and / or radioprotecting solutions, Excipient 1250 shown, which can be configured to provide at least one of long-term stabilization, radioprotection, heat protection, cryoprotection, optimization of thermal properties for lyophilization and / or other enhancement of a product. Excipient 1250 can comprise an excipient selected from the group consisting of: sucrose; ascorbic acid, glycerol, glycine, sodium ascorbate; vitamin E; EDTA; mannitol; glycine; dextran; and combinations of these. Excipient 1250 is not intended to have a therapeutic function, but only aid in the storage, stability, formulation, protection, and / or other property during processing, manufacturing, sterilization, and / or shipping.

[0124] System 1000 can further comprise one or more raw material holding, handling, transporting, and / or processing containers, Cassette 1090 shown, and as described herein in reference to Fig. 19. Cassette 1090 can comprise one or more of the following materials: stainless steel; chemically resistant plastics such as polyolefins (e.g., PP, polyethylene (PE), etc.), polysulfone (PSU), polytetrafluoroethylene (PTFE), fluorinated ethylene propylene (FEP), etc., and combinations thereof. Cassette 1090 can be configured to hold and compartmentalize one or more segments of Raw Material 1010 by internal compartments, dividers, etc. In some embodiments, each Cassette 1090 comprises an inner grid of multiple separated slots to receive segments of Raw Material 1010, such as between ten and twenty slots. In some embodiments, Cassette 1090 can comprise external dimensions of 2” x 3” x 3 / 4”. Cassette 1090 can comprise different external, internal, and slot dimensions and slot numbers to adapt to the size and quantity of the raw material segments being processed. Cassette 1090 can be configured to comprise one or more openings to allow for the exchange (diffusive and / or convective) of air, gas, and / or liquid through Cassette 1090 and each of its slots. Cassette 1090 can be configured in a reversible open status to allow the insertion of segments and configured in a reversible closed status to prevent the escape of such segments during transporting, processing, and the like. In some embodiments, Cassette 1090 can be configured to hold and compartmentalize between ten and twenty segments of Raw Material 1010 during a chemical treatment process. In some embodiments, Cassette 1090 can be coupled with other components (including as described below) during a chemical treatment process. Attorney Docket No. REN-008-PCT

[0125] System 1000 can further comprise one or more Mixing Devices 1100 shown, which can be configured to agitate a fluid disposed within a component of Mixing Device 1100 while a portion of tissue is contained within Mixing Device 1100. In some embodiments, Mixing Device 1100 contains a rotating Bioreactor Shaft Assembly 1110 that can firmly couple one or more Cassettes 1090 containing tissue segments, such as six Cassettes 1090, while the shaft is rotating into a fluid field of Mixing Device 1100, as described herein in reference to Figs. 20-21. Mixing Device 1100 can comprise a stirring mechanism and / or a bioreactor vessel to hold a fluid and allow chemical, physical, and / or mechanical processes to occur within Mixing Device 1100. Mixing Device 1100 can be configured to stir a fluid at a speed between approximately 50 rpm and 2,000 rpm, such as between 80 rpm and 150 rpm. Mixing Device 1100 can be configured to maximize and / or optimize mass transport within the tissue to facilitate desired chemical, physical, and / or mechanical processes. For example, Mixing Device 1100 can produce a turbulent flow (Re > 4,000) or transitional flow (2,000 < Re < 4,000) configured to improve mass transport over laminar flow (Re < 2,000). A temperature within Mixing Device 1100 can be controlled to be between 0 °C and 100 °C. Cyclic mechanical deformations can be applied within Mixing Device 1100 to improve mass transport. In some embodiments, the motion created within Mixing Device 1100 can be cylindrical (rotational around the axis, and / or translational along the axis), time varying or non-time varying, and periodic or non-periodic. For example, the motion can be cylindrical around the axis and accelerate until it reaches an upper rotational speed in one direction, at which point it decelerates until an inversion of rotational direction is obtained, followed by an acceleration until an upper rotational speed in the opposite direction is obtained, and so on, similarly to a washing machine cycle.

[0126] Mixing Device 1100 can include a reaction vessel comprising an internal liquid volume between 1 and 10 liters. This reaction vessel can comprise a glass beaker, a spinner flask, a bioreactor bag, or other suitable container. In some embodiments, Mixing Device 1100 includes a stir plate and stirs a fluid by a rotating magnetic stir bar. In some embodiments, Mixing Device 1100 includes a fixed overhead stirrer attached to an impeller configured to rotate, thereby agitating a fluid disposed within Mixing Device 1100. In some embodiments, Mixing Device 1100 consists of an ultrasonic mixing device, such as a device configured to provide mechanical shock waves. In some embodiments, Mixing Device 1100 consists of a rocking platform with variable rocking speed and rocking angle designed to agitate a fluid in a container secured to the rocking platform surface. In some embodiments, =97- Attorney Docket No. REN-008-PCT Mixing Device 1100 consists of an orbital shaker platform designed to agitate a fluid contained within Mixing Device 1100 at a specified speed and / or radius. In some embodiments, Mixing Device 1100 includes a peristaltic pump that defines a set fluid flow rate through Mixing Device 1100. In some embodiments, Mixing Device 1100 includes a perfusion bioreactor in which a pump, heat exchanger, and / or other components including temperature, pressure, flow, and / or chemical sensors can be used to control a specific steady or periodic flow pattern around or within the tissue samples.

[0127] System 1000 can further comprise one or more Heating Devices 1120 shown. Heating Device 1120 can be configured to warm and / or maintain the temperature of an object (e.g., Raw Material 1010). Heating Device 1120 can comprise a hotplate comprising electric heating elements. In some embodiments, Heating Device 1120 comprises a stirring hotplate comprising a rotating magnetic field configured to rotate a corresponding magnetic bar that is positioned in fluid proximate a surface of Heating Device 1120. In some embodiments, Heating Device 1120 can comprise an incubator with or without an incorporated agitation system (e.g., a system configured to shake, rock, mix, stir, and / or otherwise agitate one or more fluids and / or other materials). Heating Device 1120 can comprise an electrical resistance with a controller directly or indirectly in contact with the fluid within Mixing Device 1100.

[0128] System 1000 can further comprise one or more Purified Waters 1130 shown, which can comprise water that has been filtered, or otherwise processed, to remove one or more impurities. In some embodiments, Purified Water 1130 comprises demineralized water, Type I water, or water for injection (WFI).

[0129] System 1000 can further comprise one or more nerve size discrimination tools, Tool 1135 shown. Tool 1135 can comprise hemi-cylindrical grooves of increasing diameter configured to temporarily hold nerve segments and rapidly discriminate their sizes to identify a suitable size of Support Assembly 1140. Each hemi-cylindrical groove comprises a known diameter associated with a specific size Support Assembly 1140. In some embodiments, Tool 1135 can be used to perform image-based quantification of a nerve segment held within the grooves. In such embodiments, Tool 1135 further comprises an adjustable mount for a digital imaging device and a known width to be used as a scale for images taken.

[0130] System 1000 can further comprise one or more Support Assemblies 1140 shown, which can be configured to receive and / or secure an object (e.g., segment of Raw Attorney Docket No. REN-008-PCT Material 1010 after Decellularization Step 2300). Support Assembly 1140 can be constructed and arranged as described herein in reference to Fig. 24.

[0131] System 1000 can further comprise a Jacketing Assembly 1150 shown, to controllably insert an object (e.g., approximately cylindrical segment of Raw Material 1010 after Decellularization Step 2300) into a Support Assembly 1140. Jacketing Assembly 1150 can comprise a system to generate a negative relative pressure (i.e., vacuum), such as a vacuum pump, vacuum source with regulator, syringe, or other means of creating a controlled vacuum. Jacketing Assembly 1150 may comprise one or more in-line air filters to prevent particulate contamination during the process. Jacketing Assembly 1150 may comprise one or more attachments, connectors, and / or accessories configured to facilitate the insertion of an object of different size in Support Assemblies 1140 of different sizes. In some embodiments, Jacketing Assembly 1150 is configured to compress the diameter of a cylindrical object within Support Assembly 1140 to a certain compression ratio, defined as the original equivalent diameter of the object being inserted (e.g., nerve segment) divided by the inner equivalent diameter of the Support Assembly 1140. The object within Support Assembly 1140 may be compressed to a ratio of between 1.0 and 3.0, such as between 1.3 and 1.8, such as approximately 1.5. In some embodiments, Jacketing Assembly 1150 applies a controlled steady and / or time-variable vacuum pressure at the proximal end of a Support Assembly 1140 for each object insertion. In some embodiments, Jacketing Assembly 1150 applies a controlled vacuum pressure of between -1 and -30 inHg, such as between -5 and -15 inHg. In some embodiments, the controlled vacuum pressure applied is different for different size objects, Support Assemblies 1140, and compression ratios. Jacketing Assembly 1150 can be constructed and arranged as described herein in reference to Fig. 25.

[0132] System 1000 can further comprise one or more Holding Racks 1160 shown, which can be configured to receive, hold, and handle one or more products (e.g., segments of Raw Material 1010 after insertion into Support Assembly 1140) throughout further processing, such as freezing, lyophilization, feature creation, and the like. Rack 1160 can comprise a material selected from the group consisting of: corrosion-resistant metals (e.g., stainless steel; titanium; etc.); glass; chemically resistant plastics (e.g., PP, PTFE); and autoclave and UV radiation resistant plastics (e.g., PSU); ionizing radiation resistant plastics (e.g., polyether ether ketone (PEEK), polyimide, amorphous sulfur-containing polymers); cryo-resistant plastics (e.g., PTFE, polychlorotrifluoroethylene (PCTFE), FEP) and combinations of these. Additionally, Rack 1160 can be sterilized and depyrogenated, such as Attorney Docket No. REN-008-PCT to prevent contamination of the product from bioburden during manufacturing. In some embodiments, Rack 1160 can be placed onto a holding tray for transport, storage, and to provide a contact sterility barrier during manufacturing. In some other embodiments, Rack 1160 can be mechanically coupled with an Alignment Assembly 1190 to hold Rack 1160 in a well-defined spatial position for further processing. In other embodiments, Rack 1160 can be inserted into a storage element providing a sterile barrier, such as a sealable pouch, that can be used to store Rack 1160 and intermediate material within either before or after lyophilization for an extended period of time. In some embodiments, storage is maintained at ultra-low temperatures (e.g., -80 °C or lower). Holding Rack 1160 may further comprise a thermally conductive or insulative material element to control the heat flow during freezing (i.e., rate, direction) and respectively promote or prevent directional crystal formation and / or controlled crystal volume, leading to equivalently directional and size-distributed pores following lyophilization, critical point drying, or other dehydration techniques. In some embodiments, Rack 1160 compresses one or more regions of the product contained therein in order to create a density and / or porosity gradient along the length of the product or other features.

[0133] In some embodiments, Rack 1160 is configured to secure multiple products, such as between | and 1,000 products, such as 100 products. In some embodiments, Rack 1160 can be configured to secure a specific single size product, with separate Racks 1160 designated for each product size. In other embodiments, Rack 1160 can be configured to hold multiple specific size products in separate sections. In some embodiments, Rack 1160 can be configured to secure the product in fully or partially enclosed compartments, or by compressing a portion of the product. In some embodiments, Rack 1160 can be configured to facilitate subsequent feature creation. For example, Rack 1160 may be configured to hold devices vertically to facilitate the creation of a proximal socket 120 feature after lyophilization. In some embodiments, Rack 1160 comprises a element that provides longitudinal compression from one or more directions to aid in subsequent feature creation in one or more locations of Nerve Cap 100. In some embodiments, Rack 1160 can be configured to create a tab feature by compressing the distal portion of the product during lyophilization. In some embodiments, Rack 1160 comprises a two-piece system wherein the upper plate holds the product, while the lower plate can be positioned as “open state” to allow product loading or “closed state” to compress the distal tab feature and secure the product within. Rack 1160 may be engraved on the upper and lower plates to indicate the “open” Attorney Docket No. REN-008-PCT and / or “closed” positions. Holding Rack 1160 may comprise features to aid in alignment and calibration procedures, such as those described in reference to Alignment Assembly 1190. Holding Rack 1160 can also include a network of ports and channels to communicate / equilibrate pressure among all internal portions of the Holding Rack 1160. Holding Rack 1160 can be constructed and arranged as described herein in reference to Fig. 27.

[0134] System 1000 can further comprise one or more Lyophilization Devices, 1170 shown, such as a device configured to preserve a product (e.g., Matrix 110) via a freeze- drying process. In some embodiments, Lyophilization Device 1170 is configured to dehydrate the product to obtain a residual moisture content of between 0.1% and 10%, such as a residual moisture content between 0.1% and 2%, such as a residual moisture content of less than 2% (e.g., the moisture content as measured via the Karl-Fischer moisture content test). In some embodiments, Lyophilization Device 1170 is configured to create porosity between 35% and 90% porosity, such as a porosity between 40% and 70% in Nerve Cap 100. In some embodiments, Lyophilization Device 1170 is configured to create directionally aligned pores, such as longitudinally aligned pores, radially aligned pores, circumferentially aligned pores, and / or combinations of these. In some embodiments, Lyophilization Device 1170 is configured to create porosity and / or pore size gradients along the longitudinal, radial, and / or circumferential directions of Nerve Cap 100. For example, the porosity may decrease along the length of the Nerve Cap 100. The process executed by Lyophilization Device 1170 can comprise four primary phases: freezing, annealing, primary drying (sublimation), and secondary drying (adsorption). First, the freezing phase can be configured to cool the product within Lyophilization Device 1170 to a temperature below its triple point to ensure later sublimation, thereby preserving the product’s physical form. Freezing parameters, such as rate and temperature, can be configured to influence product pore size, pore size distribution, porosity, pore isotropy / anisotropy, pore directionality, etc. In some embodiments, a freezing rate of between 1°C and 5°C per minute is ideal to create lamellar ice formation, which after the later phases of lyophilization forms interconnected longitudinal pores. In some embodiments, a faster freezing rate, such as snap freezing using liquid nitrogen, is ideal to form small discrete pores. A specific freezing rate may be obtained by added thermal insulation, physical distance from the cold source, monitoring with an array of thermocouples, and / or one or more heating elements. Secondly, the annealing phase stabilizes the molecular crystalline structure to minimize disruptions during drying. During the primary Attorney Docket No. REN-008-PCT drying phase, a lower pressure with or without an increase in temperature within Lyophilization Device 1170 can be configured to promote water sublimation until most of the water has been sublimated from the product. Finally, the secondary drying phase can be configured to maintain or further increase the temperature of the product to promote removal of ionically bound water molecules (e.g., break the bonds between the product and the water molecules).

[0135] System 1000 can further comprise one or more material removal devices, Material Device 1180 shown, which can be configured to shape or otherwise alter the geometry and / or surface properties of a product. Material Device 1180 can be configured to perform a function selected from the group consisting of: drill; grind; rout; plane; sand; bore; cut; ablate; and combinations of these. Material Device 1180 can comprise a drill press, micro drill press, dental pin drilling unit, longitudinal file, cylindrical punch die, laser cutter, ultrasonic cutter, electrosurgery unit, hot wire cutter, CNC milling machine, powder blasting, and combinations of these.

[0136] System 1000 can further comprise one or more Alignment Assemblies 1190 shown. In some embodiments, Alignment Assembly 1190 comprises a keyless drill chuck and / or a collet chuck configured to concentrically secure a cylindrical object therein (e.g., Nerve Cap 100). In some embodiments, Alignment Assembly 1190 comprises a four-axis micrometer-driven linear stage configured to allow controlled and precise movement in X, Y, Z, R directions of an object therein. In some embodiments, the linear stage is incorporated into Material Device 1180, such as a CNC laser cutting system. In some embodiments, Alignment Assembly 1190 comprises a jig comprising the same dimensions of a device platform (e.g., Material Device 1180, etc.) and is configured to secure an object therein (e.g., Nerve Cap 100; Holding Rack 1160; Nerve Cap 100 secured within keyless drill chuck, collet chuck, etc.) in a precise location for alteration of the object by the device. In some embodiments, Alignment Assembly 1190 includes a visible laser alignment beam at the central axis to directly visualize the concentricity of an object therein in relation to Alignment Assembly 1190, device (Material Device 1180, etc.), and / or other platform.

