Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
  • A61L27/3633—Extracellular matrix [ECM]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0618—Cells of the nervous system
  • C12N5/0619—Neurons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2430/00—Materials or treatment for tissue regeneration
  • A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction

Definitions

• Lorenzo Soletti a citizen of the USA, residing at: 217 Vine Street, Pittsburgh, PA 15218
• the present inventive concepts relate generally to improved nerve injury treatment systems, devices, and methods.
• Peripheral nerve injuries caused by laceration, compression, stretch, or iatrogenic injuries, such as those caused by tumor resection, have severe and wide-ranging impacts on the quality of life, productivity, and interpersonal relationships of those affected.
• PNI in the upper extremities can prevent patients from performing basic daily activities (e.g. getting dressed, working, or feeding themselves), while facial nerve injuries can impede vocalization and are associated with social stigma and withdrawal.
• Existing FDA-approved nerve products are primarily indicated for use as passive support or to prevent complications (e.g. mechanical instability, neuroma, or donor site morbidity associated with autograft). None of these products has shown clinical improvement in functional outcomes.
• neuroma formation is common following traumatic or oncologic nerve transections.
• Neuromas result from aberrant and disorganized axonal outgrowth through Schwann cell proliferation ahead of an injury site as the cells attempt to restore axonal continuity of the disrupted nerve end(s).
• the regenerating axons cannot reach their target tissue and instead form a tangled bulbous mass which causes pain, likely due to pathological interactions between axons within the neuroma as well as traction between the nerve and scar tissue or ischemic necrosis of the nervous tissue.
• a system for treating a patient comprises at least one of a nerve graft-conduit or nerve cap-graft comprising a nerve segment derived from a tissue source.
• the system is configured to provide a therapeutic benefit to the patient.
• the at least one nerve graft-conduit or nerve cap-graft is configured to be remodeled over time into a native tissue of the patient.
• the at least one nerve graft-conduit or nerve cap-graft is configured to inhibit growth of tissue of the patient.
• the at least one nerve graft-conduit or nerve cap-graft comprises a decellularized extracellular matrix.
• the decellularized extracellular matrix can comprise structural and/or non-structural biomolecules.
• the decellularized extracellular matrix can comprise endogenous and/or exogenous growth factors.
• the decellularized extracellular matrix can be configured to promote and/or sustain the growth of tissue and/or associated tissue properties.
• the decellularized extracellular matrix can be configured to inhibit the growth of tissue and/or other associated tissue properties.
• the at least one nerve graft-conduit or nerve cap-graft comprises raw material harvested from a tissue source.
• the tissue source can comprise sensory, motor, and/or mixed nerve tissue.
• the tissue source can comprise autonomic nerve tissue.
• the tissue source can be selected and harvested from a specific animal species of a specific age, sex, and/or weight.
• the tissue source can be harvested from a Landrace, Landrace X, or Yorkshire pig.
• the at least one nerve graft-conduit or nerve cap-graft is designed and/or manufactured with one, two, or more structural and functional qualities intended to match or mismatch the structural and functional qualities of a nerve site in the patient.
• the at least one nerve graft-conduit or nerve cap-graft comprise different degrees of decellularization.
• the at least one nerve graft-conduit or nerve cap-graft is configured regenerate nerve tissue following an injury.
• the at least one nerve graft-conduit or nerve cap-graft is configured inhibit nerve growth following an injury.
• the at least one nerve graft-conduit or nerve cap-graft comprises a degradation rate in vivo of between 24 hours and 6 months.
• the nerve cap-graft is configured to be at least partially placed over one, two, or more nerve endings.
• the nerve graft-conduit is constructed and arranged as a nerve connector configured to align and/or connect two or more nerve endings.
• the nerve graft-conduit is constructed and arranged to at least partially replace and/or supplement one, two, or more nerves.
• the at least one nerve graft-conduit or nerve cap-graft is configured to exhibit one, two, or more cell adhesion properties selected from the group consisting of: integrins; laminin; immunoglobulins; cadherins; selectins; and combinations thereof.
• the nerve graft-conduit comprises a lumen surrounded by a luminal wall and a conduit wall.
• the conduit wall can comprise one or more design variables selected from the group consisting of: porosity; pore size; pore interconnectedness; pore alignment; degradation rate; swell ratio; and combinations thereof.
• a method for producing a device comprising a nerve segment comprises: harvesting and/or preparing a nerve segment; decellularizing the harvested nerve segment; providing external support to the decellularized nerve segment; lyophilizing the externally supported nerve segment; creating one, two, or more desired features through the nerve segment; decellularizing and/or performing other chemical, physical, and/or mechanical treatments to the lyophilized nerve segment; stabilizing the treated nerve segment; packaging the stabilized nerve segment within a container; sterilizing the container comprising the packaged nerve segment; and shipping and/or storing the container comprising the packaged nerve segment.