[0137] In some embodiments, Alignment Assembly 1190 comprises Holding Rack 1160 configured to hold multiple products (e.g., Nerve Cap 100) in a precise orientation and location, an Alignment Base 1193 permanently coupled with Material Device 1180, a removable Base Cradle 1194 acting as an intermediary component (aseptic contact barrier) between Holding Rack 1160 and Alignment Base 1193, and disposable Calibration Chips Attorney Docket No. REN-008-PCT 1191 hosted and mechanically coupled within a designated Calibration Socket 1192 on the top surface of Holding Rack 1160. In this embodiment, Alignment Assembly 1190 allows the accurate and precise alignment and offset between the reference frame of Material Device 1180 and that of Holding Rack 1160 (and Nerve Caps 100 within). In some embodiments, Alignment Assembly 1190 includes a calibration system to verify / calibrate / adjust the alignment and offset between the reference frame of Material Device 1180 and that of Holding Rack 1160 (and Nerve Caps 100 within). The calibration system in this embodiment leverages the disposable Calibration Chips 1191 that are hosted and mechanically coupled within Holding Rack 1160 with the purpose of allowing Material Device 1180 to create features in the intended accurate and precise position on Nerve Cap 100. In other embodiments, the calibration system may include a calibration mask plate simulating the position of the target product, with the purpose of testing and verifying that Material Device 1180 creates features in the intended accurate and precise position on Nerve Cap 100. In other embodiments, the calibration system may include an opto-electronic measuring system mechanically coupled within Holding Rack 1160 that provides an output in response to the level of alignment of the secondary visible laser beam of Material Device 1180 to the Holding Rack 1160 coordinate system. In this configuration, alignment can be tested and adjusted based on the output of the opto-electronic measuring system, without the need for ablation tests on disposable calibration chips.

[0138] In some embodiments, Alignment Assembly 1190 includes an algorithm to calibrate the alignment of the Holding Rack 1160 in relation to Material Device 1180 by aligning, offsetting, rotating, scaling, (or combinations thereof) the reference frame of Material Device 1180 to that of Holding Rack 1160 based on calibration results. The algorithm may include homogeneous or heterogenous position scaling for each axis, translation in positioning for each axis, and / or rotation around each axis of the rack location within the Material Device 1180 reference frame. In some embodiments, Alignment Assembly 1190 can include a Z-alignment block 1195 configured to calibrate the distance between the head of Material Device 1180 and the top surface of Holding Rack 1160. Components of Alignment Assembly 1190 can be constructed and arranged as described herein in reference to Fig. 30.

[0139] System 1000 can further comprise one or more Feature Trimming Devices, Device 1200 shown and as described herein in reference to Figs. 29 and 31, which can be configured to trim or otherwise alter the geometry of a product. Device 1200 can comprise a Attorney Docket No. REN-008-PCT scalpel, biopsy punch, razor blade, surgical shears, skin grafting knife (dermatome), hot wire cutter, peeler, precision drill / drill press, press and die system, filer and / or sander, slicer, saw, laser cutter, ultrasonic cutter, electrosurgery unit, hot wire cutter, CNC milling machine, powder blasting, and combinations of these.

[0140] System 1000 can further comprise one or more Imaging Holders 1260 shown and as described herein in reference to Fig. 28, which can be configured to allow handling and identification / discrimination of one or more Nerve Caps 100, and maintenance of stable sample orientation and alignment within a sample holder of a MicroCT (Microcomputed Tomography) apparatus. Imaging Holder 1260 can comprise a main body of a radiotransparent material with a cross-section consistent with the internal cross-section of the MicroCT sample holder to enable the slide-fit insertion of Imaging Holder 1260 within a MicroCT sample holder. Imaging Holder 1260 can further comprise one or more bores, each of a diameter and depth consistent with those of a specific size Nerve Cap 100 to be scanned. Imaging Holder 1260 can further comprise additional features such as an elongated handle and orientation identification markers to aid in the process of MicroCT scanning.

[0141] Referring now to Fig. 2, a graphical representation of Nerve Cap 100 is illustrated, consistent with the present inventive concepts. In some embodiments, the Nerve Cap 100 comprises a cylindrical device with a proximal Socket 120, central Matrix 110, and distal Anchoring Tab 130. The proximal end of Nerve Cap 100 can be secured to a severed nerve stump through Socket Wall 125, and the distal end of Nerve Cap 100 can be also secured to the surrounding / adjacent tissue through Anchoring tab 130. Nerve Cap 100 can isolate / protect the severed nerve stump and a portion of the upstream nerve providing nociceptive innervation to surrounding tissues from the inflammatory signaling milieu and other signaling causing nociceptive sensitization. Nerve Cap 100 can further prevent the outgrowth of axons from the severed nerve stump into the surrounding environment. Nerve Cap 100 can provide a neuro-inhibitory Matrix 110 to the severed nerve stump which permits the longitudinally-aligned growth of the axons, but progressively inhibits their growth to a stable configuration without formation of neuroma.

[0142] Matrix 110 of Nerve Cap 100 can comprise one or more internal interconnected or non-interconnected longitudinal pores, these can be intertwining tunnels constructed and arranged to mimic the natural plexus structure of a peripheral nerve. Nerve Cap 100 can comprise internal longitudinally-aligned pores that incrementally narrow over the length of Nerve Cap 100 to allow limited axonal extension and sprouting into the device and Attorney Docket No. REN-008-PCT > subsequent slowing and stopping of growth. Nerve Cap 100 can comprise internal longitudinally-aligned pores, each of which braces and / or compartmentalizes individual axons entering into the device, and prevents these axons from significant extension / sprouting and from entangling with themselves and / or other axons. This entanglement of axons can become a precursor to neuroma formation, which can be characterized by a bulb of entangled axons. These pores can be configured to offer a progressively resistive path to these axons along the Nerve Cap 100 length until the axons’ growth potential is exhausted. As Nerve Cap 100 degrades and is progressively replaced by a healthy remodeled connective tissue, these compartmentalized and aligned axons embedded in connective tissue can remain ‘frozen’ in a stable configuration, which is immune to the structural formation of neuroma. As a result, Nerve Cap 100 can prevent the onset of neuroma pain and residual limb pain, which are both associated with the formation of neuroma at the nerve stump. Previous studies utilizing acellular autografts have shown that, when axon-permeable materials are used, a significant graft length (e.g., 5cm) is needed to terminate axonal growth. However, applicant has demonstrated axonal exhaustion begins at the proximal end of the Nerve Cap 100 with axonal growth termination in < 10mm length. This growth restriction can ultimately result in retraction of axons unable to reach their target tissue, providing long-term prevention of neuroma.

[0143] Socket 120 can comprise an empty cylindrical bore cut into the proximal end of Nerve Cap 100. Socket 120 can comprise multiple 3-dimensional hollow geometries, including multiple extruded hollow shapes, including circle, ellipse, oval, regular or irregular polygonal, etc. The shape extrusion can be incrementally or continuously tapered to obtain straight or rounded internal surfaces.

[0144] Socket 120 can be configured to accept a free nerve ending (severed nerve) with a sufficient longitudinal overlap and secure it via standard surgical sutures or glues within Socket 120 and Socket Wall 125. In some embodiments, Socket 120 can be configured to directly appose the internal surface(s) of Socket 120 to the free nerve ending cross-section. In other embodiments, Socket 120 can be configured such that a hollow volume / spacing exists between the internal surface(s) of Socket 120 and the free nerve ending cross-section. Such volume / spacing can be configured to induce a high degree of alignment for the axonal sprouting as they enter Nerve Cap 100 and prevent axonal misalignment and / or entanglement at the nerve-device interface. Attorney Docket No. REN-008-PCT

[0145] Tab 130 can comprise a mechanically compressed region of Matrix 110 at the distal end of Nerve Cap 100. Tab 130 can be configured to increase surgical usability to Nerve Cap 100 by providing a handling mechanism to Nerve Cap 100 and / or the nerve stump tethered to Nerve Cap 100 for surgeons during implantation. Tab 130 can further increase usability by acting as a visual aid for distinguishing the proximal end of Nerve Cap 100 from the distal end. In some embodiments, Tab 130 can be configured to anchor Nerve Cap 100 to the surrounding tissue, such as muscle or connective tissues.

[0146] Nerve Cap 100 can comprise a Socket 120 equivalent diameter (ED) (i.e., the diameter of the circle with the same area as the Socket 120 cross-section) that allows it to host the outer diameter of at least one of the anatomical elements (e.g., a nerve stump). Nerve Cap 100 can comprise a Socket 120 equivalent diameter (ED) that is smaller than the outer diameter of at least one of the anatomical elements (e.g., a nerve stump). Nerve Cap 100 can comprise an outer diameter (OD) that allows it to host the outer diameter of at least one of the anatomical elements (e.g., a nerve stump). In some embodiments, Nerve Cap 100 comprises an OD that is consistent along its length. In some embodiments, Nerve Cap 100 comprises an OD that varies along its length (e.g., tapers). Nerve Cap 100 can comprise a fixed length (L) independent of its ED and / or OD. In other embodiments, length (L) is a function of its ED and / or OD (e.g., larger ED and / or OD devices have longer L). In some embodiments, Nerve Cap 100 comprises a longer device that can be split into two or more smaller devices of desired length L. The length of Nerve Cap 100 can be modified prior to, during, and / or after the procedure in which Nerve Cap 100 is implanted in the patient.

[0147] In some embodiments, at least a portion of Nerve Cap 100 comprises a material that is impermeable or selectively permeable to cells, axons, and / or nutrients.

[0148] Prior to being degraded and replaced by remodeled tissue, Nerve Cap 100 can be constructed and arranged to withstand tension, flexion, and / or torsion forces exhibited between the two or more anatomical elements (¢.g., nerve stump proximal tethering, distal tab tethering), such as to withstand motion attributed to characteristic body and / or surrounding tissue movement. In the embodiments in which Socket Wall 125 is tethered to the proximal nerve stump via suture, Socket Wall 125 can possess sufficient suture retention strength to withstand motion attributed to characteristic body and / or surrounding tissue movement. In the embodiments in which Anchoring Tab 130 is tethered to the adjacent tissue via suture, Anchoring Tab 130 can possess sufficient suture retention strength to withstand Attorney Docket No. REN-008-PCT motion attributed to characteristic body and / or surrounding tissue movement and mechanical forces resulting therefrom.

[0149] Nerve Cap 100 can be configured to degrade at a rate compatible with the rate of nerve regenerative potential exhaustion. In some embodiments, Nerve Cap 100 can be configured to reach full degradation in a period between 4 weeks and 24 weeks, such as a period between 8 weeks and 16 weeks. In some embodiments, Nerve Cap 100 can be configured such that different locations in the device have different degradation rates (e.g., the proximal end of Nerve Cap 100 degrades faster than the distal end).

[0150] Referring now to Fig. 3, a time-lapsed representation of device degradation / remodeling and axon infiltration into Nerve Cap 100, including axonal extension, growth interruption, and ultimate die-back, is illustrated, consistent with the present inventive concepts. Fig. 3 illustrates how Nerve Cap 100 can reach a complete degradation / remodeling and stable interruption of axonal growth within 16 weeks from implantation. In this period, Nerve Cap 100 is progressively degraded / remodeled by the local host inflammatory and tissue repair process. Between 0 and 4 weeks post-implant, a diffuse infiltration of macrophages and other healthy inflammatory cells is accompanied by angiogenesis, leading to a mild swelling of the device. In the same period, a limited quantity of myelinated axons extends and is formed within the first few millimeters of device depth. These axons extend within the longitudinal porosity of Matrix 110, which directs them and prevents them from becoming entangled. Between 4 and 8 weeks post-implant, Matrix 110 is progressively degraded / remodeled by the inflammatory process into healthy connective tissue. In this period, the axons further extend into the depth of the device, but with limited myelination, and surrounded by connective tissue. Between 8 and 16 weeks post-implant, complete degradation of Matrix 110 is obtained; axon growth is fully interrupted within the surrounding connective tissue and the onset of axonal demyelination and die-back takes place. Following 16 weeks post-implant, Nerve Cap 100 is no longer visible; the proximal nerve stump tapers into a stable short tail of remodeled connective tissue with negligible internal axonal content, which will no longer extend.

[0151] Referring now to Figs. 4A-F, schematic views of Nerve Cap 100 implanted at a nerve injury site are illustrated, consistent with the present inventive concepts. Nerve Cap 100 can be implanted at the site of a severed nerve end in multiple possible configurations.

[0152] In some embodiments, and as shown in Fig. 4A, the implant site comprises a single severed nerve end fitted with one Nerve Cap 100 of size based on the size of the Attorney Docket No. REN-008-PCT severed nerve end. Alternatively, and as shown in Fig. 4B, the size of Nerve Cap 100 and Socket 120 can be larger than the size of the severed nerve end.

[0153] In some embodiments, and as shown in Fig. 4C, the implant site comprises two or more severed nerve ends, such as in the case of extremity amputation. Each nerve end can be fitted with a Nerve Cap 100 of size based on the size of each severed nerve end.

[0154] In some embodiments, and as shown in Fig. 4D, the implant site comprises two or more adjacent severed nerve ends (e.g., those derived from a bifurcation point). Multiple adjacent nerve ends can be fitted with a single Nerve Cap 100 of size sufficiently large to fit them. Alternatively, and as shown in Fig. 4E, a Nerve Cap 100 with two or more Sockets 120 can be used to fit the multiple adjacent nerve ends.

[0155] In some embodiments, and as shown in Fig. 4F, the implant site comprises a single large, severed nerve end, which can be fitted with multiple smaller Nerve Caps 100, similar to a cable graft configuration.

[0156] Referring now to Fig. 5, a schematic view of a sizing tool is illustrated, consistent with the present inventive concepts. Sizing tool 220 can be constructed and arranged to measure and / or alter one or more dimensions (e.g., length, diameter, etc.) of Nerve Cap 100. Sizing tool 220 can be constructed and arranged to measure one or more of the implant site dimensions (e.g., surgical pocket, nerve stump diameter, etc.).

[0157] Sizing tool 220 can comprise elements with which the outer diameter of Nerve Cap 100 and / or the diameter of the nerve stump at the implant site can be measured. In some embodiments, Sizing tool 220 comprises two, three, or more circular openings, bores 221a-c as shown. Each bore 221 can comprise a unique outer diameter OD. For example, bore 221a can comprise an outer diameter smaller than bore 221b, which comprises an outer diameter smaller than bore 221c. A sterile operator (e.g., scrub nurse, surgeon, etc.) can insert Nerve Cap 100 through each bore 221 to measure or otherwise confirm the device’s outer diameter before or after rehydration. A sterile operator can insert the proximal nerve stump(s) through each bore 221 to measure or otherwise confirm the nerve diameter to be matched with the correct size of Nerve Cap 100.

[0158] Sizing tool 220 can further comprise elements with which the length of Nerve Cap 100 is measured. In some embodiments, sizing tool 220 comprises two, three, or more slots, slots 222a-c as shown. Each slot 222 can comprise a unique width. For example, slot 222a comprises a width smaller than slot 222b, which comprises a width smaller than slot 222c. A Attorney Docket No. REN-008-PCT sterile operator can compare the width of each slot 222 to the length of Nerve Cap 100 and compare it to the available intended surgical pocket site to confirm the availability of sufficient length to implant Nerve Cap 100. In some embodiments, the operator can trim or otherwise alter the length of Nerve Cap 100 to achieve the desired length prior to implantation.

[0159] Sizing tool 220 can further comprise one, two, or more elements with which the diameter of the Socket 120 is measured. In some embodiments, sizing tool 220 comprises two, three, or more cylindrical protrusions, protrusions 223a-c as shown. Each protrusion 223 can comprise a unique outer diameter OD. For example, protrusion 223a can comprise a diameter that is smaller than the diameter of protrusion 223b, which comprises a diameter that is smaller than the diameter of protrusion 223c. A sterile operator can insert each protrusion 223 into at least a portion of Socket 120 to measure or otherwise confirm the diameter of Socket 120 before or after rehydration. In some embodiments, at least a portion of protrusion 223 is rounded / tapered to allow the operator to expand the diameter of Socket 120 by pressing the device against the rounded / tapered portion of protrusion 223 before or after rehydration to slightly enlarge the existing diameter to better fit the intended nerve stump size at the implant site.

[0160] Sizing tool 220 can further comprise a length measurement element, ruler 224, comprising two or more markings at regular intervals configured to measure a distance between two reference points. In some embodiments, ruler 224 is utilized to measure a length of Nerve Cap 100, or a feature such as Anchoring Tab 130.

[0161] Sizing tool 220 can further comprise one or more flexible marked probes, probe 225, which can be configured to measure the depth of the surgical pocket and represent different outer diameters of Nerve Cap 100. A surgeon can insert probe 225 into the surgical pocket to measure or otherwise confirm the adequacy of the surgical pocket diameter and depth to fit a specific size of Nerve Cap 100 for implantation. Probe 225 can further be used to aid in the tissue dissection during the creation of a surgical pocket or tunnel of adequate diameter and depth to fit a specific size of Nerve Cap 100.