• a method for obtaining an ideal degree of decellularization of a nerve segment comprises: defining one, two, or more features of a desired nerve segment; selecting a nerve segment having one, two, or more the desired features and/or beginning at a known distance from a branching point; defining a desired degree of tissue processing and/or decellularization of the nerve segment; setting one, two, or more tissue processing and/or decellularization parameters; analyzing the degree of tissue processing and/or decellularization of the nerve segment during and/or following the tissue processing and/or decellularization.
• a method for treating a patient comprises: deploying a device comprising at least one of a nerve graft- conduit or nerve cap-graft at a deposit site in the patient, wherein the device is configured to provide a therapeutic benefit at a treatment site.
• FIG. 1 illustrates a schematic view of a system for producing and deploying a medical device comprising an extracellular matrix, consistent with the present inventive concepts.
• Fig. 2 illustrates a perspective view of a medical device comprising a nerve graftconduit or nerve cap-graft, consistent with the present inventive concepts.
• Fig. 3 illustrates a cutaway side view of a nerve graft-conduit or nerve cap-graft graft, consistent with the present inventive concepts.
• FIGs. 4A-E illustrate cutaway side views of a nerve graft-conduit or nerve capgraft graft implanted at a nerve injury site, consistent with the present inventive concepts.
• FIGs. 5A-C illustrate perspective views of a nerve graft-conduit or nerve cap-graft graft, connecting a nerve ending with muscle tissue, consistent with the present inventive concepts.
• Fig. 6 illustrates a method for producing a nerve graft-conduit or nerve cap-graft graft from tissue, consistent with the present inventive concepts.
• Fig. 7 illustrates a method for harvesting and/or preparing nerve tissue for further manipulation, consistent with the present inventive concepts.
• FIGs. 8 and 8A illustrate methods for decellularizing a nerve segment, consistent with the present inventive concepts.
• Fig. 9 illustrates a method for inserting a nerve segment into a support assembly, consistent with the present inventive concepts.
• Fig. 10 illustrates a method for lyophilizing a nerve segment contained within a support assembly, consistent with the present inventive concepts.
• Fig. 11 illustrates a method for creating channels through a nerve segment, consistent with the present inventive concepts.
• Fig. 12 illustrates a method for decellularizing a nerve graft-conduit or nerve capgraft is illustrated, consistent with the present inventive concepts.
• Fig. 13 illustrates a method for stabilizing a nerve graft-conduit or nerve cap-graft, consistent with the present inventive concepts.
• Fig. 14 illustrates a method for packaging a nerve graft-conduit or nerve cap-graft, consistent with the present inventive concepts.
• FIG. 15 illustrates a method for an irradiation based sterilization of a container comprising a nerve graft-conduit or nerve cap-graft consistent with the present inventive concepts.
• FIG. 16 illustrates a method for shipping and/or storing a container comprising a nerve graft-conduit or nerve cap-graft, consistent with the present inventive concepts.
• Fig. 17 illustrates a method for deploying a device comprising a nerve graftconduit or nerve cap-graft consistent with the present inventive concepts.
• Fig. 18 illustrates a perspective view of a sizing tool, consistent with the present inventive concepts.
• Fig. 19 illustrates a graphical representation of the rehydration of a nerve graftconduit or nerve cap-graft, consistent with the present inventive concepts.
• Figs. 20A and B illustrate photographs of unprocessed and processed nerve tissue, respectively, consistent with the present inventive concepts.
• FIGs. 21A and B illustrate photographs of unprocessed and processed nerve tissue, respectively, consistent with the present inventive concepts.
• Figs. 22A and B illustrate photographs of an untrimmed nerve branch system and trimmed nerve segments, consistent with the present inventive concepts.
• FIGs. 23A and B illustrate photographs of a nerve segment prepared for insertion into a nerve jacket, consistent with the present inventive concepts.
• FIGs. 24A and B illustrate photographs of a nerve segment inserted into a nerve jacket, consistent with the present inventive concepts.
• FIGs. 25A and B illustrate photographs of a nerve graft-conduit or nerve cap-graft implanted at a nerve injury site, consistent with the present inventive concepts.
• Fig. 26 illustrates a photograph of an alignment assembly positioned below a material removal assembly, consistent with the present inventive concepts.
• Fig. 27 illustrates a schematic view of container to hold, transport, and/or treat one or more nerve segments, consistent with the present inventive concepts.
• Fig. 28 illustrates a perspective view of a support assembly for manufacturing a nerve graft-conduit or nerve cap-graft, consistent with the present inventive concepts.