[0162] Referring now to Fig. 6, a method for producing a nerve cap graft device from tissue is illustrated, consistent with the present inventive concepts. The nerve cap graft device can comprise Nerve Cap 100 as described herein. Method 2000 comprises a sequence of sub- methods, Methods 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, and 2900 as described herein in reference to Figs. 7-15, respectively. Method 2100 comprises a method for selecting Attorney Docket No. REN-008-PCT and harvesting tissue for further processing. Method 2200 comprises a method for cleaning and segmenting harvested tissue for further processing. Method 2300 comprises a method for treating the tissue obtained from Method 2200 to remove cells and other immunogenic components and other treatments. Method 2400 comprises a method for inserting processed tissue segments produced in Method 2300 into correctly sized individual Support Assemblies 1140. Method 2500 comprises a method for mounting the processed tissue segments produced in Method 2400 into a Holding Rack 1160 for further processing including lyophilization. Method 2600 comprises a method for creating one, two, or more desired features (e.g., Socket 120, Anchoring Tab 130, etc.) in the processed tissue segments produced in Method 2500. Method 2700 comprises a method for kitting and packaging the nerve cap graft device produced in Method 2600. Method 2800 comprises a method for bulk sterilizing the multiple packaged nerve cap graft devices produced in Method 2700. Method 2900 comprises a method for storing the multiple packaged nerve cap graft devices produced in Method 2800. A method of producing a nerve cap graft device may include the addition and / or omission of one, two or more sub-methods as described herein. In some embodiments, the order of one, two, or more sub-methods can be altered depending on a desired feature of Nerve Cap 100.

[0163] Referring now to Fig. 7, a method for selecting and harvesting tissue for further processing is illustrated, consistent with the present inventive concepts. Method 2100 can be configured to harvest Raw Material 1010 from a selected Tissue Source 1020 as described herein.

[0164] In STEP 2101, Raw Material 1010 is selected from a tissue source (e.g., Tissue Source 1020) comprising one, two, or more desired structural and / or functional features, whose compositions / sources and specific desired criteria for selection were disclosed extensively earlier in application. In some embodiments, the desired structural and / or functional features are configured to provide a desired structure and / or function to Nerve Cap 100.

[0165] In STEP 2102, Raw Material 1010 is harvested from a tissue source (e.g., Tissue Source 1020). In some embodiments, Raw Material 1010 is harvested aseptically.

[0166] In STEP 2103, Raw Material 1010 is frozen or preserved for storage and / or transportation. For short-term storage (e.g., for a duration less than 24 hours), harvested Raw Material 1010 can be at least partially immersed in Buffer Solution 1030. In some embodiments, Raw Material 1010 can be stored in Chamber 1040 at a temperature between Attorney Docket No. REN-008-PCT approximately 2°C and 8°C. For long-term storage and / or transportation (e.g., for a duration of more than 24 hours), Raw Material 1010 can be rapidly frozen in Buffer Solution 1030. In some embodiments, Raw Material 1010 is rapidly frozen via Cooling Agent 1050. Raw Material 1010 can be stored and / or transported in Chamber 1040 at a temperature of approximately -80°C (or lower temperatures such as those afforded by dry ice or liquid nitrogen storage). In some embodiments, Raw Material 1010 can be stored in Chamber 1040 at a temperature of approximately -80°C for a period of up to 12 months. In some embodiments, Raw Material 1010 is stored at a temperature of approximately -20°C for a limited period, such as 3-6 months in a sealed environment devoid of air via vacuum (e.g., to protect from oxygen) with a preservation solution. In some embodiments, Raw Material 1010 is stored at a temperature of between 2°C and 8°C for up to 48 hours in a sealed environment devoid of air via vacuum (e.g., to protect from oxygen) with a preservation solution comprising germostatic or germicidal agents consistent with that used for human organ procurement and transport for transplantation. For preservation, Raw Material 1010 can be placed into a preservation solution for the duration of storage and / or transportation. In some embodiments, the preservation solution comprises a gaseous phase consisting of nitrogen gas.

[0167] Referring now to Fig. 8, a method for aseptically cleaning and segmenting tissue for further manipulation is illustrated, consistent with the present inventive concepts. Method 2200 can be configured to prepare Raw Material 1010 harvested in Method 2100 described herein in reference to Fig. 7.

[0168] In STEP 2201, frozen and / or preserved Raw Material 1010 is prepared for processing. In some embodiments, frozen Raw Material 1010 is thawed in Chamber 1040 at a temperature of between 2°C and 8°C. In some embodiments, frozen Raw Material 1010 is thawed in Chamber 1040 for at least 48 hours, such as at least 72 hours. In some embodiments, preserved Raw Material 1010 is removed from the preservation solution.

[0169] In STEP 2202, Raw Material 1010 is transferred into an aseptic environment (e.g., clean room, biosafety cabinet, etc.) and further aseptically processed (e.g., cleaned) to remove excess connective and / or accessory tissue (e.g., remove non-nerve tissue), and as described hereinbelow in reference to Figs. 17A-B. From this Step through the remainder of processing, Raw Material 1010 is only in contact with cleaned and sterilized surfaces and tools. Raw Material 1010 can be processed at a temperature of between 2°C and 25°C, In some embodiments, Raw Material 1010 comprises a portion of nerve tissue comprising one, two, or more side branches. The side branches can be removed and processed as described -4]- Attorney Docket No. REN-008-PCT herein. In some embodiments, Raw Material 1010 is processed manually (e.g., by hand, via surgical instruments such as scalpels and / or forceps). In some embodiments, Raw Material 1010 is processed via a mechanical treatment using an industrial machine designed similarly to those used for peeling fruit, cleaning vegetables, or preparing meat fillet out of mammals or fish. In some embodiments, Raw Material 1010 is processed via a chemical treatment (e.g., acid spray plus wash) to partially remove external layers of tissue from the nerve. In some embodiments, Raw Material 1010 is processed via physical treatment involving heat, cold, radiation, laser, and / or other physical forces to remove external layers of tissue and / or excess tissue. In some embodiments, Raw Material 1010 is processed via a combination of manual, mechanical, chemical, and / or physical treatments.

[0170] In STEP 2203, cleaned Raw Material 1010 is cut, or otherwise divided, into smaller segments, and as described hereinbelow in reference to Figs. 17C and 18A-C. Cleaned Raw Material 1010 can be cut into segments comprising a length of between 0.5cm and 20cm, such as between Icm and 5cm, such as approximately 2cm. Cleaned Raw Material 1010 can comprise segments comprising a diameter of between 0.5mm and 10mm, such as between 1mm and 10mm. In some embodiments, cleaned Raw Material 1010 is selected from a specific nerve branch and at a specific distance from the proximal branching point in order to obtain defined features, such as nerve area, fascicular density, axonal density, etc. In some embodiments, individual nerve branches are isolated before segmentation. In some embodiments, branching points are excised from the cleaned raw material before segmentation. In some embodiments, nerve segments of larger diameter are cut into longer segments than nerve segments of smaller diameter. In some embodiments, a Sizing Tool 220 is used to measure the length and diameter of each segment, and as described hereinbelow in reference to Fig. 5. In some embodiments, a Segmentation Tool 1025 is used to cut Raw Material 1010 into segments of a precise pre-defined length.

[0171] In STEP 2204, Raw Material 1010 is transferred to one, two, or more Vessels 1060. Cleaned Raw Material 1010 can be transferred at a temperature of between 2°C and 37°C. In some embodiments, each Vessel 1060 comprises Buffer Solution 1030 and can be configured to store segmented tissue temporarily until the completion of Method 2200.

[0172] Referring now to Fig. 9, a method for chemically treating tissue segments is illustrated, consistent with present inventive concepts. Method 2300 can be configured to chemically treat / decellularize (e.g., remove cells, DNA, myelin, and / or other immunogenic Attorney Docket No. REN-008-PCT components) tissue segments prepared in Method 2200 described herein in reference to Fig. 8.

[0173] In STEP 2301, Detergent Solution 1070 and Disinfecting Solution 1080 are prepared and filtered to remove impurities and contaminants. Detergent Solution 1070 and Disinfecting Solution 1080 can be prepared at a temperature of between 2°C and 25°C.

[0174] In STEP 2302, cleaned raw material from STEP 2204 is removed from Vessel 1060 and transferred to one or more Cassettes 1090, as described herein in reference to Fig. 19. One or more portions of Raw Material 1010 may be anchored to Cassette 1090, such as to secure Raw Material 1010 to and / or within Cassette 1090. In some embodiments, Cassette 1090 can be divided into subcompartments, such as to store multiple portions of Raw Material 1010 individually.

[0175] In STEP 2303, Cassettes 1090 are secured within Mixing Device 1100, as described hereinbelow in reference to Figs. 20-21. In some embodiments, Mixing Device 1100 comprises a spinner flask which contains a Bioreactor Shaft Assembly 1110 attached to the central shaft. One or more Cassettes 1090, such as six Cassettes 1090, can be secured into the Bioreactor Shaft Assembly 1110. The bioreactor shaft assembly may hold the Cassettes 1090 throughout decellularization and allow mixing / agitation of the fluid field surrounding Cassettes 1090. Mixing Device 1100 can comprise an internal volume between 500mL and 1,000L, such as 5L, depending on the amount of tissue to be treated.

[0176] In STEP 2304, cleaned Raw Material 1010 is treated (e.g., washed or otherwise treated) with Detergent Solution 1070. In some embodiments, Detergent Solution 1070 is brought in Chamber 1040 to a temperature of between 2°C and 37°C, such as approximately 20°C. Cleaned Raw Material 1010 and Detergent Solution 1070 can comprise a mass-to- volume (g / mL) ratio between 1:20 and 1:500, such as between 1:100 and 1:200. Detergent Solution 1070 is added to Mixing Device 1100. In some embodiments, at least 3-6 L of Detergent Solution 1070 are added to Mixing Device 1100 to treat 20-60g of Raw Material 1010. Mixing Device 1100 is stored in Chamber 1040 at a temperature of between 2°C and 37°C, such as between 18°C and 25°C, such as approximately 20°C. Mixing Device 1100 is combined with Heating Device 1120 configured to mix / agitate Detergent Solution 1070 at a speed between 10 rpm and 1,000 rpm, such as 90 + 10 rpm, for between 6 hours and 10 hours, such as 8 hours + 20 minutes, thereby treating cleaned Raw Material 1010 within Cassette 1090. Attorney Docket No. REN-008-PCT

[0177] In some embodiments, a real-time in-process control is used to indirectly assess the degree of tissue chemical treatment obtained by Detergent Solution 1070 in order to inform the required process duration. The in-process control can assess the solution over time for the direct or indirect presence of analytes or byproducts released in the solution from nerve tissue or generated during the process. For example, samples may be measured over time for turbidity (light absorbance via spectrophotometer), surface tension, conductivity, pH, temperature, chemical species, or other quantifiable metrics. These direct or indirect metrics tracked over time can be used to identify or predict the correct process duration based on the attainment of a certain value, slope (relative maximum), inflection point or other qualitative or quantitative aspect of the aforementioned metrics. In a non-limiting embodiment, the Detergent Solution 1070 process can be interrupted when turbidity or conductivity reaches the value that was previously established to be correlated with the desired degree of tissue chemical treatment. Detergent Solution 1070 is decanted aseptically from Mixing Device 1100. In some embodiments, Detergent Solution is decanted via a dedicated purging port. {178] In STEP 2305, cleaned Raw Material 1010 is washed with Purified Water 1130. In some embodiments, Purified Water 1130 is brought in Chamber 1040 to a temperature of between 2°C and 37°C, such as approximately 20°C. Cleaned Raw Material 1010 is washed with Purified Water 1130 at least six times for at least 1 minute, such as for 5 minutes. Cleaned Raw Material 1010 and Purified Water 1130 can comprise a mass-to-volume (g / mL) ratio between 1:20 and 1:500, such as between 1:100 and 1:200. Purified Water 1130 is added to Mixing Device 1100. In some embodiments, at least 3-6L of Purified Water 1130 are added to Mixing Device 1100 to treat 20-60g of Raw Material 1010. Mixing Device 1100 is stored in Chamber 1040 at a temperature of between 2°C and 37°C, such as between 18°C and 25°C, such as approximately 20°C. Mixing Device 1100 is combined with Heating Device 1120 configured to mix / agitate Purified Water 1130 at a speed between 10 rpm and 1,000 rpm, such as 90 + 10 rpm, for at least 5 minutes, thereby washing cleaned Raw Material 1010 within Cassette 1090. Purified Water 1130 is decanted aseptically from Mixing Device 1100 via a dedicated purging port and replaced with fresh Purified Water 1130. In some embodiments, this process is repeated at least five additional times, thereby washing cleaned Raw Material 1010 within Vessel 1060 at least six times. In some embodiments, a real-time in-process control is used to indirectly assess the degree of tissue wash obtained by Purified Water 1130 in order to inform the required process duration. The in-process control can assess the solution over time for the direct or indirect presence of analytes or byproducts Attorney Docket No. REN-008-PCT released in the solution from nerve tissue or generated during the process. For example,samples may be measured over time for turbidity(light absorbance via spectrophotometer),surface tension, conductivity, pH, temperature, chemical species, or other quantifiable metrics. These direct or indirect metrics tracked over time can be used to identify or predict the correct process duration or number of wash repetitions based on the attainment of a certain value, slope (relative maximum), inflection point or other qualitative or quantitative aspect of the aforementioned metrics. In a non-limiting embodiment, the wash duration and / or number of washes can be interrupted when turbidity, conductivity, or surface tension reach the values that were previously established to be correlated with the desired degree of tissue washing.

[0179] In STEP 2306, cleaned Raw Material 1010 is washed with Purified Water 1130 overnight. In some embodiments, Purified Water 1130 is brought in Chamber 1040 to a temperature of between 2°C and 37°C, such as approximately 20°C. Cleaned Raw Material 1010 and Purified Water 1130 can comprise a mass-to-volume (g / mL) ratio between 1:20 and 1:500, such as between 1:100 and 1:200. Purified Water 1130 is added to Mixing Device 1100. In some embodiments, at least 3-6 L of Purified Water 1130 are added to Mixing Device 1100 to treat 20-60g of Raw Material 1010. Mixing Device 1100 is stored in Chamber 1040 at a temperature of between 2°C and 37°C, such as between 18°C and 25°C, such as approximately 20°C. Mixing Device 1100 is combined with Heating Device 1120 configured to mix / agitate Purified Water 1130 at a speed between 10 rpm and 1,000 rpm, such as 90 + 10 tpm, for between 18 hours and 36 hours, such as between 18 hours and 20 hours, thereby washing cleaned Raw Material 1010 within Cassette 1090. Purified Water 1130 is decanted aseptically from Mixing Device 1100 via a dedicated purging port. This process can be repeated between one and three times, such as two times.

[0180] In STEP 2307, cleaned Raw Material 1010 is treated with Disinfecting Solution 1080. In some embodiments, Disinfecting Solution 1080 is brought in Chamber 1040 to a temperature of between 2°C and 37°C, such as approximately 20°C. Cleaned Raw Material 1010 and Disinfecting Solution 1080 can comprise a mass-to-volume (g / mL) ratio between 1:20 and 1:500, such as between 1:100 and 1:200. Disinfecting Solution 1080 is added to Mixing Device 1100. In some embodiments, at least 3-6 L of Disinfecting Solution 1080 are added to Mixing Device 1100 to treat 20-60g of Raw Material 1010. Mixing Device 1100 is stored in Chamber 1040 at a temperature of between 2°C and 37°C, such as between 18°C and 25°C, such as approximately 20°C. Mixing Device 1100 is combined with Heating Attorney Docket No. REN-008-PCT Device 1120 configured to mix / agitate Disinfecting Solution 1080 at a speed between 10 rpm and 1,000 rpm, such as 90 + 10 rpm, for between 30 minutes and 240 minutes, such as 120 + 5 minutes, thereby washing cleaned Raw Material 1010 within Vessel 1060. Disinfecting Solution 1080 is decanted aseptically from Mixing Device 1100 via a dedicated purging port.

[0181] In STEP 2308, cleaned Raw Material 1010 is washed with Buffer Solution 1030. In some embodiments, Buffer Solution 1030 is brought in Chamber 1040 to a temperature of between 2°C and 37°C, such as approximately 20°C. Raw Material 1010 and Buffer Solution 1030 can comprise a mass-to-volume (g / mL) ratio between 1:20 and 1:500, such as between 1:100 and 1:200. Buffer Solution 1030 is added to Mixing Device 1100. In some embodiments, at least 3-6 L of Buffer Solution 1030 are added to Mixing Device 1100 to treat 20-60g of Raw Material 1010. Mixing Device 1100 is stored in Chamber 1040 at a temperature of between 2°C and 37°C, such as between 18°C and 25°C, such as approximately 20°C. Mixing Device 1100 is combined with Heating Device 1120 configured to mix / agitate Buffer Solution 1030 at speed between 10 rpm and 1,000 rpm, such as 90 + 10 rpm, for between 5 minutes and 60 minutes, such as at least 15 minutes, thereby washing cleaned Raw Material 1010 within Vessel 1060. Buffer Solution 1030 is decanted aseptically from Mixing Device 1100 via a dedicated purging port.