• Figs. 29A-E illustrate histological images of native nerve tissue versus decellularized nerve tissue and assay results for the decellularized nerve tissue, consistent with the present inventive concepts.
• Figs. 30A-F illustrate a generalized nerve jacketing process, consistent with the present inventive concepts.
• Figs. 31A-D illustrate scanning electron microscopy (SEM) images for morphological assessment of nerve graft-conduits following lyophilization in tubing with various porosities, consistent with the present inventive concepts.
• FIGs. 32A-C illustrate photographs of a nerve graft-conduit lumen creation, or nerve cap-graft socket creation consistent with the present inventive concepts.
• Figs. 33A-G illustrate in-vitro and histological images of a nerve graft-conduit, consistent with the present inventive concepts.
• Figs. 34A and B illustrate photographs of a nerve graft-conduit and a nerve capgraft following implantation at a sciatic nerve injury, respectively, consistent with the present inventive concepts.
• FIGs. 35A-H illustrate histological images of a neuroma control, a nerve cap-graft at 8 weeks post-injury in a rodent model, consistent with the present inventive concepts.
• Figs. 36A-T illustrate results demonstrating nerve tissue maintaining multiple structure and functional components following decellularization, consistent with the present inventive concepts.
• Figs. 37A and B illustrate a photograph and graphical representation of a gel derived from porcine sciatic nerve promoting Schwann cell (SC) proliferation and axon regrowth at the site of a nerve injury, respectively, consistent with the present inventive concepts.
• SC Schwann cell
• Fig. 38 illustrates a graphical representation of a nerve graft-conduit comprising a peripheral nerve matrix, consistent with the present inventive concepts.
• Fig. 39 illustrates a graphical representation of a nerve cap-graft comprising a peripheral nerve matrix, consistent with the present inventive concepts.
• Fig. 40 illustrates a table containing feature development parameters of a nerve cap-graft, consistent with the present inventive concepts.
• Fig. 41 illustrates a table containing feature development parameters of a nerve graft-conduit, consistent with the present inventive concepts.
• Fig. 42 illustrates a table containing nerve graft-conduit or nerve cap-graft feature creation strategies, consistent with the present inventive concepts.
• Fig. 43 illustrates a table containing nerve graft-conduit or nerve cap-graft matrix morphology control strategies, consistent with the present inventive concepts.
• Fig. 44 illustrates a table containing nerve graft-conduit or nerve cap-graft clinical requirements, consistent with the present inventive concepts.
• Figs. 45 through 53 illustrate various graphical representations, photographs, histological images, and graphs regarding the development of an acellular nerve cap-graft xenograft for neuroma prevention.
• Figs. 54A-D illustrate various theories of operation, framework, and timeline for both nerve graft-conduit or nerve cap-graft, consistent with the present inventive concepts.
• Fig. 55 illustrates a method for obtaining an ideal degree of decellularization in order to obtain desired structure and function in a nerve graft-conduit or nerve cap-graft, consistent with the present inventive concepts.
• first element 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.
• 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.
• a component positioned proximate an anatomical site e.g. a target tissue location
• 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.
• 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.
• 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.
• a quantifiable parameter when 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.
• 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.
• 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.
• the expression “a device configured to” may mean that the device “can” operate together with another device or component.
• threshold refers to a maximum level, a minimum level, and/or range of values correlating to a desired or undesired state.
• 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).
• a system parameter is maintained above a first threshold (e.g.
• a threshold value is determined to include a safety margin, such as to account for patient variability, system variability, tolerances, and the like.
• “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.
• 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.
• 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.
• a 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.
• 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).
• a functional element e.g. a functional element comprising a sensor
• a sensor or other functional element is configured to perform a diagnostic function (e.g.
• a functional element is configured to perform a therapeutic function (e.g. to deliver therapeutic energy and/or a therapeutic agent).
• 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.
• 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.
• 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.
• System 10 comprises medical device 100 shown, as well as various components used to manufacture, package, sterilize, and/or deploy device 100.
• Device 100 is configured to be deployed (e.g. injected, inserted, delivered, implanted, and the like) at one, two, or more “deposit sites”, such as to provide a therapeutic benefit at one, two, or more “treatment sites”.
• Each treatment site can comprise a location that is proximate to and/or remote from the associated deposit site.
• a treatment site comprises a location that is relatively the same location as the associated deposit site.
• Device 100 can be deployed at the deposit site to promote, and/or otherwise support, tissue growth of a patient (e.g. support tissue growth and/or regeneration at locations proximate and/or remote from the deposit site). In some embodiments, device 100 is remodeled over time into native tissue of the patient. In some embodiments, device 100 is deployed at the deposit site to inhibit growth of patient tissue to prevent one, two, or more conditions derived from such tissue growth. As used herein, the deposit site can comprise one, two, or more locations on and/or within the patient.