[0182] In STEP 2309, cleaned Raw Material 1010 is washed with Purified Water 1130. In some embodiments, Purified Water 1130 can be used in place of Purified Water 1130. In some embodiments, Purified Water 1130 is brought in Chamber 1040 to a temperature of between 2°C and 37°C, such as approximately 20°C. Cleaned Raw Material 1010 and Purified water 1130 can comprise a mass-to-volume (g / mL) ratio between 1:20 and 1:500, such as between 1:100 and 1:200. Purified Water 1130 is added to Mixing Device 1100. In some embodiments, at least 3-6L of Purified Water 1130 are added to Mixing Device 1100 to treat 20-60g of Raw Material 1010. Mixing Device 1100 is stored in Chamber 1040 at a temperature of between 2°C and 37°C, such as between 18°C and 25°C, such as approximately 20°C. Mixing Device 1100 is combined with Heating Device 1120 configured to mix / agitate Purified Water 1130 at a speed between 10 rpm and 1,000 rpm, such as 90 + 10 rpm, for between 5 minutes and 60 minutes, such as at least 15 minutes, thereby washing cleaned Raw Material 1010 within Vessel 1060. Purified Water 1130 is decanted aseptically from Mixing Device 1100 via a dedicated purging port.

[0183] In STEP 2310, cleaned Raw Material 1010 is washed with Buffer Solution 1030. In some embodiments, Buffer Solution 1030 is brought in Chamber 1040 to a temperature of Attorney Docket No. REN-008-PCT between 2°C and 37°C, such as approximately 20°C. Raw Material 1010 and Buffer Solution 1030 can comprise a mass-to-volume (g / mL) ratio between 1:20 and 1:500, such as between 1:100 and 1:200. Buffer Solution 1030 is added to Mixing Device 1100. In some embodiments, at least 3-6L of Buffer Solution 1030 are added to Mixing Device 1100 to treat 20-60g of Raw Material 1010. Mixing Device 1100 is stored in Chamber 1040 at a temperature of between 2°C and 37°C, such as between 18°C and 25°C, such as approximately 20°C. Mixing Device 1100 is combined with Heating Device 1120 configured to mix / agitate Buffer Solution 1030 at speed between 10 rpm and 1,000 rpm, such as 90 + 10 rpm, for between 5 minutes and 60 minutes, such as at least 15 minutes, thereby washing cleaned Raw Material 1010 within Vessel 1060. Buffer Solution 1030 is decanted aseptically from Mixing Device 1100 via a dedicated purging port.

[0184] In STEP 2311, cleaned Raw Material 1010 is washed with Purified Water 1130. In some embodiments, Purified Water 1130 can be used in place of Purified Water 1130. In some embodiments, Purified water 1130 is brought in Chamber 1040 to a temperature of between 2°C and 37°C, such as approximately 20°C. Cleaned Raw Material 1010 and Purified Water 1130 can comprise a mass-to-volume (g / mL) ratio between 1:20 and 1:500, such as between 1:100 and 1:200. Purified Water 1130 is added to Mixing Device 1100. In some embodiments, at least 3-6L of Purified Water 1130 are added to Mixing Device 1100 to treat 20-60g of Raw Material 1010. Mixing Device 1100 is stored in Chamber 1040 at a temperature of between 2°C and 37°C, such as between 18°C and 25°C, such as approximately 20°C. Mixing Device 1100 is combined with Heating Device 1120 configured to mix / agitate sterile water solution at a speed between 10 rpm and 1,000 rpm, such as 90 + 10 rpm, for between 5 minutes and 60 minutes, such as at least 15 minutes, thereby washing cleaned Raw Material 1010 within Vessel 1060. Purified Water 1130 is decanted aseptically from Mixing Device 1100 via a dedicated purging port.

[0185] In STEP 2312, cleaned Raw Material 1010 is washed with Purified Water 1130. In some embodiments, Purified Water 1130 is brought in Chamber 1040 to a temperature of between 2°C and 37°C, such as approximately 20°C. Cleaned Raw Material 1010 is washed with Purified Water 1130 at least six times. Cleaned Raw Material 1010 and Purified Water 1130 can comprise a mass-to-volume (g / mL) ratio between 1:20 and 1:500, such as between 1:100 and 1:200. Purified Water 1130 is added to Mixing Device 1100. In some embodiments, at least 3-6L of Purified Water 1130 are added to Mixing Device 1100 to treat 22g of Raw Material 1010. Mixing Device 1100 is stored in Chamber 1040 at a temperature Attorney Docket No. REN-008-PCT of between 2°C and 37°C, such as between 18°C and 25°C, such as approximately 20°C. Mixing Device 1100 is combined with Heating Device 1120 configured to mix / agitate Purified water 1130 at a speed between 10 rpm and 1,000 rpm, such as 90 + 10 rpm, for at least 5 minutes, thereby washing cleaned Raw Material 1010 within Cassette 1090. Purified Water 1130 is decanted aseptically from Mixing Device 1100 via a dedicated purging port and replaced with Purified water 1130. This process is repeated at least five additional times, thereby washing cleaned Raw Material 1010 within Vessel 1060 at least six times.

[0186] In STEP 2313, comprising an optional step, a verification analysis can be performed on the nerve segments following the completion of chemical treatment. This verification analysis can sample one or more nerve segments from each Mixing Device 1100 to test inter- device (between multiple Mixing Devices 1100) variability and / or different locations within the same Mixing Device 1100 to test intra-device (within a single Mixing Device 1100) variability and tissue size / type variability. In some embodiments, the verification analysis comprises an assay to determine the sufficient removal of any residual chemical species introduced during the chemical treatment. In some embodiments, this includes chromatography to identify chemical species within devices, washing solutions, or extracts thereof. In some embodiments, this analysis includes a cytotoxicity assay, such as Neutral Red Uptake (NRU) Assay, etc., as the chemical species can be cytotoxic and elicit an adverse response upon implantation / use. In some embodiments, the verification analysis examines the degree, homogeneity, and reproducibility of chemical treatment, such as decellularization and selective removal from nerve tissue of any combination of myelin, glycosaminoglycans (GAGs), laminin, and / or other biologic components that can affect the functionality of the final product. In such embodiments, standard histologic fixation and preparation can be performed to assess and verify the desired degree of tissue modification at different longitudinal lengths and radial depths, as illustrated in Fig. 22.

[0187] In some embodiments, each of the aforementioned chemical treatments and rinses can be modeled as a function describing the gradient of concentration for each reagent and rinsing agent between the outer tissue surface and its surrounding fluid over time. The integral of such function over time can be used to characterize the mass transport requirements to obtain the desired result for that specific reagent. (e.g., decellularize the tissue to the desired level and sufficiently remove the reagents used to obtain such decellularization). Attorney Docket No. REN-008-PCT

[0188] Upon the conclusion of Method 2300, cleaned Raw Material 1010 comprises a chemically treated segmented nerve tissue (referred to as “nerve segment” herein).

[0189] Referring now to Fig. 10, a method for inserting processed nerve segments into correctly sized individual Support Assemblies 1140 is illustrated, consistent with the present inventive concepts. Method 2400 can be configured to insert a tissue segment decellularized in Method 2300 into the correct sized Support Assembly 1140. Inserting a nerve segment into a Support Assembly 1140 according to Method 2400, referred herein as jacketing, can be an iterative process. The process starts by attempting to jacket the nerve segment in a defined initial size jacket. If the nerve segment is considered correctly jacketed, it remains in such initial jacket for subsequent processing. If the nerve segment appears too big for the jacket, then iteratively larger jackets are tried until a size is found to correctly jacket the nerve segment. Similarly, if instead the nerve segment appears too small for the jacket, then iteratively smaller jackets are tried until a size is found to correctly jacket the nerve segment.

[0190] In STEP 2401, a defined initial size Support Assembly 1140, herein referred to as a jacket and as described hereinbelow in reference to Fig. 24, is inserted into Jacketing Assembly 1150. The proximal end of the jacket can be concentrically press-fitted to the jacket adapter, or otherwise coupled in a manner to communicate internal pressure without loss between the jacket adapter of Jacketing Assembly 1150 and the jacket.

[0191] In STEP 2402, a nerve segment is removed from Cassette 1090.

[0192] In STEP 2403, comprising an optional step, a Tool 1135, as described hereinbelow in reference to Fig. 23, is used to quickly identify the most appropriate initial nerve jacket size to use to optimize the speed and accuracy of the jacketing procedure. In this embodiment, each cylindrical nerve segment is placed longitudinally into incrementally larger hemi-cylindrical grooves of known cross-sectional area (and equivalent diameter) in the Tool 1135 until the deformable nerve segment cross-section fully fills one of the grooves, indicating the nerve equivalent diameter associated with a specific jacket size. In embodiments where this step is performed, the iterative jacketing process begins with the initial jacket size identified by Tool 1135. In some embodiments, this step reduces the number of iterations required or eliminates the need for an iterative process.

[0193] In STEP 2404, one end of such nerve segment is placed adjacent to the distal end of the jacket coupled with Jacketing Assembly 1150. Attorney Docket No. REN-008-PCT

[0194] In STEP 2405, Jacketing Assembly 1150 is activated to apply a controlled relative negative pressure (vacuum) to slowly draw the nerve segment into the jacket until the desired overlap is achieved.

[0195] In STEP 2406, the compatibility between size of the nerve segment and jacket size is assessed. A nerve segment is deemed to be incompatibly too large if the nerve segment will not enter the jacket despite the generation of sufficient vacuum by the full occlusion of the adapter opening achieved by the nerve segment. If a nerve segment is deemed to be too large, STEP 2407 is performed. A nerve segment is deemed to be incompatibly too small if a vacuum cannot be formed to pull the segment into the jacket because the nerve segment cannot achieve full occlusion of the adapter opening, or if the segment is pulled into the jacket but does not circumferentially contact all portions of the jacket walls. If a nerve segment is deemed to be too small, STEP 2407 is also performed. If a nerve segment is deemed to be of compatible size, STEP 2408 is performed.

[0196] In STEP 2407, comprising an optional step to be performed if the nerve segment is deemed incompatible (i.e., too large or too small), the jacket is removed from Jacketing Assembly 1150 and replaced by the next larger (or smaller) jacket size. STEPs 2404 and 2405 are repeated using the larger (or smaller) jacket size. In some embodiments, STEP 2407 is repeated in an iterative fashion with incrementally larger (or smaller) jacket sizes until the nerve segment is correctly drawn into the jacket.

[0197] In STEP 2408, the vacuum pressure is released via a dedicated pressure release valve and the jacket containing the nerve segment is removed from Jacketing Assembly 1150. In some embodiments, the jacket comprises a length of approximately 1 cm and uncompressed portions of the nerve segment extend beyond the jacket on either one or both sides (referred to as “flares” herein).

[0198] In STEP 2409, the jacketed nerve segment is placed into a bin containing Purified Water 1130 labeled according to the jacket size. In some embodiments, each jacket size is distinguishable by a distinct color or other visual aid to aid in the identification and segregation of the jacket sizes.

[0199] Upon the conclusion of Method 2400, nerve segments are compressed into jackets and segregated according to jacket sizes. {200] Referring now to Fig. 11, a method for mounting Support Assemblies 1140 comprising nerve segments in a Holding Rack 1160 and lyophilizing the rack and nerve segments therein is illustrated, consistent with the present inventive concepts. Method 2500 Attorney Docket No. REN-008-PCT can be configured to secure and lyophilize a nerve segment contained within a Support Assembly 1140 produced in Method 2400 described herein in reference to Fig. 10.

[0201] In STEP 2501, Holding Rack 1160, as described hereinbelow in reference to Fig. 27, is configured into the “open state” position by adjusting the relative position between the upper and lower plates to allow an open geometrical configuration to receive Support Assemblies 1140.

[0202] In STEP 2502, Support Assemblies 1140 comprising nerve segments are inserted into a Holding Rack 1160 configured in the “open state” position. In some embodiments, Support Assemblies 1140 and Holding Rack 1160 have mutual features enabling the correct positioning / alignment, coupling, engagement, and locking of Support Assemblies 1140 comprising nerve segments within Holding Rack 1160. These features allow the nerve segment flaring to position itself into its intended location within the Holding Rack 1160. In some embodiments, one Holding Rack 1160 can hold up to 100 Support Assemblies 1140. Each Holding Rack 1160 may comprise bores configured to fit one or more specific size of Support Assembly 1140.

[0203] In STEP 2503, Holding Rack 1160 is configured into the “closed state” position by adjusting the relative position between the upper and lower plates to allow a closed geometrical configuration to secure Support Assemblies 1140. In some embodiments, the “closed state” position of the Holding Rack 1160 facilitates the creation of a distal Anchoring Tab 130 feature during lyophilization.

[0204] In STEP 2504, Holding Racks 1160 comprising nerve segments can be transferred to, and stored within, Chamber 1040 comprising heat flow controls and methods to freeze the nerve segment at a defined rate of freezing and with a defined directionality of the freezing front. Such rate and directionality of freezing are intended to affect the size and orientation of the water crystals inside the nerve tissue. Water crystals geometry affect the geometry of induced macroporosity, pore size, pore orientation, tissue permeability, rehydration kinetics and degree of swelling following rehydration, and in vivo degradation rate resulting after lyophilization. In a nonlimiting embodiment, the nerve can be placed into a thermally controlling system placed inside Chamber 1040. Such thermally controlling system can comprise thermally insulating materials, selective openings to the external environment into Chamber 1040, internal electrical resistances connected to a controller to provide heating patterns within the geometry of nerve segment, or channels for the flow of warmed (or cooled) fluid heated by a heat exchanger connected to a controller to also provide Attorney Docket No. REN-008-PCT heating patterns within nerve segment. Such thermally controlling system can be programmed to control the directionality and speed of the freezing front within nerve segments. For example, such system can create a longitudinally oriented freezing front able to move at a selected speed throughout the main axis of the nerve segment to induce the growth of the water crystal along the main axis of the nerve segment. In some embodiments, Holding Racks 1160 comprising nerve segments are processed in Chamber 1040 for at least 60 minutes. In some embodiments, Holding Racks 1160 comprising nerve segments are processed in Chamber 1040 for 12 hours.

[0205] In STEP 2505, one or more Holding Racks 1160 comprising nerve segments are loaded into Lyophilization Device 1170. In some embodiments, Holding Racks 1160 comprising nerve segments are loaded into a preconditioned Lyophilization Device 1170.

[0206] In STEP 2506, Holding Racks 1160 comprising nerve segments are lyophilized via Lyophilization Device 1170. In some embodiments, Holding Racks 1160 comprising nerve segments are lyophilized for 72 hours.

[0207] In STEP 2507, Holding Racks 1160 comprising nerve segments are removed from Lyophilization Device 1170.

[0208] In STEP 2508, comprising an optional step, Holding Racks 1160 comprising nerve segments are transferred to, and stored within, Chamber 1040 comprising a temperature between 2°C and 8°C.

[0209] Upon the conclusion of Method 2500, the nerve segments comprise lyophilized nerve segments (referred to as “lyophilized nerve segment” herein).

[0210] Referring now to Fig. 12, a method for creating desired features within a nerve segment is illustrated, consistent with the present inventive concepts. Method 2600 can be configured to create one, two, or more features (e.g., Socket 120, Anchoring Tab 130, etc.) in lyophilized nerve segments produced in Method 2500 described herein in reference to Fig. 11.

[0211] In STEP 2601, comprising an optional step (in reference to STEP 2508), loaded Holding Racks 1160 (i.e., comprising lyophilized nerve segments) are removed from Chamber 1040.

[0212] In STEP 2602, the “flaring” of lyophilized nerve segments outside the Holding Rack 1160 is trimmed by Device 1200 to produce flat nerve ends that are flush with the upper surface of Holding Rack 1160, as shown in Fig. 29. In some embodiments, only the proximal flare is trimmed to allow the creation of an Anchoring Tab 130 feature at the opposite end of Attorney Docket No. REN-008-PCT the lyophilized nerve segments. STEP 2602 may be performed before or after STEP 2605 depending on the method of material removal / displacement.

[0213] In STEP 2603, the disposable calibration chips of Alignment Assembly 1190 are pressure fitted (or otherwise fitted) into their dedicated slots within Holding Rack 1160 to ensure exact alignment between the two components.

[0214] In STEP 2604, the removable base cradle component of Alignment Assembly 1190 is pressure fitted (or otherwise fitted) into the alignment base component of Alignment Assembly 1190 that is permanently secured to Material Device 1180 to ensure exact alignment between the two components.

[0215] In STEP 2605, the loaded Holding Rack 1160 is pressure fitted (or otherwise fitted) to ensure exact alignment into the removable base cradle component of Alignment Assembly 1190, thereby securing Holding Rack 1160 in a defined orientation and position within Material Device 1180, as described in reference to Fig. 30.

[0216] In STEP 2606, an iterative calibration process is performed using Material Device 1180 to create reference features (e.g., small markings of defined size) within two or more calibration chips of Alignment Assembly 1190. The relative position between the reference features created and existing pre-defined markings on the calibration chips are used as inputs for the Alignment Assembly 1190 algorithm which outputs roto-translational reference adjustment in the Material Device 1180 coordinate system. The process ends after one or more iterations when sufficient coherence (e.g., accuracy and precision) between Material Device 1180 and calibration chip reference frames is achieved.

[0217] In STEP 2607, the automated feature creation program of Material Device 1180 is executed to create features in all lyophilized nerve segments within the Holding Rack 1160. In some embodiments the features comprise a concentric cylindrical bore of defined diameter and depth at the proximal end of the lyophilized nerve segments (i.e., Socket 120).

[0218] In STEP 2608, Holding Rack 1160 is removed from the base cradle component of Alignment Assembly 1190 and Material Device 1180.

[0219] In STEP 2609, the upper and lower plates of Holding Rack 1160 are disengaged, releasing the lyophilized nerve segments from Holding Rack 1160. All lyophilized nerve segments are subsequently removed from Support Assemblies 1140.

[0220] In STEP 2610, the Anchoring Tab 130 feature is trimmed by Device 1200 as described hereinbelow in reference to Fig 31. In some embodiments, Anchoring Tab 130 is cut longitudinally on each side to the approximate width of the lyophilized nerve segment. Attorney Docket No. REN-008-PCT Anchoring Tab 130 is cut horizontally at a defined length away from the base of Anchoring Tab 130. Two chamfers or fillets may be cut at the two top corners of the device. In some other embodiments, the shape of the perimeter of Anchoring Tab 130 can be achieved using Device 1200 comprising a punch die press tool. (221] Upon the conclusion of Method 2600, the lyophilized nerve segment comprises a nerve cap graft device (referred to as “Nerve Cap 100” herein).

[0222] Referring now to Fig. 13, a method for kitting and packaging one or more nerve cap graft devices into a full product unit (Device 10) and subsequent bulk packaging for terminal sterilization and / or storage is illustrated, consistent with the present inventive concepts. Method 2700 can be configured to package Nerve Cap 100 produced in Method 2600 described herein in reference to Fig. 12. Method 2700 is configured to be performed within an environment suitable for aseptic processing or otherwise entailing appropriate environmental / particulate controls (e.g., clean room).

[0223] In STEP 2701, an inspection of the features of Nerve Cap 100 is performed. The inspection may include confirmation of the presence / accuracy of Anchoring Tab 130 geometry of Nerve Cap 100 including but not limited to: vertical longitudinal walls of Anchoring Tab 130; horizontal Anchoring Tab 130 end at defined length away from device main body; confirmation of pre-defined tab geometry. The inspection may include confirmation of Socket 120 geometry of Nerve Cap 100 including but not limited to: Socket 120 presence; full containment of socket 120 within the device circular cross-sectional surface without breaching the device outer wall; sufficient Socket Wall 125 wall thickness; correct Socket 120 depth. The inspection may include confirmation of color appearance of Nerve Cap 100 and absence of particulates.

[0224] In STEP 2702, Nerve Cap 100 is inserted into a pre-labeled Inner Sterile Barrier 200. The Inner Sterile Barrier 200 label can include device name and assembly number, lot number, manufacturing date, expiration date, storage conditions, number of devices, and any other pertinent information. Nerve Cap 100 is confirmed to be positioned away from the open edge of Sterile Barrier 200. In some embodiments, two or more Nerve Caps 100 of the same size can be inserted into a single Inner Sterile Barrier 200.

[0225] In STEP 2703, the Inner Sterile Barrier 200 containing Nerve Cap 100 is sealed along the open edge. In some embodiments, the Inner Sterile Barrier 200 is sealed using a pouch sealer with an attached tray table and the following parameters: sealing temperature of 280°C, sealing time of 1 second, and sealing pressure of 45 psi. Attorney Docket No. REN-008-PCT

[0226] In STEP 2704, the sealed Inner Sterile Barrier 200 containing Nerve Cap 100 is inserted into an Outer Sterile Barrier 300 with the label visible through the clear window in Outer Sterile Barrier 300. In some embodiments, Inner Sterile Barrier 200 or Outer Sterile Barrier 300 may include one or more of the following components: Protective Bracket 205, Rehydrating Solution 210, Sizing Tool 220, and / or Trimming Tool 230. In some embodiments, two or more Inner Sterile Barriers 200 containing Nerve Caps 100 of the same or different sizes can be inserted into a single Outer Sterile Barrier 300.

[0227] In STEP 2705, the Outer Sterile Barrier 300 containing Nerve Cap 100 is sealed along the open edge. In some embodiments, the Outer Sterile Barrier 300 is sealed using a Pouch sealer with an attached tray table and the following parameters: sealing temperature of 150°C, sealing time of 1 second, and sealing pressure of 45 psi.

[0228] In STEP 2706, the sealed Outer Sterile Barrier 300 containing Nerve Cap 100 is inserted into a pre-labeled Protective Packaging 400. The Outer Sterile Barrier 300 is curled but not creased to fit in the Protective Packaging 400, create a protective buffer for the internal Nerve Cap 100, and to prevent movement within Protective Packaging 400. The Protective Packaging 400 label can match the Inner Sterile Barrier 200 label and include the same pertinent information listed above in STEP 2702. In some embodiments, Protective Packaging 400 may include IFU 410 and / or Item 420. At the completion of STEP 2706, the fully manufactured and packaged Nerve Cap 100 comprises Device 10. In some embodiments, a larger Protective Packaging 400 can include multiple Nerve Caps 100 of the same or different sizes included into their respective sterile packaging. For example, an ‘amputation pack’ can be provided to include multiple devices and sizes most commonly used for amputation of a given extremity.

[0229] In STEP 2707, units of Device 10 are packaged into a sterilization shipper box measuring 24 inches by 14 inches by 6 inches. In some embodiments a sterilization shipper box can hold between 10 and 100 Devices 10, such as 46 Devices 10. All Devices 10 are oriented in the same direction. The outside of the sterilization shipper box is labeled to indicate the beam face for e-beam sterilization to obtain a single layer irradiation (i.e., the irradiation flow through the sterilization shipper box has only unit of Device 10 in its path).

[0230] In STEP 2708, two sterilization shipper boxes containing Device 10 are placed in an insulated shipper measuring 30 4 inches by 14 % inches by 16 inches, and comprising an internal Styrofoam insulation liner, an internal Styrofoam insulation lid, and an outer cardboard box. Attorney Docket No. REN-008-PCT

[0231] In STEP 2709, at least 6 preconditioned Gel Packs are added to the insulated shipper. Gel packs may be conditioned to between -15°C and -25°C. Gel packs may be conditioned to between 2°C and 8 °C. In some embodiments, a temperature logger may also be added to the insulated shipper to monitor temperature changes during shipping. The full packaging configuration of PNM-CAP 2200 is described hereinabove in reference to Fig. 32.

[0232] Referring now to Fig. 14, a method for an irradiation-based sterilization of a container comprising a nerve cap graft device is illustrated, consistent with the present inventive concepts. Method 2800 can be configured to sterilize the Device 10 produced in Method 2700 described herein in reference to Fig. 13.

[0233] In STEP 2801, the sterilization shipper boxes comprising Device 10 are sterilized. The containers comprising Device 10 can be sterilized via electron-beam irradiation (“e-beam irradiation” herein), such as that the sterilization shipper boxes are exposed to a stream of electrons. In some embodiments, the containers comprising Device 10 can be sterilized via e- beam irradiation at an internal dose between 16kGy and 40kGy, such as 31kGy + 10%. In some embodiments, the selected dose of irradiation can be used to modulate the degradation rate of Nerve Cap 100 in vivo. In particular, higher doses of irradiation can accelerate the degradation rate of Nerve Cap 100. Different degradation rates of Nerve Cap 100 for different Nerve Cap 100 sizes.

[0234] Referring now to Fig. 15, a method for storing a container comprising a nerve cap graft device is illustrated, consistent with the present inventive concepts. Method 2900 can be configured to store the sterilized Device 10 produced in Method 2800 described herein in reference to Fig. 14.

[0235] In STEP 2901, the sterilization shipper boxes comprising Device 10 are stored. The sterilization shipper boxes comprising Device 10 can be stored at a temperature of between -25°C and +30°C, such as at a temperature of approximately 20°C.

[0236] Referring now to Fig. 16, a method for implanting a device comprising a nerve cap graft device is illustrated, consistent with the present inventive concepts. Method 3000 can be configured to implant Nerve Cap 100 as described herein.

[0237] As described herein in reference to Method 3000, and for non-limiting purposes, the implant site comprises one, two, or more severed nerve stumps.

[0238] In STEP 3010, the Device 10 comprising Nerve Cap 100 is retrieved from a non- sterile field (e.g., storage) by a first, non-sterile operator (e.g., circulating nurse, etc.) and is Attorney Docket No. REN-008-PCT transported to the room within which the clinical procedure is to be performed (e.g., treatment room, operating room).

[0239] In STEP 3020, the non-sterile operator opens Protective Packaging 400 and retrieves Outer Sterile Barrier 300.

[0240] In STEP 3030, the non-sterile operator peels open Outer Sterile Barrier 300 in proximity to the sterile field where a sterile operator (e.g., scrub nurse, surgeon, etc.) assists with the transfer of the internal components (e.g., Inner Sterile Barrier 200) to the sterile field using aseptic techniques.

[0241] In STEP 3040, the sterile operator peels open Inner Sterile Barrier 200 and transfers the internal Nerve Cap 100 to Rehydrating Solution 210.

[0242] In STEP 3045, comprising an optional step, the surgeon alters the size of Nerve Cap 100 to a desired length using Trimming Tool 230 or other surgical tool by trimming or otherwise reducing the length of Nerve Cap 100 prior to rehydrating and implanting Nerve Cap 100 at the implant site. In some embodiments, the surgeon can trim Nerve Cap 100 to remove the proximal Socket 120 and / or the Anchoring Tab 130 feature prior to rehydration and implantation.

[0243] In STEP 3050, the sterile operator rehydrates Nerve Cap 100 for a duration of between 1 minute and 10 minutes, such as at least 5 minutes. In some embodiments, Nerve Cap 100 can be implanted after up to 6 hours of rehydration. The rehydration kinetics of Nerve Cap 100 can be configured to allow a sufficient degree of dimensional swelling within the surgically compatible rehydration time of approximately 5 minutes. In some embodiments, Nerve Cap 100 comprises a rehydration swell ratio (e.g., rehydrated diameter / initial diameter) of between 105% and 115%, such as approximately 110% following 5 minutes of rehydration.

[0244] In STEP 3060, the sterile operator transfers the rehydrated Nerve Cap 100 to the surgeon or other sterile personnel, or directly into the surgical field.

[0245] In STEP 3070, comprising an optional step, the surgeon alters the size of Nerve Cap 100 to a desired length using Trimming Tool 230, or other surgical tool by trimming or otherwise reducing the length of Nerve Cap 100 prior to implanting Nerve Cap 100 at the implant site. In some embodiments, the surgeon can trim Nerve Cap 100 to remove the proximal Socket 120 and / or Anchoring Tab 130 feature prior to implantation. The surgeon can further use Sizing Tool 220 to assist throughout the sizing, trimming, and implant procedure. Attorney Docket No. REN-008-PCT

[0246] In STEP 3080, the implant site comprises one or more amputated nerves, such as those derived from extremity amputation and Nerve Cap 100 is apposed and secured to the proximal stump of an amputated nerve. The surgeon can create two or more stitches between the nerve stump epineurium and Socket Wall 125 of Nerve Cap 100 using appropriate sutures (e.g., 9-0 nylon monofilament suture) to secure Nerve Cap 100 to the amputated nerve stump. Alternatively, the surgeon can use fibrin glue or other surgical glue in combination with or replacing the stitches to secure Nerve Cap 100 to the amputated nerve stump. Alternatively, in some embodiments, sutures or fibrin glue can be used to connect the amputated nerve stump to the proximal wall of Nerve Cap 100 in which a Socket 120 is not present.

[0247] Referring now to Figs. 17A-C, diagrams of raw nerve tissue and cleaned nerve tissue derived from the porcine sciatic nerve tree are illustrated, respectively, consistent with the present inventive concepts, and as described in STEPS 2202 and 2203. Raw nerve tissue comprises excess connective tissue, as shown in Fig. 17A, that is physically removed to result in cleaned nerve tissue, as shown in Fig. 17B. Cleaned nerve tissue, which in some embodiments includes multiple branches and sub-branches, is segmented into individual branches, as shown by Fig. 17C.

[0248] Referring now to Fig. 18A-C, diagrams of cleaned nerve tissue branch undergoing segmentation are illustrated, consistent with the present inventive concepts, and as described in STEP 2203. The sub-branching points are identified and excised from the main nerve branch, as shown in Fig. 18A. The remaining main branch portions are segmented to a length of between 0.5cm and 5cm, such as 2cm, as shown in Fig. 18B. Segments may be discarded if the segment diameter is unsuitable or if the segment length is lower than the specific length sought. The excised sub-branches may also be segmented if the sub-branch has a suitable diameter and length, as shown in Fig. 18C. Referring now to Fig. 19, a schematic view of Cassette 1090 for securing one or more nerve segments is illustrated, consistent with the present inventive concepts, and as described in STEP 2302. In some embodiments, Cassette 1090 comprises a chemically resistant polymer cassette. In some embodiments, Cassette 1090 comprises two separate parts, a housing body 1091 and a housing lid 1092. Cassette 1090 can comprise material that is permeable to diffusive and convective mass transport to allow the passage of a fluid throughout Cassette 1090. In some embodiments, Cassette 1090 comprises walls that have an open structure, such as a mesh, with mesh voids small enough to prevent the escape of nerve segments housed within. Cassette 1090 can comprise two or more internal compartments 1093, such as 14 Attorney Docket No. REN-008-PCT compartments 1093, each configured to receive and compartmentalize an individual nerve segment, such as Raw Material 1010 described herein. Each internal compartment 1093 in cassette 1090 can comprise one or more separate interlocking internal dividers 1094 configured to create a matrix of adjacent internal compartments 1093. In some embodiments, such dividers 1094 can be pressure fit. In some embodiments, dividers 1094 are permanently incorporated into housing body 1091. In some embodiments, dividers 1094 comprise chemically resistant plastics with similar fluid permeability as Cassette 1090.

[0249] Referring now to Fig. 20, an exploded view of an embodiment of Bioreactor Shaft Assembly 1110 for securing one or more Cassettes 1090 during decellularization into Mixing Device 1100 is illustrated, consistent with the present inventive concepts, and as described in STEP 2303. Bioreactor Shaft Assembly 1110 can comprise two or more Outer Cassette Brackets 1111a and one or more Inner Cassette Brackets 1111b which are mechanically coupled via collets and set screws with the Central Shaft Assembly 1112 of Mixing Device 1100 to prevent relative sliding and rotation between the Central Shaft Assembly 1112 and the brackets 1111a and 1111b. The Central Shaft Assembly 1112 can comprise a Cap 1112a with an internal collect hosting and anchoring a Metal Shaft 1112b, a PTFE Shaft Extension 1112c, and a PTFE-coated magnetic cylindrical bar 1112d, and a Shaft Collet 1112e. Brackets 1111a and 1111b can securely hold one or more Cassettes 1090 in a uniform orientation around the Central Shaft Assembly 1112 of Mixing Device 1100. In some embodiments, the orientation of Cassettes 1090 is designed to create a defined flow pattern within Mixing Device 1100, or a defined mass transport within Cassettes 1090, or combinations thereof. In some embodiments, Brackets 1111a and 1111b can hold three Cassettes 1090 between each pair of Brackets 1111a and 1111b oriented around the Central Shaft Assembly 1112 main axis. In some embodiments, one or more additional Brackets 1111b are added to multiply the number of Cassettes 1090 between the two Outer Cassette Brackets 1111a by the number of additional Inner Cassette Brackets 1111b added. For example, in one embodiment, a total of 6 Cassettes 1090 are used into Bioreactor Shaft Assembly 1110 with the use of a single Bracket 1111b, as shown in Fig. 20. In some embodiments, Bioreactor Shaft Assembly 1110 comprises one or more of the following materials: stainless steel; plastics such as PP, PTFE, PSU, high-density polyethylene (HDPE) etc.