• Device 100 comprises a decellularized extracellular matrix, ECM 120 shown.
• ECM 120 can comprise structural and non-structural biomolecules, including, but not limited to, collagens, elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractant, cytokines, matrix bound vesicles, and endogenous (i.e. naturally present in ECM 120) and/or exogenous (i.e. added to one, two, or more components of the device 100) growth factors.
• ECM 120 can be configured to promote and/or sustain the growth of tissue and/or associated tissue properties (e.g. structural proteins, growth factors, etc.) proximate to and/or remote from the deposit site.
• ECM 120 can be configured to inhibit the growth of tissue and/or other associated tissue properties (e.g. structural proteins, growth factors, etc.) proximate to and/or remote from the deposit site.
• ECM 120 can be derived, or otherwise produced, from one, two, or more raw material 65 as described herein.
• ECM 120 is derived from a raw material as described in applicant’s copending International PCT Patent Application Serial Number PCT/US2020/053570, entitled “Extracellular Matrix Devices and Methods of Manufacture”, filed September 30, 2020.
• ECM 120 can comprise a concentration of native protein between 2mg/mL and 50mg/mL, such as a concentration between 5mg/mL and 20mg/mL, such as a concentration of approximately 10 mg/mL.
• the protein concentration can be configured to improve a parameter of ECM 120, such as to improve solubility, reconstitution, solution viscosity, solution injectability, solution mixability, gelation kinetics, working time, and gel mechanical/structural properties, such as storage modulus, stiffness, suture retention strength, micro structure, porosity, pore size distribution, liquid or gas permeability, cell penetration, cell phenotype, gene expression, protein expression, degradation rate, and/or durability.
• Device 100 can further comprise a neutralizing element 140 and/or a reconstituting element 160, each configured to interact (e.g. physically, chemically interact) with ECM 120.
• neutralizing element 140 and/or reconstituting element 160 interact with ECM 120 to cause a physical, mechanical, and/or chemical change to ECM 120 and/or other component of system 10.
• Neutralizing element 140 can be configured to counteract, or otherwise offset, a property (e.g. physical, mechanical, chemical property) of ECM 120, reconstituting element 160, and/or other component of system 10.
• Neutralizing element 140 can comprise an element selected from the group consisting of: water; buffer solutions, such as phosphate buffer solution (PBS), buffer amino acids (e.g.
• Neutralizing element 140 can be configured to extend its ability to function as a neutralizing element over time (i.e. neutralizing element 140 shelf life). In some embodiments, the alteration of the buffer concentration of neutralizing element 140 prevents the natural pH drift over time of the solution due to the interaction with environmental CO2.
• ECM 120 comprises a fluid and neutralizing element 140 comprises a concentration of PBS that is configured to modify (e.g. increase, decrease) the mechanical strength of ECM 120, modify (e.g. increase, decrease) a gelation time of ECM 120, modify (e.g.
• neutralizing element 140 comprises a buffer solution in water with the components and ranges of concentration shown hereinbelow in Table 1 :
• Reconstituting element 160 can be configured to change, or otherwise modify, a property (e.g. physical, chemical, mechanical, biological, and/or shelf-life property) of ECM 120, neutralizing element 140, and/or other component of system 10.
• a property e.g. physical, chemical, mechanical, biological, and/or shelf-life property
• neutralizing element 140 comprising PBS can affect physical, chemical, mechanical, and/or biological properties of ECM 120.
• Reconstituting element 160 can comprise water.
• reconstituting element 160 and neutralizing element 140 are combined to comprise a single reconstituting and neutralizing solution.
• Raw material 65 can comprise sensory, motor, and/or mixed nerve tissue.
• raw material 65 comprises autonomic nerve tissue.
• raw material 65 comprises spinal cord nerve tissue.
• raw material 65 comprises ventral and/or dorsal root ganglion.
• raw material 65 comprises somatic nerve tissue.
• raw material 65 comprises sciatic nerve tissue, such as bilateral sciatic nerves.
• raw material 65 comprises brachial plexus tissue. Tissue harvested from multiple (e.g. two, three, or more) nerve types can be pooled to provide a larger quantity and/or heterogenous raw material 65.
• raw material 65 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 device 100.
• this specific anatomic location includes a specific nerve branch, as well as a specific position within a specific nerve branch (e.g. a position at a defined distance from a prior and/or neighboring nerve branch).
• Desired features of device 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 fascicles (e.g.
• fascicles 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 thickness, and/or axonal density, such as can be as measured by the total axonal area divided by the total nerve cross-sectional area
• axons e.g. average axonal cross-sectional area, average myelin thickness, and/or axonal density, such as can be as measured by the total axonal area divided by the total nerve cross-sectional area
• device 100 is designed and/or manufactured with one, two, or more structural and functional qualities intended to match or mismatch the structural and functional qualities of the nerve site at the deposit site.
• device 100 can be intentionally designed and/or manufactured to have a similar size and/or number of fascicles as the nerve at the deposit site, in order to support nerve regeneration.
• device 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.
• a convergence e.g. funneling excessive axonal growth into an insufficient distal space
• a divergence e.g. excessive splicing of axonal growth into multiple distal recipients
• device 100 comprises nerve tissue segments that have different degrees of decellularization depending on the radial depth.
• nerve segments are submerged in a detergent solution.
• the radial distance from the edge of the nerve can define the mass transport of detergent solution into each specific area and the mass transport of cellular content out of the area.
• the outer segments of the nerve can be exposed to more detergent solution and are subsequently more decellularized than the inner segments, resulting in a consistent gradient of cellular content (e.g. myelin) along the radial depth into the nerve segment, with the highest level of cellular content proximate the center of the nerve segment and the lowest level of cellular content proximate the outer edge of the nerve segment.
• cellular content e.g. myelin
• Raw material 65 can comprise tissue harvested from a tissue source 60 selected from the group consisting of: a mammal, such as pig, human, cow, horse, and the like; an amphibian, such as salamander (e.g. an axolotl) 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.
• raw material 65 comprises tissue harvested from a Landrace, Landrace X, or Yorkshire pig.
• raw material 65 can comprise sciatic nerve tissue, such as sciatic nerve tissue harvested from a tissue source 60 comprising a pig (e.g. a male pig), such as a pig with a weight between libs and 4001bs, such as a weight between 501bs and 3001bs, such as a weight of approximately 250 lbs.
• a pig e.g. a male pig
• a weight between libs and 4001bs such as a weight between 501bs and 3001bs, such as a weight of approximately 250 lbs.
• Raw material 65 can comprise tissue harvested from one, two, or more similar and/or dissimilar tissue sources 60. Tissue harvested from multiple (e.g. two or more) tissue sources 60 can be pooled to provide a larger quantity of homogeneous raw material 65.
• Raw material 65 can comprise tissue harvested from a uniform sex, such as tissue harvested from all male tissue sources 60 or all female tissue sources 60. Tissue harvested from a uniform sex can increase tissue consistency.
• Raw material 65 can comprise tissue harvested from both male and female tissue sources 60.
• Raw material 65 can comprise tissue harvested from an adult and/or juvenile tissue source 60, such as tissue harvested from all adult tissue sources, all juvenile tissue sources, or both adult and juvenile tissue sources.
• Raw material 65 can comprise tissue harvested from a genetically uniform tissue source 60 (e.g.
• raw material 65 can comprise tissue harvested from a genetically modified tissue source 60.
• raw material 65 can comprise tissue harvested from an a 1,3 -galactosyltransferase knockout pig .
• Raw material 65 can comprise tissue harvested from one, two, or more tissue sources 60 that provide an increased potency and/or altered mechanical, physical, and/or chemical characteristics of raw material 65.
• Raw material 65 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.
• Raw material 65 can comprise tissue harvested from one, two, or more tissue sources 60 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 65.
• raw material 65 is cross-linked to alter its degradation rate and/or orient its microstructure.
• Raw material 65 can comprise tissue harvested from one, two, or more tissue sources 60 comprising an animal that adhered to a pre-determined diet, exercise, chemical, pharmacological, physical, psychological stimulation, and/or other regimen.
• the predetermined regimen can be configured to modify the anatomical and/or physiological characteristics of tissue source 60.
• 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.
• 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.
• Device 100 can be constructed and arranged to be used in the field of nerve injury and repair including applications in which nerve regeneration is desirable (e.g. nerve transection, nerve gap injury, nerve transfer, etc.) and/or applications in which nerve growth inhibition following nerve injury is desirable (e.g. nerve/limb amputation, prevention of painful neuroma, prevention of other neuromas, etc.).
• applications in which nerve regeneration is desirable e.g. nerve transection, nerve gap injury, nerve transfer, etc.
• nerve growth inhibition following nerve injury e.g. nerve/limb amputation, prevention of painful neuroma, prevention of other neuromas, etc.
• Device 100 can be constructed and arranged to be used in a therapeutic, diagnostic, and/or other clinical applications in one or more medical fields, such as dentistry (e.g. endodontics, orthodontics), dermatology, ophthalmology, obstetrics, gynecology, cardiology and cardiac electrophysiology, gastroenterology, orthopedic, oncology, neurology, neurosurgery, endocrinology, lymphology, surgery (e.g. plastic aesthetic, plastic reconstruction, otolaryngology, and oral and maxillofacial surgery), and the like.