[0250] Referring now to Figs. 21A-B, schematic views of an embodiment of Mixing Device 1100 are illustrated, consistent with the present inventive concepts, and as described Attorney Docket No. REN-008-PCT in STEP 2303. A glass spinner flask enclosure with side caps comprising polypropylene can host interchangeable liquids, as described in Method 2300. The glass spinner flask can host the Bioreactor Shaft Assembly 1110 described in Fig. 20 and be magnetically or mechanically coupled while aseptically insulated from a motor / actuator which creates a defined motion in the shaft.

[0251] Referring now to Fig. 22, methods and results of an in-process control analysis are illustrated, consistent with the present inventive concepts. Standard histologic fixation and preparation can be performed to assess and verify the desired degree of tissue modification obtained following the tissue processing described hereinabove in reference to Fig. 9. In some embodiments, an in-process verification is performed to examine the degree, homogeneity, and reproducibility of chemical treatment, such as decellularization and selective removal from nerve tissue of any combination of myelin, glycosaminoglycans (GAGs), laminin, and / or other biologic components that can affect the functionality of the final product. This in-process control analysis can sample one or more nerve segments from each Mixing Device 1100 to test inter-device (between multiple Mixing Devices 1100) variability and / or different locations within the same Mixing Device 1100 to test intra-device (within a single Mixing Device 1100) variability at the completion of Method 2300. In some embodiments, this method can be used to verify that different tissue sizes and / or type populations are chemically processed to the desired level. Cross-sections from sampled nerve segments can be assessed at different longitudinal lengths and different radial depths. In particular, these cross-sections can be stained or immuno-labeled for myelin and / or cellular content and / or other elements, whose appearance is correlated to the degree of chemical tissue modification obtained. These stained or immunolabeled cross-sections can be analyzed via a semi-quantitative scoring specific for each stain / immunolabel used that assigns a discrete score (e.g., between 0 and 3) based on the appearance of the element labeled by the particular stain / immunolabel. For example, the semi-quantitative scoring for myelin content ranges from 0, indicating no myelin and a full chemical treatment, to 3, indicating a native myelin appearance and no chemical treatment. Each cross-section can be scored as a whole, or the distribution of the scores for each cross-section can be obtained by analyzing different regions of interest, such as individual fascicles. In some embodiments, the regions of interest can be a function of one or more variables, such as longitudinal position and radial depth. In some embodiments, an average myelin score of less than 1 for the region of interest distribution across different longitudinal and radial positions can verify that a chemical Attorney Docket No. REN-008-PCT treatment has led to a suitable tissue modification process. In other embodiments, a myelin score of less than 1 in a large percentage of the region of interest distribution, such as 90% of the regions of interest across different longitudinal and radial positions, can verify that a chemical treatment has led to suitable and / or desired tissue characteristics.

[0252] Referring now to Figs. 23A-B, a schematic of a nerve size discrimination tool, Tool 1135, is illustrated, consistent with present inventive concepts. Tool 1135 comprises hemi- cylindrical grooves of increasing diameter configured to temporarily hold nerve segments and rapidly discriminate their sizes to identify a suitable size of Support Assembly 1140. Each hemi-cylindrical groove comprises a known equivalent diameter associated with a specific size Support Assembly 1140. By inserting a nerve segment into each groove and observing which groove the collapsible nerve segment fills such that it sits flush with the upper edge of the groove, applicant can ascertain the most appropriate size support assembly in which the nerve segment should be inserted, as shown in Fig. 23A. In some embodiments, Tool 1135 can be used to perform image-based quantification of a nerve segment held within the grooves. In such embodiments, Tool 1135 further comprises a known width 2301 to be used as a scale for images taken. Tool 1135 further comprises an adjustable mount 2302 for a digital imaging device 2303 to be positioned orthogonally to the cross-section of a nerve segment positioned at the front edge of a groove, as shown in Fig, 23B. The set distance between the imaging device and Tool 1135 allows the applicant to take a single measurement of scale and propagate it through multiple nerve images. Images taken using Tool 1135 can be analyzed to determine nerve segment cross-sectional area, equivalent diameter, etc. in image-based analysis software.

[0253] Referring now to Fig. 24, a perspective view of a Support Assembly 1140 for manufacturing a Nerve Cap 100 is illustrated, consistent with the present inventive concepts. Support Assembly 1140 can be constructed and arranged as a Cylindrical Tube 1147 with a lumen 1141 therethrough. Support Assembly 1140 comprises a proximal end 1142 and a distal end 1143. Support Assembly 1140 can comprise different outer diameters, inner diameters, wall thicknesses, and lengths depending on the application. In some embodiments, Support Assembly 1140 comprises a permanent or Detachable Locking Feature 1144 configured to secure Support Assembly 1140 in a defined position and orientation within an external element (e.g., within Holding Rack 1160). In some embodiments, Support Assembly 1140 comprises a permanent or detachable Tab 1145 to enable handling of Support Assembly 1140. In some embodiments, Support Assembly 1140 can be configured to temporarily open Attorney Docket No. REN-008-PCT or expand its closed circumferential perimeter to allow or facilitate the insertion of a component therein (e.g., nerve segment). For example, Support Assembly 1140 can comprise two hemicylindrical shells that can engage and disengage with each other to form a closed circumference. Alternatively, Support Assembly 1140 can be obtained by shaping a flat sheet of material (e.g., wrap, ribbon, etc.) around a cylindrical template to create a wrap. In this embodiment, the temporary unraveling of the cylindrical wrap allows for the geometrical expansion and / or opening of the circumferential perimeter required to insert the nerve segment therein. In some embodiments, Support Assembly 1140 further includes a component to be inserted into a feature (e.g., Socket 120) of the contents therein. This component can maintain (e.g., reinforce) the feature during rehydration, disinfection, and / or storage of Nerve Cap 100. In some embodiments, Support Assembly 1140 further includes a component to cap one end of Support Assembly during pre-freezing and lyophilization processing, in order to affect the formation of internal porosity.

[0254] Support Assembly 1140 walls can comprise a plurality of Pores 1146 constituting interconnected and / or non-interconnected wall porosity. Pores 1146 can have different cross- sectional area shapes and sizes. In some embodiments, pores 1146 are non-interconnected, circular in shape, and comprise diameters of between 0.1 micron and 500 microns. Support Assembly 1140 walls can comprise a porosity ratio between void area and total area of between 10% and 90%. Pores 1146 shape and size can be distributed uniformly or heterogeneously as a function of the longitudinal, circumferential, and / or radial directions of Support Assembly 1140. In some embodiments, pores 1146 are configured to allow the transmural passage of a fluid, vapor, and / or gas between Support Assembly 1140 inner and outer walls. Support Assembly 1140 can comprise a controlled surface roughness to modulate the surface interaction (e.g., increase / reduce friction and / or coaptation) between Support Assembly 1140 and its contents (e.g., nerve segment, etc.), and Support Assembly 1140 and external components (e.g., Holding Rack 1160, etc.). In some embodiments, Support Assembly 1140 comprises non-porous walls.

[0255] Support Assembly 1140 can comprise a permeable / impermeable material to modulate the transmural passage of a fluid, vapor, and / or gas between Support Assembly 1140 inner and outer walls. Support Assembly 1140 can comprise thermally conductive / insulating properties to modulate the transmural heat flow between the inner and outer walls. In some embodiments, Support Assembly 1140 can comprise electrically conductive / insulating properties to modulate the transmural current flows between the inner Attorney Docket No. REN-008-PCT and outer walls. In some embodiments, Support Assembly 1140 can comprise optically transparent / translucent or semi-opaque walls to enable visualization of the internal contents. In some embodiments, Support Assembly 1140 can comprise acoustic properties to modulate vibrational flow between the inner and outer walls.

[0256] In some embodiments, Support Assembly 1140 can comprise a ngid, elastic, or viscoelastic wall. In some embodiments, Support Assembly comprises hydrophobic (nonpolar), hydrophilic (polar), or semipolar surface properties. In some embodiments, Support Assembly 1140 comprises a surface property of a certain lubricity to enable the insertion of nerve segments into Support Assembly 1140 and the insertion of Support Assembly 1140 into the Holding Rack 1160. The material properties of Support Assembly 1140 described above can have isotropic or anisotropic behavior.

[0257] In some embodiments, Support Assembly 1140 can comprise a material selected from the group consisting of: expanded and non-expanded PTFE; FEP; polydimethylsiloxane (PDMS); PP; PE; and combinations of these. In some embodiments, Support Assembly 1140 comprises a hydrophobic, expanded polyfluorotetraethylene (ePTFE) tube comprising a porosity of between 50% and 60%, a wall thickness of between 0.3mm and 0.8mm, and / or a length of between 5mm and 100mm. In some embodiments, Support Assembly 1140 comprises a non-porous hydrophobic FEP tube.

[0258] Support Assembly 1140 can be configured to receive either Raw Material 1010 or a nerve segment following chemical treatment as described in Method 2300 (collectively “contents” herein). Support Assembly 1140 can be constructed and arranged to slidingly receive contents therein. For example, a nerve segment can be pulled into Support Assembly 1140 using relative negative pressure applied at one end of Support Assembly 1140 (e.g., Lumen 1141) until a desired portion of the nerve segment is drawn within Support Assembly to the desired position.

[0259] Support Assembly 1140 can further comprise one, two, or more elements configured to impart structural modifications to the contents therein. For example, Support Assembly 1140 can be configured with outer walls / masks of a defined porosity to impart a plurality of channels to a nerve segment when laser ablation processes are performed through such a wall / mask.

[0260] Support Assembly 1140 can be constructed and arranged as a compressive, tubular structure configured to radially constrict or restrict the contents therein. Support Assembly 1140 can be configured to promote the homogenous and / or unidirectional Attorney Docket No. REN-008-PCT lyophilization of the contents therein. Support Assembly 1140 can be configured to affect the degree of porosity of the contents herein (e.g., Matrix 110) following lyophilization. Support Assembly 1140 can be configured to homogenize the diameter of the contents therein. Support Assembly 1140 may comprise several sizes (i.e., internal diameters) depending on the diameter of the nerve segments to be held therein.

[0261] Support Assembly 1140 may be configured to compress within the nerve segment to a certain compression ratio, defined as the original diameter of the nerve segment divided by the inner diameter of Support Assembly 1140. This compression ratio may comprise a range of between 1.1 and 2.0, such as approximately 1.5. In some embodiments, higher compression ratios lead to a progressively lower degree of porosity in Nerve Cap 100. Ensuring a sufficient degree of porosity, such as between 40% and 70%, provides the necessary conditions for effective neuro-inhibition, which requires axonal ingress into the matrix to occur.

[0262] In some embodiments, after attaining a certain critical compression ratio, a non- continuous abatement in degree of porosity in Nerve Cap 100 is obtained. For example, a compression ratio approximately 2.0 and higher causes a significantly lower porosity than 40%, converting the neuroinhibitory properties of the matrix into a ‘neuro-prohibiting’ one, which can deflect axons rather than entrapping and hindering their growth, possibly fostering the conditions for neuroma formation. In some embodiments, a significantly higher porosity than 70% converts the neuroinhibitory properties of the matrix into neuro-inductive ones, which allows axonal growth through the full length of Matrix 110.

[0263] Support Assembly 1140 can be configured to undergo dimensional change during lyophilization (e.g., due to the thermal expansion / compression properties of the material comprising Support Assembly 1140) and subsequently change the diameter of the contents within according to a diameter compression factor. Support Assembly 1140 can be configured to alter the length of the contents therein according to a conversion factor (e.g., radial compression and assuming incompressibility). Support Assembly 1140 may comprise increasing lengths according to the diameter of the nerve segments to be held therein. For example, nerve segments of larger diameter may be cut to a longer length and therefore necessitate a longer Support Assembly 1140. Support Assembly 1140 and the related methods can be configured to manufacture at least a portion of Nerve Cap 100 as described herein. Attorney Docket No. REN-008-PCT

[0264] Referring now to Fig. 25, a schematic view of a Jacketing Assembly 1150 to insert and secure an object (e.g., Raw Material 1010, nerve segment, etc.) within a Support Assembly 1140, herein referred to as a jacket. Jacketing Assembly 1150 can comprise a handheld vacuum pump 1151 configured to apply a progressive and controlled relative negative pressure to one end of the jacket to slidingly draw a nerve segment from the opposite to a desired position within the jacket. Vacuum pump 1151 can be triggered manually to apply the vacuum pressure through the nozzle 1152 of vacuum pump 1151. Vacuum pump 1151 can further comprise a pressure gauge 1153 to visualize and control the vacuum pressure applied. Vacuum pump 1151 can further comprise a pressure relief safety valve 1154 to ensure a prescribed level of vacuum is not exceeded (to prevent tissue damage). Vacuum pump 1151 can further comprise a secondary vacuum release trigger 1155, which can be utilized to modulate the vacuum during the insertion process and relieve the vacuum after correct positioning of the nerve segment within the jacket has been achieved to release the now-jacketed nerve segment from the Jacketing Assembly 1150. Vacuum pump 1151 can further comprise a pressure inverter 1156, which can be configured to switch to positive pressure to push out the nerve segment from the jacket if the vacuum pulls the nerve segment further into the jacket than desired. Jacketing Assembly 1150 can further comprise an air filter assembly 1157 in-line with the nozzle 1152 to prevent passage of particulates between vacuum pump 1151 and a nerve segment. Air filter assembly 1157 may also prevent the nerve segment from being pulled into vacuum pump 1151. Air filter assembly 1157 can comprise, in addition to the air filter itself, tubing and connectors (e.g., Luer lock) to airtightly couple the air filter to nozzle 1152 and jacket adapter 1158. Jacketing Assembly 1150 can further comprise one or more jacket adapters 1158. Jacket adapters 1158 may comprise tubing with a connector on one end to couple to air filter assembly 1157. Jacket adapters 1158 may be configured to pressure fit one or more sizes of jacket at the distal end. Jacket adapters 1158 may be configured to easily attach to and detach from air filter assembly 1157 in order to facilitate the iterative jacketing process.

[0265] Referring now to Figs. 26A-F, schematics of different types of jacket-to-nerve coupling are illustrated (in bi-sectional views), consistent with the present inventive concepts. In some embodiments, a nerve segment is held symmetrically within a jacket such that there is equal tissue exiting either end of the jacket, as shown in Fig. 26A. In some embodiments, a nerve segment is held asymmetrically within a jacket such that there is more tissue exiting one end of the jacket than the other, as shown in Fig. 26B. In some embodiments, nerve Attorney Docket No. REN-008-PCT segments of equivalent size (diameter) are coupled with jackets of different internal diameters to produce different compression ratios, as shown in Fig. 26C. In some embodiments, a longer or shorter jacket can be applied to leave less or more tissue exiting either end of the jacket, respectively, as shown in Fig. 26D. In some embodiments, a nerve segment is held within a jacket of increasing internal diameter along the length to produce a compression ratio gradient, as shown in Fig 26E. In some embodiments, a nerve segment is held within a jacket with an internal regular or irregular geometry (e.g., corrugated geometry, etc.) feature along the inner surface to provide a template for Nerve Cap 100 outer surface, as shown in Fig 26F. Such a geometrical template could aid in conferring certain properties to Nerve Cap 100 such as kink resistance, enhanced surface area to modulate degradation rate, improved integration with surrounding tissue, support in anchoring of Nerve Cap 100, creation of markings to aid in orientation and positioning of Nerve Cap 100, and enhanced surgical handleability.

[0266] Referring now to Figs. 27A-H, a schematic view of a Holding Rack 1160 for handling, holding, and storing one or more intermediate materials (e.g., jacketed nerve segments) during the manufacturing process of Nerve Cap 100 is illustrated, consistent with the present inventive concepts. Each Holding Rack 1160 can be configured to hold between 1 and 1,000 jacketed processed nerve segments, such as 100 jacketed processed nerve segments. Holding Rack 1160 can be used during the lyophilization, feature creation, trimming, intermediate transport, and storage of Nerve Cap 100 during its manufacturing process.