• device 100 is constructed and arranged as a bulking agent for administration during a surgical procedure (e.g. a plastic reconstruction, aesthetic procedure).
• device 100 is constructed and arranged as an embolic, clotting, and/or obstructive agent for administration during a surgical, minimally invasive, and/or percutaneous procedure.
• Device 100 can be constructed and arranged for treatment of male and female sexual dysfunction or for sexual enhancement applications, such as male erectile dysfunction, retarded or premature ejaculation, lack of sensation, penile enlargement, and female genital cosmetic, reconstructive, and enhancement surgery.
• sexual enhancement applications such as male erectile dysfunction, retarded or premature ejaculation, lack of sensation, penile enlargement, and female genital cosmetic, reconstructive, and enhancement surgery.
• Device 100 can be constructed and arranged for a therapeutic and/or clinical application in a veterinary field, such as large animal surgery, small animal surgery, farm animal surgery, competitive animal conditioning, farm animal conditioning, and military and training animal conditioning.
• Device 100 can be constructed and arranged for a cosmetic application, such as a tissue bulking agent, restoration of facial animation or improvement of facial expressivity.
• Device 100 can comprise a topical formulation configured for the treatment of skin conditions.
• Device 100 can be constructed and arranged for a food supplement application, such as to improve skin health, improve hair health, improve nail health, relieve joint pain and increase motility, prevent bone loss, improve heart health, increase muscle mass, and increase physical performance.
• Device 100 comprising ECM 120, can comprise a configuration selected from the group consisting of: a fluid and/or semi-fluid (either or both, “fluid” herein), such as a hydrogel, cream, ointment, or the like; a spongy material; a compressed material, such as a film; a solid material, such as a wrap, nerve conduit, nerve graft, nerve cap, suture, or the like; an aerosolized material, such as a spray; a flowable particulate, such as a micronized and flowable particulate; a powder; a fibrous material; and combinations of these.
• device 100 is configured to deliver one, two, or more therapeutic agents (e.g. agent 70 described herein) to the patient (e.g. pharmaceutical drugs, stem cell therapies, etc.), such was when device 100 further comprises a plurality of microspheres comprising a therapeutic agent.
• a fluid and/or semi-fluid such as a hydrogel, cream, oin
• Device 100 can comprise a mechanical strength and/or degradation rate/durability that is modified via at least one of chemical cross-linking or physical cross-linking.
• Device 100 can comprise a degradation rate in vivo of between 24 hours and 6 months, such as a degradation rate in vivo of between 2 weeks and 2 months, such as a degradation rate in vivo of approximately 4 weeks.
• device 100 comprises a fluid comprising a dynamic viscosity between 20cP and 200cP.
• Device 100 can comprise a lower viscosity for injectable applications, such as a viscosity of between IcP and lOOcP.
• Device 100 can comprise a greater viscosity for topical applications, such as a viscosity of between lOOOcP and 45,000cP.
• device 100 comprises a fluid having an osmolarity of between 100 and 365mOsmol/L, such as an osmolarity of between 200 and 280mOsmol/L.
• the osmolarity can be adjusted to modify (e.g. increase, decrease) the mechanical properties, gelation kinetics, working time, and/or responsiveness to temperature of device 100.
• device 100 comprises a fluid that transitions to a gel.
• Device 100 can be configured to transition from a fluid to a gel via self-assembly, fibrillogenesis, chemical cross-linking, physical cross-linking, and combinations of these.
• Device 100 can transition to a gel prior to its deployment at the deposit site, such that device 100 transitions to a gel ex vivo.
• device 100 is configured to transition to a gel at a time between 0.5 minutes and 2 hours prior to deployment of device 100 at the deposit site.
• Device 100 can transition to a gel during and/or after its deployment at the deposit site, such as transition to a gel in situ.
• device 100 is configured to transition to a gel at a time between 0.5 minutes and 10 minutes within the time of deployment of device 100 at the deposit site.
• Device 100 can comprise a volume that decreases as it transitions from a fluid to a gel.
• device 100 can contract over a period of time as it transitions from a fluid to a gel.
• device 100 further comprises a plurality of expansion elements configured to compensate for the decrease in volume. The expansion elements can be configured to maintain a relatively constant volume of device 100, such that the expansion elements correspondingly expand as device 100 contracts.
• device 100 comprises a semi-fluid and/or solid that is molded, or otherwise manipulated, into a geometric shape prior to, during, and/or after deployment at the deposit site.
• device 100 is constructed and arranged as a coating configured to at least partially cover one, two, or more surfaces of the deposit site.
• Device 100 can be configured to coat a surface of the deposit site via an atomization process, such as an atomization process performed using tool 80.
• Device 100 can be configured to coat a surface of the deposit site via a brushing process, such as a brushing process performed using tool 80.
• Device 100 can be configured to coat a surface of the deposit site via a dipping process, such as a dipping process performed using tool 80.
• device 100 is constructed and arranged as a “nerve capgraft” configured to be at least partially placed over one, two, or more nerve endings, such as peripheral nerve matrix cap, PNM-CAP 2200, described herein.
• the nerve cap-graft comprises a cylindrical device open at one end and closed or open at the opposite end, and into which a nerve end can be inserted and secured.
• device 100 can be placed over one or more nerve ending/s following an amputation.
• device 100 can be configured to embed a severed nerve directly into a muscle body to promote reinnervation of the muscle.
• device 100 is constructed and arranged as a nerve connector configured to align and/or connect two or more nerves (e.g. end-to-end; end-to- side, side-to-side nerve connections, etc.) at a treatment site.
• device 100 can connect two or more nerve stumps and promote nerve regrowth between the nerve stumps.
• device 100 is constructed and arranged as a nerve conduit, graft (e.g. allograft, autograft, xenograft), or a combination of a graft and a conduit (e.g. graft-conduit herein), and can be configured to at least partially replace and/or supplement one, two, or more nerves at a treatment site.
• Device 100 can comprise one or more internal longitudinal tunnels, these can be intertwining tunnels constructed and arranged to mimic the natural plexus structure of a peripheral nerve.
• device 100 is constructed and arranged to fill the lumen of a conduit, graft, graft-conduit combination (“nerve graft-conduit” herein, and such as implant 20, and/or PNM-G 2100 described herein), or cap-graft (such as PNM-CAP 2200 described herein).
• conduit, graft, or graft-conduit can comprise polymeric materials, such as polycaprolactone, PLLA, PGA, silicone, polyurethane, PET, or PTFE.
• such conduit or graft can comprise naturally derived materials such as collagen, elastin, GAG, keratin, chitosan, and/or combinations of synthetic and naturally derived materials.
• device 100 is comprised of a segment of decellularized nerve, with or without modification to create one or more internal longitudinally oriented tunnels.
• device 100 is constructed and arranged as a suture material, such as a nerve suture material.
• ECM 120 can be extruded or drawn using textile manufacturing techniques to fabricate a biodegradable mono- or multi- stranded filament configured to connect or repair nerve tissues.
• Device 100 can be constructed and arranged as a suture material comprising a straight strength and a knot strength, such that the knot strength is between 70% and 80% of the straight strength.
• Device 100 can be constructed and arranged as a suture comprising a size between 6-0 and 10-0.
• Device 100 can be constructed and arranged as a suture comprising a tensile strength greater than 0.5N, such as greater than IN, such as greater than 2N.
• device 100 is constructed and arranged as an electrode coating.
• Device 100 can be configured to reduce inflammation, infection, scarring, and/or facilitate nerve growth and/or integration at the tissue-electrode interface of an implantable device. Growth and/or integration of nerve tissue at the tissue-electrode interface can reduce impedance and improve conductivity, such as to improve signal to noise reductions. Reduction of inflammation and/or scarring can prolong the useful life of the electrode, lead, or sensor.
• device 100 is configured to enhance the performance of a nerve stimulation device.
• device 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, and/or tissue development at a treatment site.
• Device 100 can be incorporated into (e.g. embedded in, combined with, used in conjunction with, and the like) an existing medical device and/or material. In some embodiments, device 100 is incorporated into a patch and/or film. In some embodiments, device 100 is incorporated into suture material. In some embodiments, device 100 is incorporated into an adhesive, such as fibrin glue.
• Device 100 can be incorporated into a nerve graft-conduit or nerve cap-graft, such as implant 20 as described herein in reference to Figs. 2, or such as PNM-G 2100 as described herein in reference to Fig. 3.
• device 100 is incorporated into the nerve conduit prior to implantation into a patient, such as during a manufacturing process of the nerve conduit and/or ECM 120.
• ECM 120 comprising neutralizing element 140 can be injected into the lumen of the nerve conduit.
• the nerve conduit can be submerged in a bath of ECM 120 comprising neutralizing element 140.
• the nerve conduit can be spray coated with ECM 120 comprising neutralizing element 140.
• the nerve conduit comprising ECM 120 and neutralizing element 140 can be dehydrated via vacuum drying or lyophilization.
• the dehydrated nerve conduit Prior to implantation, the dehydrated nerve conduit can be rehydrated upon immersion into a saline solution.
• ECM 120 comprising a lyophilized cake can be press-fit into the nerve conduit, such as when ECM 120 comprises a micronized lyophilized cake.
• device 100 can be injected via a needle into the lumen of the nerve conduit and allowed to gel at a temperature of approximately 37°C prior to implantation into the patient.
• the nerve conduit can be impregnated with device 100 via a vacuum source.