[0267] Holding Rack 1160 can comprise plastics such as HDPE, PP, PTFE, FEP, PSU; metals such as stainless steel, anodized aluminum, titanium; or combinations of these. Holding Rack can comprise an upper plate 11601, a lower plate 11602, handles 11603, a locking system 11604 to couple and secure the two plates together and / or to an external structure, a calibration system 11605, one or more networks of pressure equalizing channels 11606, a matrix of bores 11607 to hold individual jacketed nerve segments, a system 11608 to create controlled compression in a portion of Nerve Cap 100, an extractor system 11609 to facilitate nerve segment removal, a system 11610 to identify Holding Rack 1160 orientation and a system 11611 to lock / release jacketed nerve segments from bores. The upper plate 11601 can comprise a matrix of throughbores 11607 to secure jacketed nerve segments within, as shown in the top view of the upper plate 11601 in Fig. 27A. In some embodiments, the bores 11607 comprise the same length as the nerve segment jackets. In some Attorney Docket No. REN-008-PCT embodiments, the bores 11607 comprise the same diameter as the nerve segment jacket outer diameter. In some embodiments, system 11604 to lock / release jacketed nerve segments can be engaged with Detachable Locking Feature 1144 to lock the jacketed nerve segments into bores 11604 in a defined position and orientation and prevent rotational or translational movement during subsequent processing. System 11604 can be disengaged to release the jackets from the locked position. The top of the upper plate 11601 can further comprise a calibration system 11605 including two or more calibration sockets to secure disposable calibration chips, which can be used to perform a multi-point calibration of Material Device 1180 before feature creation. The bottom of the upper plate 11601 can further comprise a matrix of vertical concave features 11612 aligned and contiguous with the bottom of each bore 11607, as shown in the bottom view of the upper plate 11601 in Fig. 27C. The lower plate 11602 may comprise a matrix of corresponding convex features 11613 aligned with the aforementioned concave features 11612, as shown in the top and bottom views of the lower plate 11602 in Figs. 27B and D, respectively. The upper and lower plates in a closed configuration may facilitate the formation of a compressed tab feature between each concave feature 11612 in the upper plate 11601 and each convex feature 11613 in the lower plate 11602.

[0268] The upper plate 11601 and lower plate 11602 can be configured to slide against each other and be secured in an “open state” and / or “closed state" position. When the Holding Rack is in its “open state” position, the space between the concave features of the upper plate and the corresponding convex features of the lower plate is maximized, as shown in Fig. 27E and Fig. 27F (cross-sectional view). When the Holding Rack is in its “closed state” position, the space between the concave features of the upper plate and the corresponding convex features of the lower plate is minimized, as shown in Fig. 27G and Fig. 27H (cross-sectional view).

[0269] Referring now to Fig. 28, a method for in-process control or quality control of the geometry and / or micromorphology of Nerve Cap 100 is illustrated, consistent with the present inventive concepts. Nerve Cap 100 devices can be loaded into a custom-made Imaging Holder 1260. Imaging Holder 1260 can allow handling of one or more Nerve Caps 100, identification / discrimination of individual samples within Imaging Holder 1260, maintenance of stable sample orientation and alignment within a sample holder of a MicroCT (Microcomputed Tomography) apparatus, as shown in Fig. 28. Imaging Holder 1260 can comprise a radiotransparent material, such as polypropylene, other polyolefins, etc. Imaging Attorney Docket No. REN-008-PCT Holder 1260 can further comprise an elongated handle 1261 to enable the handling, insertion, securing, acquisition / imaging, and extraction of the samples from the MicroCT sample holder and scanner. Imaging Holder 1260 can further comprise a main body 1262 with a cross- section consistent with the internal cross-section of the MicroCT sample holder to enable the slide-fit insertion of Imaging Holder 1260 within the MicroCT sample holder. Imaging Holder 1260 can comprise an additional feature 1263 to identify the rotational orientation of samples within Imaging Holder 1260. This feature may comprise a radio-opaque component, such as a paint or inset component. The Imaging Holder 1260 main body 1262 can comprise one or more bores 1264, each of a diameter and depth consistent with those of a specific size Nerve Cap 100 to be scanned. MicroCT scanning can be used to produce stacks of images along the longitudinal axis of Nerve Cap 100 within Imaging Holder 1260.

[0270] The percent porosity of each Nerve Cap 100 cross-sectional image can be analyzed via an automated image-analysis software, yielding porosity data for the full length of each Nerve Cap 100. In some embodiments, the automated software can further identify the pore size distribution, pore planar and volumetric interconnectedness, pore direction- dependent features (i.e., anisotropy), etc. In some embodiments, a percent porosity between 20% and 80%, such as between 40% and 70% can indicate suitable properties for neuro- inhibitory behavior. MicroCT images can also be used to identify and quantify additional geometry parameters of Nerve Cap 100 including external dimensions; Socket 120 position / alignment, shape, perimeter, area, diameter, and depth; Socket 120 wall thickness; Anchoring Tab 130 position / alignment, width, length, and thickness; Matrix 110 length, volume, etc.

[0271] Referring now to Figs. 29A-B, a schematic view of a Device 1200 to be used during feature creation in conjunction with Holding Rack 1160 is illustrated, consistent with the present inventive concepts. Device 1200 may comprise a skin grafting knife (dermatome). Device 1200 can be used to remove the flared nerve ends (deflare) from the top of lyophilized nerve segments within Holding Rack 1160. To remove the flared nerve ends, Device 1200 is laid with the flat blade edge on the top surface of the Holding Rack 1160 to one side of the matrix of bores containing lyophilized nerve segments, as shown in Fig. 29A. To perform deflaring, Device 1200 is slid across the top surface of the Holding Rack 1160, as shown in Fig. 29B. Following deflaring, lyophilized nerve segments should comprise a proximal end that sits flush with the top of Holding Rack 1160. In some embodiments, deflaring occurs before feature creation via Material Device 1180. Attorney Docket No. REN-008-PCT

[0272] Referring now to Figs. 30A-B, schematic views of Alignment Assembly 1190 components, including a calibration chip 1191, calibration socket 1192, alignment base 1193, removable base cradle 1194, and z alignment block 1195 for alignment of feature creation by Material Device 1180 within Holding Rack 1160 are illustrated, consistent with the present inventive concepts.

[0273] In some embodiments, a calibration chip 1191 may be configured as a cut-out from a flat sheet of material, as shown in Fig. 30A. The calibration chip 1191 may comprise one or more of the following materials: nylon, polymethyl methacrylate (PMMA), PSU, other plastics, glass, anodized aluminum, etc. One or more calibration chips 1191 may be configured to be pressure fit into the Calibration Socket 1192 of Holding Rack 1160 in only one orientation to clearly identify one or more reference points of Holding Rack 1160. Calibration Chip 1191 may comprise an etched circle, point, or other features identifying the center point of the chip, which overlays the respective reference point of Holding Rack 1160. Calibration Chip 1191 may further comprise a grid of equally spaced lines (0.5mm spacing) etched onto its top surface. Material Device 1180 can be programmed to create one or more etched reference features onto Calibration Chip 1191 top surface. The grid can be used to calculate the offset between the actual mark and the intended location at the center point. Such offset may be further measured via image-based quantification or other measurement methods. In some embodiments, the settings of Material Device 1180 are adjusted accordingly to calibrate the mark to the center point of Calibration Chip 1191 (and by extension Holding Rack 1160). In some embodiments, the Holding Rack 1160 is physically moved to calibrate the mark to the center point of Calibration Chip 1191 (and by extension Holding Rack 1160). In some embodiments, Calibration Chip 1191 is reusable to allow multiple calibrations on a single chip or an iterative calibration. In some embodiments, two Calibration Chips 1191 can be fit into two separate locations on Holding Rack 1160 to provide a two-point calibration to precisely align the reference frame of Material Device 1180 to that of Holding Rack 1160 via a two-dimensional roto-translational solid body movement calculation. In some embodiments, 2-4 calibration chips can be fit into multiple separate locations on Holding Rack 1160 to provide a multi-point calibration to precisely align the reference frame of Material Device 1180 to that of Holding Rack 1160 via a two- dimensional scaling calculation in addition to the aforementioned two-dimensional roto- translational solid body movement calculation. Attorney Docket No. REN-008-PCT

[0274] A schematic view of the coupling of Holding Rack 1160, a removable base cradle 1194, and an alignment base 1193 permanently secured to Material Device 1180 is illustrated in Fig. 30B. Base Cradle 1194 can comprise a material selected from the group consisting of: anodized aluminum; stainless steel; glass; plastics such as PMMA, polyoxymethylene (POM), or PSU; and combinations of these. Base Cradle 1194 may be configured such that the Holding Rack 1160 is pressure fit (or otherwise fit) within and firmly held for the duration of feature creation. In some embodiments, Base Cradle 1194 can be used to secure multiple Holding Racks 1160 with equal and / or different sized nerve segments. The base cradle may be configured such that it is pressure fit (or otherwise fit) in a precise position and orientation within a larger Alignment Base 1193. Alignment Base 1193 can comprise a material selected from the group consisting of: anodized aluminum; stainless steel; glass; plastics such as PMMA, POM, or PSU; and combinations of these. Alignment Base 1193 may be configured such that it is permanently anchored in a precise position and orientation within Material Device 1180.

[0275] Z-alignment Block 1195 may comprise one or more of the following materials: nylon, PMMA, PSU, other plastics, anodized aluminum, stainless steel, etc. Z-alignment Block 1195 can comprise a known height. In some embodiments in which Material Device 1180 comprises a laser, the height of Z-alignment Block 1195 is equal to the optimal focal height for a given laser and lens setup. In some embodiments in which Material Device 1180 comprises a CNC milling machine, Z-alignment Block 1195 comprises a standard CNC multi-axis sensor. Z-alignment Block 1195 can be configured to calibrate the distance between the head of Material Device 1180 and the top surface of Holding Rack 1160. In some embodiments, when using the laser cutter, the laser cutter z-axis focal distance can be dynamically modified throughout the cut to maintain the ideal focal height throughout the different depths of the cut.

[0276] Referring now to Fig. 31, a schematic view of the Anchoring Tab 130 trimming process to obtain a desired shape of Anchoring Tab 130 during Step 2610 is illustrated, consistent with the present inventive concepts. In some embodiments, Feature Trimming Device 1200 comprises surgical scissors (Iris, Metzenbaum, etc.), and each step of Anchoring Tab 130 trimming is performed separately, as shown in Fig. 31. In some embodiments, Device 1200 comprises a mechanical punch die that trims Anchoring Tab 130 to a desired shape in a single step. Using Device 1200, Anchoring Tab 130 is cut longitudinally on each side to the approximate width of the lyophilized nerve segment. Anchoring Tab 130 is cut Attomey Docket No. REN-008-PCT horizontally at a defined distance from the base of the tab. In some embodiments, thisdistance is comprised between1 and 10mm, such asbetween 2 and 5mm.

[277] Referring now to Fig. 32,a schematic view of thepackaging configuration ofNerve Cap 100 is illustrated, consistent with the present inventive concepts. Nerve Cap 100can be packaged within a sealed and labeled InnerSterile Barrier 200. Inner Sterile Barrier200 can be packaged within a sealedOuter Sterile Barrier 300provided with a transparentwindow. Outer Sterile Barrier 300 can be curled around one or more edges to providemechanical protection to itscontents and enclosedin a labeled Protective Packaging 400.

[278] Referring now to Fig. 33, a table containing arange of Nerve Cap 100dimensions and other features is illustrated, consistent with the present inventive concepts.Nerve Cap 100 may comprise multiple sizes intended for different applications, such as tocap nerve sizes comprised between 0.5mm and 9.0mm in equivalent diameter.

[279] The outer diameter of Nerve Cap100 is established by the sum of the Socket 120diameter and the double of the Socket 120 wall thickness. The Socket 120 diameter can beadjusted to enable the insertion of multiple sizes of free nerve endings into Nerve Cap 100.The Socket 120 wall thickness of Nerve Cap 100can be configured to provide a sufficientpermeability barrier to and fromthe surrounding environment and sufficient mechanicalstrength to allow secure alignment and anchoring between nerve stump and device, withoutcompromising permeabilityto nutrients or creatingexcessive bulk. The total length of NerveCap 100 is established by the sumof the Socket 120 depth, neuroinhibitory Matrix 110length, and Tab 130 length. The depth ofSocket 120 can be configured to allow sufficientoverlap with the proximal free nerve ending to align and secure the nerve in position, toprotect it from the chemical signaling and cell ingress derived from the surroundingenvironment, to prevent chemical signaling from the nerve stump to the surroundingenvironment leading to nociceptive sensitization, andto prevent axon radial and retrogradeoutgrowth into surrounding tissue. Thelength of neuroinhibitoryMatrix 110 can be adjustedto provide sufficient physicalspacing between the nerve stump and surrounding tissue toprevent chemoattraction, and / or toenable sufficient axonalelongation into Matrix 110 toexhaust the natural axonal growth potential (passive neuro-inhibition), and / or to providesufficient mass of Matrix 110 toactively inhibit axonaloutgrowth into Matrix 110 byproviding sufficient and / or sufficientlydistributed cues (activeneuro-inhibition), and / or toprovide sufficient volume of Matrix 110 to modulate the desired degradation rate to ensurestable neuro-inhibition. The lengthof Tab 130 can be configured to allow effective surgical Attorney Docket No, REN-008-PCT handling of Nerve Cap 100 and sufficient area to enable optional surgical fixation of Nerve Cap 100 to the surrounding tissue. Matrix 110 porosity and pore size can be adjusted to modulate the degradation rate of Nerve Cap 100, and / or the extent and depth of axonal ingress into Matrix 110, and / or the modality (e.g., passive or active neuro-inhibition, etc.), degree, and / or progression of neuro-inhibition.

[0280] The Nerve Cap 100 outer diameter and length can be controlled via source tissue selection, tissue segmentation parameters, compression ratio and use of Support Assembly 1140, and combinations thereof. Other geometric variables of Socket 120 diameter, Socket 120 wall thickness, Socket 120 depth, and Tab 130 length can be controlled by manufacturing processing parameters. Neuroinhibitory Zone porosity and pore size can be controlled by freezing and lyophilization parameters.

[0281] In some embodiments, Socket 120 is created via laser ablation. In this embodiment, Material Device 1180 comprises a laser cutting and engraving apparatus, and the specific area and shape of Socket 120 created can be controlled by a user-defined vector graphic. In this embodiment, Socket 120 dimensions and surface properties (e.g., degree of charring, roughness, surface porosity, etc.) are controlled by altering parameters of the laser beam, including: wavelength; power; pulse width and frequency; beam diameter; beam polarization; beam depth of field; speed of laser head; number of ablation passes; beam focal distance; and combinations of these. In some embodiments, a CO laser beam is used with parameters as follows: wavelength between 9.31.m and 10.6um; power between 2W and 50W; pulse frequency between 1,000Hz and 50,000Hz; beam diameter between 70 tum and 250 um; speed of laser head between 10mm / sec and 3,000mm / sec; Dots Per Inch (DPI) between 500 and 1,000.

[0282] Referring now to Fig. 34, a method for obtaining ideal decellularization in order to obtain desired structure and function in the nerve cap graft device, consistent with the present inventive concepts. Method 4000 can be configured to customize the choice of Raw Material 1010 for the intended application. Method 4000 can be configured to obtain an ideal (e.g., preferred) degree of decellularization in different sized nerve segments. It has been previously demonstrated that decellularization decreases with radial depth of the nerve segment. Thus, a nerve segment comprising a larger diameter requires a longer incubation period in detergent solution (STEP 2304) and an associated longer rinse protocol (STEPs 2305-6) to obtain the same level of decellularization as a nerve segment comprising a small diameter. In addition to diameter, fascicular complexity can also impact the degree of Attorney Docket No. REN-008-PCT decellularization. As previously discussed, Nerve Cap 100 can be selected from a specific nerve branch at a specific distance from the proximal branching point, which can result in a device with defined features (e.g., external diameter and fascicular complexity). Based on these parameters and the degree of decellularization desired for the target application, the decellularization process can be tailored to each individual nerve segment to produce equivalently decellularized grafts with different features (e.g., external diameter and fascicular complexity).

[0283] In some embodiments, Nerve Cap 100 can comprise a large nerve segment, such as that from the main sciatic trunk, comprising a large diameter (e.g., 8-13mm) and high fascicular count (e.g., 50-150). This nerve segment can require a longer incubation in detergent solution, such as an incubation period of 8-16 hours. This nerve segment can further require the enhancement of the convective fluid dynamic environment, as well as the introduction of cyclic mechanical deformation of the tissue during the process. In some embodiments, Nerve Cap 100 can comprise a small nerve segment, such as that from the sural branch of the sciatic nerve, comprising a smaller diameter (e.g., 1-3mm) and low fascicular count (e.g., 10-30). This nerve segment can undergo a shorter incubation in detergent solution, such as an incubation period of 4-8 hours. To supplement this approach to decellularization, the cellular content in the detergent solution may be monitored through one, two, or more features, such as turbidity, absorbance, conductivity, and pH.

[0284] In STEP 4010, the nerve segment external diameter, length, and structure / fascicular complexity are defined based on the intended use. For example, if a graft is intended to cap a nerve stump of 3 mm in diameter, the graft would be derived from a nerve segment sourced from anatomic locations of similar / slightly larger external diameter (e.g., intended nerve stump diameter plus 0.5mm - 2mm) and with high fascicular complexity (e.g., 10+ fascicles).

[0285] In STEP 4020, Based on the desired features above, the source nerve tissue would be selected from a nerve branch and location known to possess the desired features.

[0236] In STEP 4030, still based on the desired features above, the source nerve tissue would be selected starting at a known distance from its proximal branching point in order to have the desired features and allowing sufficient graft length.

[0287] In STEP 4040, a desired degree of tissue processing / decellularization of the source nerve tissue for the intended size nerve stump is identified. In some embodiments, Attorney Docket No. REN-008-PCT tissue can be treated / decellularized to a high degree to completely remove adhesive molecules, such as laminin, known to support nerve regeneration.

[0288] In STEP 4050, one, two, or more treatment / decellularization process parameters are set. In some embodiments, the treatment / decellularization parameters include the incubation time, the temperature, the concentration of the reagent use, the degree of washing following treatment with the reagents, parameters affecting mass transport, etc.

[0289] In STEP 4060, one, two, or more treatment / decellularization process parameters are used to treat / decellularize the source nerve tissue.

[0290] In STEP 4070, the degree of tissue processing in the source nerve tissue segment can be analyzed during or following the tissue treatment / decellularization to confirm that the desired features have been obtained. For example, the degree of tissue processing, can be measured / tracked during the process by measuring the absorbance to visible light and / or the electrical conductivity of the solution used for tissue treatment / decellularization and / or the solution(s) used for washing the tissue following treatment. The degree of tissue processing / decellularization and the structural and fascicular complexity of the graft can be also confirmed by performing histological analysis of a sample of the graft. The diameter and length of the graft can be confirmed with standard tools after completion of the process.

[0291] The above-described embodiments should be understood to serve only as illustrative examples; further embodiments are envisaged. Any feature described herein in relation to any one embodiment may be used alone, or in combination with other features described, and may also be used in combination with one or more features of any other of the embodiments, or any combination of any other of the embodiments. Furthermore, equivalents and modifications not described above may also be employed without departing from the scope of the present inventive concepts, which are defined in the accompanying claims.

Claims

Attorney Docket No. REN-008-PCT Anerve cap devicecomprising an acellular nerve graft configured to inhibit nerve growthand neuroma formation or pain, providing a therapeutic benefit to patients. The device according to claim 1, wherein the acellular nerve graft comprises at least onesensory, motor, mixed, and / or autonomic nerve tissues. The device according to claim 1, wherein the acellular nerve graft is treated to removepotentially immunogenic components. The device according to claim 1, wherein the acellular nerve graft retains the decellularized extracellular matrix comprising structural and / or non-structural biomolecules including endogenous growth factors. The device according toclaim 1, wherein the device comprises one or more of thefollowing features: a socket; asocket wall; a neuro-inhibitory matrix, and an anchoringtab. The device according to claim 1, wherein the device comprisesone, two, or morestructural and functional qualities intended to match or mismatch the structural and functional qualities of a nerve injury site in the patient. The device according to claim 1, wherein the device achieves an effective isolation of thenociceptive innervation between the implant site and the downstream nerve stump at theamputation site, leading to an elimination / reduction of pain. The device according to claim1, wherein the device is configured to accept, protect, andbe secured to a nerve ending of a specific size range. The device according to claim 5, wherein the device anchoring tab feature is configuredto allow connection of the device to surrounding tissues. 10.The system according to claim 1, wherein the nerve cap device is configured to be at leastpartially placed over one, two, or more nerve endings.

11. The device according to claim 1, wherein the acellular nerve graft is configured to comprise a porosity of 20-90%.

12. The device according to claim 1, wherein the acellular nerve graft is configured to comprise average pore size of 3-50pm.

13. The device according to claim 1 wherein the acellular nerve graft can comprise longitudinally oriented pores.Attorney Docket No. REN-008-PCT 14. The device according to claim 1 wherein the acellular nerve graft comprises interconnected pores, non-interconnected pores, or combinations thereof.

15. The device according to claim 1 wherein the acellular nerve graft comprises intertwining pores constructed and arranged to mimic the natural plexus structure of a peripheral nerve.

16. The device according to claim 1 wherein the acellular nerve graft comprises pores which provide a resistive path constructed and arranged to exhaust the growth capacity of axons.

17. The device according to claim 1 wherein the acellular nerve graft comprises pores which provide a resistive path constructed and arranged to exhaust the growth capacity of axons within a 10mm distance from the nerve stump.

18. The device according to claim 1 wherein the acellular nerve graft comprises pores that compartmentalize and separate axons, preventing them from entangling with themselves and / or with other axons.

19. The device according to claim 1, wherein the acellular nerve graft comprises a degradation rate in vivo between 2 weeks and 1 year, such as a degradation rate of between 8 and 16 weeks.

20. The device according to claim 1, wherein acellular nerve graft is configured to be remodeled over time into a native tissue of the patient.

21. The device according to claim 1, wherein acellular nerve graft is configured to be remodeled over time into a native connective tissue of the patient which maintain aligned, compartmentalized, and non-entangled axons embedded within in a stable configuration, which is immune to the structural formation of neuroma.

22. The device according to claim 5, wherein the socket wall is configured to possess sufficient suture retention strength to withstand forces generated by its connection to a nerve end during characteristic body and / or surrounding tissue motions.

23. The device according to claim 5, wherein the anchoring tab is configured to possess sufficient suture retention strength to withstand forces generated by its connection to surrounding tissue during characteristic body and / or surrounding tissue motions.

24. A method for deploying a nerve cap device comprising positioning the nerve cap device at one or more implant sites comprising a peripheral nerve stump or stumps for whichAttorney Docket No. REN-008-PCT functional recovery is not desirable or possible, and wherein the nerve cap device is configured to inhibit nerve growth and neuroma formation or pain.

25. The method according to claim 24, wherein deploying the nerve cap device comprises attaching one or more of the nerve cap devices to one or more transected nerve ends, orienting each of the nerve cap devices within the one or more implant sites, and optionally anchoring each of the nerve cap devices within the one or more implant sites.

26. The method according to claim 24, where the nerve cap device is attached to a nerve stump, to a portion of a nerve stump such as at one or more fascicular stumps, to fractions of fascicular nerve stumps, or to combinations thereof within the peripheral nervous system.

27. The method according claim 24, wherein the nerve cap device is placed in any natural or surgically created anatomic location that can host the dimensions of the nerve cap device and optionally anchoring the nerve cap device to any tissue type present within the anatomic location.

28. The method according to claim 24, wherein the nerve cap device can be used for applications involving neuropathic conditions causing pain following nerve injury or condition for which nerve functional recovery is not attainable or desirable.

29. The method according to claim 24, wherein the nerve cap device is deployed at one or more implant sites via minimally invasive methods.

30. The method according to claim 24, wherein a single nerve cap device is deployed to a single severed nerve end, wherein the single device comprises a socket, the socket being of a size based on the size of the severed nerve end.

31. The method according to claim 24, wherein a single device is deployed to a single severed nerve end, wherein the single device comprises a socket, the socket being of a size that is equal or larger than that of the severed nerve end.

32. The method according to claim 24, wherein multiple devices are deployed to multiple severed nerve ends, wherein the multiple devices each device comprising a socket, each socket being of a size based on each of the sizes of the multiple severed nerve ends.

33. The method according to claim 24, wherein a single device is deployed to multiple severed nerve ends, wherein the single device is comprised of a plurality of sockets, and each socket being sized based on each of the sizes of the multiple severed nerve ends.Attorney Docket No. REN-008-PCT 34. The method according to claim 24, wherein multiple nerve cap devices are deployed to a single large severed nerve end.

35. A method for manufacturing one or more nerve cap devices comprising: harvesting and preparing a one or more nerve segments; chemically, physically, and / or mechanically treating the one or more nerve segments producing one or more treated nerve segments; compressing the one or more treated nerve segments to a defined level to obtain one or more desired properties and / or features producing one or more compressed treated nerve segments; freezing and lyophilizing the one or more compressed treated nerve segments producing one or more lyophilized and compressed treated nerve segments; creating a socket and an anchoring tab feature in each of the one or more lyophilized and compressed treated nerve segments to obtain the one or more nerve cap devices; wherein each nerve cap device comprises a single lyophilized and compressed treated nerve segment and the one or more nerve cap devices are packaged producing packaged nerve cap devices; and then the packaged nerve cap devices are sterilized.

36. The method according to claim 35, where the harvesting and preparing the one or more nerve segments comprises selecting specific nerve branches having specific desired sizes to configure the nerve cap device to be of a specific desired size.

37. The method according to claim 35, wherein the chemically, physically, and / or mechanically treating the one or more nerve segments is achieved in a mixing device system.

38. The method according to claim 37, wherein the mixing device system comprises a mixing device configured to agitate a fluid disposed within the mixing device while the one or more nerve segments are contained within the mixing device.

39. The method according to claim 37, wherein the mixing device system comprises a mixing device configured to produce laminar, transitional, or turbulent flow within a fluid field of the mixing device.

40. The method according to claim 37, wherein the mixing device system comprises a mixing device configured to maximize and / or optimize mass transport within the one or more nerve segments to facilitate the chemical, physical, and / or mechanical treatment.Attorney Docket No. REN-008-PCT 41. The method according to claim 37, wherein the mixing device system comprises a mixing device, the mixing device being capable of putting into motion one or more containers containing the one or more nerve segments within a fluid field of the mixing device.

42. The method according to claim 41, wherein the one or more containers are capable of allowing mass transfer between the fluid outside an individual container and the fluid inside the individual container while holding multiple nerve segments, each nerve segment being contained within individual compartments within the individual container, maximizing mass transport between each nerve segment and the fluid inside and outside the individual container.

43. The method according to claim 37, wherein the mixing device system comprises a mixing device can be controlled to maintain a fluid temperature within the mixing device between 0°C and 100°C. AA, The method according to claim 37, wherein the mixing device system comprises a mixing device; wherein the mixing device can be controlled to deliver cyclic mechanical deformations to the one or more nerve segments contained in the mixing device to improve mass transport within each of the one or more nerve segments to facilitate the desired chemical, physical, and / or mechanical treatment.

45. The method according to claim 37, wherein the mixing device system comprises a mixing device; wherein the mixing device is capable of creating cylindrical motion that is rotational around an axis and / or cylindrical motion that is translational along an axis, time varying or non-time varying motion, periodic or non-periodic motions with a fluid field in the mixing device to facilitate the desired chemical, physical, and / or mechanical treatment.

46. The method according to claim 45, wherein the cylindrical motion that is rotational around an axis and / or the cylindrical motion that is translational along an axis comprises a first acceleration until a first upper rotational speed and / or an upper translational speed in a first direction is reached, followed by a deceleration until an inversion of the first direction is obtained, followed by a second acceleration until a second upper rotational and / or an upper translational speed in a second direction is obtained, the second direction being the opposite of the first direction, wherein the first acceleration and the second acceleration are repeated in order to enable a desired degree of mass transfer within the fluid field of the mixing device.Attorney Docket No. REN-008-PCT 47. The method according to claim 35, wherein the chemically, physically, and / or mechanically treating the nerve segment producing a treated nerve segment is achieved in a tissue mass-to-fluid volume ratio (g / mL) comprised between 1:20 and 1:

500.

48. The method of claim 35, wherein the chemically, physically, and / or mechanically treating of the one or more nerve segments producing one or more treated nerve segments is achieved in a fluid at a temperature of between 2°C and 37°C.

49. The method of claim 35 wherein the chemically, physically, and / or mechanically treating the one or more nerve segments producing the one or more treated nerve segments is comprised of immersing the one or more nerve segments in a detergent solution comprising sodium deoxycholate in water at a concentration of 4% (w / v) for a time period between 6 hours and 10 hours.

50. The method of claim 35 wherein the chemically, physically, and / or mechanically treating the one or more nerve segments producing the one or more treated nerve segments comprised of immersing the one or more nerve segments in a disinfecting co-solution comprising 0.1% peracetic acid (w / v) and 4% ethanol (v / v) for a time period between 30 minutes and 240 minutes.

51. The method of claim 35 wherein the chemically, physically, and / or mechanically treating the one or more nerve segments producing the one or more treated nerve segments occurs in a mixing device, the mixing device capable of creating cylindrical motion that is rotational around an axis and / or cylindrical motion that is translational along an axis, time varying or non-time varying motion, periodic or non-periodic motions with a fluid field.

52. The method of claim 51 where in the mixing device has an average rotational speed of between 10 rpm and 1,000 rpm.

53. The method of claim 35, wherein the compressing the one or more treated nerve segments to a defined level to obtain one or more desired properties and / or features producing one or more compressed treated nerve segments is achieved by drawing each of the one or more nerve segments into its own individual support assembly by the application of a negative relative pressure until a desired overlap between each of the one or more nerve segments and its respective support assembly is achieved.Attorney Docket No. REN-008-PCT 54. The method according to claim 53, wherein the support assembly is a segment of tubing having a defined inner diameter and length to achieve a desired level of circumferential compression and length overlap.

55. The method according to claim 53, wherein each of the one or more treated nerve segments has a longitudinal midpoint, wherein each respective support assembly has a longitudinal midpoint, wherein each of the one or more treated nerve segments and its respective support assembly has a length overlap such that each longitudinal midpoint of each of the one or more nerve segments is adjacent to the longitudinal midpoint of its respective support assembly.

56. The method of claim 35, wherein the one or more nerve segments each has a diameter, wherein at least a portion of the diameter is compressed, the at least a portion of the diameter ranging from between 50% and 91%.

57. The method of claim 35, wherein the chemically, physically, and / or mechanically treating the one or more nerve segments producing the one or more treated nerve segments comprises a holding rack.

58. The method according to claim 57 where the holding rack is configured to receive, hold, and handle the one or more treated nerve segments after each of the one or more nerve segments inserted into its respective support assembly to enable further processing, said processing being comprised of freezing, lyophilization, feature creation, intermediate transport and / or storage.

59. The method of claim 57 wherein the holding rack can comprise a material selected from the group consisting of corrosion-resistant metals, glass, chemically resistant plastics, autoclave resistant plastics, ionizing radiation resistant plastics, cryo-resistant plastics, and / or combinations of these.

60. The method of claim 57, wherein the holding rack can be sterilized and depyrogenated without damage to prevent contamination.

61. The method of claim 57, where the holding rack further comprises a thermally conductive or insulative material element to control the heat flow during freezing and to promote or prevent directional crystal formation, to control crystal volume, and to enable directional and size distributed pore formation.Attorney Docket No. REN-008-PCT 62. The method of claim 57, wherein the holding rack can compress one or more regions of the one or more treated nerve segments contained therein in order to create a density and / or porosity gradient along the length of the one or more treated nerve segments.

63. The method of claim 57, wherein the holding rack can be configured to hold the one or more treated nerve segments in a defined orientation to facilitate the creation of the socket and tab features in the nerve cap device.

64. The method of claim 57, wherein the holding rack can be configured to create a tab feature by compressing to a defined level a distal portion of the one or treated nerve segments prior to and during lyophilization.

65. The method of claim 57, the holding rack can be configured to include a network of ports and channels to communicate / equilibrate pressure between the exterior of the holding rack and all internal portions of the holding rack.

66. The method of claim 57, wherein the holding rack can be configured to include a matrix of bores to host the one or more treated nerve segments in a defined position and orientation and prevent their rotational or translational movement during subsequent processing.

67. The method of claim 57, wherein the holding rack can be configured to include a calibration system to align the position and reference frame of the one or more treated nerve segments held within the holding rack with that of a material removal device to create features in the one or more treated nerve segments.

68. The method of claim 35, wherein the one or more compressed treated nerve segments is frozen and lyophilized to obtain a residual moisture content comprised between 0.1% and 10%.

69. The method of claim 35, wherein the one or more compressed treated nerve segments is further processed to create a socket feature using a material removal device comprised of a drill press, micro drill press, dental pin drilling unit, longitudinal file, cylindrical punch die, laser cutter, ultrasonic cutter, electrosurgery unit, hot wire cutter, CNC milling machine, or powder blasting, and combinations of thereof.

70. The method according to claim 69, wherein the socket feature is created via a laser cutting and engraving apparatus in which the socket dimensions and surface properties are obtained via a CO2 laser beam of wavelength comprised between 9.3m and 10.6um;Attorney Docket No. REN-008-PCT power comprised between 2W and 50W; pulse frequency comprised between 1,000Hz and 50,000Hz; beam diameter comprised between 70 um and 250 ym; speed of laser head comprised between 10mm / sec and 3,000mm / sec; and resolution comprised between 500 DPI and 1,000 DPI.

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