• the nerve conduit can be held within a channel and the vacuum source can be applied at one end of the conduit while device 100 is injected at an opposite end.
• Device 100 can be configured to permeate (e.g. transmural permeation) through the nerve conduit.
• the nerve conduit can be impregnated with device 100 via submersion into a bath comprising device 100.
• the nerve conduit can be impregnated with device 100 via a spray coating comprising device 100.
• the nerve conduit can be inserted into a container comprising device 100 and centrifuged or otherwise agitated within the container.
• the nerve conduit can be impregnated with device 100 via immersion into a fluid flow comprising device 100.
• the nerve conduit can be printed with device 100.
• device 100 is incorporated into a conduit or into a nerve via percutaneous or minimally invasive methods.
• device 100 can be injected via a needle under ultrasound or fluoroscopy guidance into the implanted nerve conduit, such as injected into the lumen of the nerve or conduit via an end and/or through a side wall of the nerve or conduit.
• Device 100 can be delivered, injected, implanted, and/or otherwise deployed (“deployed” herein) proximate a treatment site.
• Device 100 can be deployed into, onto, and/or at the deposit site, such as a focal area of a treatment site.
• Device 100 can be deployed to extend to, or otherwise cover, one, two, or more locations beyond the deposit site (e.g. into locations of the treatment site or other locations). Device 100 can be deployed to extend longitudinally beyond the deposit site. In some embodiments, device 100 extends proximally from the deposit site, such as between 2mm and 20mm proximally from the deposit site, such as between 2mm and 5mm, such as between 5mm and 10mm, such as between 10mm and 20mm. In some embodiments, device 100 extends distally from the deposit site, such as between 2mm and 20mm distally from the deposit site, such as between 2mm and 5mm, such as between 5mm and 10mm, such as between 10mm and 20mm.
• Device 100 can be deployed at one, two, or more deposit sites positioned about the circumference of a nerve within and/or externally to the nerve. Two or more deposit sites can comprise a uniform spacing about the circumference of the nerve. The two or more deposit sites can comprise a non-uniform spacing about the circumference of the nerve. For example, device 100 can be deployed at a first deposit site representing 0°, at a second deposit site that is 120° relative to the first deposit site, and at a third deposit site that is 240° relative to the first deposit site (and 120° relative to the second deposit site).
• Device 100 can be deployed at one, two, or more deposit sites about the circumference of a nerve and can be further deployed at one, two, or more locations beyond the deposit sites, as described herein. In some embodiments, deployment of device 100 at the deposit sites and locations beyond the deposit sites comprise a matrix of device 100 along the external surface of the nerve.
• the deposit site comprises a location (e.g. one, two, three, or more locations) within the central nervous system, such as a site located within and/or surrounding the brain and/or spinal cord.
• the deposit site comprises a location within the peripheral nervous system, such as a site located outside the brain and spinal cord, including any location along and/or around the peripheral nervous system spanning from the dorsal and/or ventral root ganglia to motor, sensory, or autonomic endings (e.g. end-muscle plate, Pacinian corpuscle, Ruffini endings).
• the deposit site comprises a location within, and/or around, and/or proximate an uninjured nerve.
• the deposit site comprises a location within, and/or around, and/or proximate a diseased nerve.
• the deposit site comprises a location within, and/or around, and/or proximate a nerve injury, such as an intra-nerve and/or peri-nerve injury location.
• the deposit site comprises a location within, and/or around, and/or proximate a partial or full nerve transection, such as a transected and repaired nerve (e.g. epineural and/or fascicular repair, such as neurorrhaphy).
• the deposit site comprises the location of a nerve amputation (for example, following a limb amputation with which two or more nerves are left amputated and each of such nerve stumps represents a potential deposit site).
• device 100 can be deployed to provide an interface between two or more nerves, nerve stumps, or nerve epineural windows.
• the two or more nerve stumps can be coapted together, such as via a suture or fibrin glue configured to eliminate or otherwise reduce a gap length between the nerve stumps.
• the two or more nerve stumps are not coapted together and a calculated gap length is maintained between the nerve stumps.
• the calculated gap length can be configured to promote nerve cone sprouting and alignment from a proximal nerve stump having a greater degree of freedom to properly align toward a distal nerve stump.
• the deposit site comprises a location within, around, and/or proximate a nerve injury repaired with a nerve transfer technique, such as an end-to-end transfer, side-to-side transfer, end-to-side transfer, or supercharged end-to-side transfer.
• the deposit site comprises a location within, around, and/or proximate a nerve crush injury, such as an acutely crushed nerve.
• the deposit site comprises a location within, around, and/or proximate a nerve stretch injury, such as an acutely stretched nerve.
• the deposit site comprises a location within, around, and/or proximate a compression nerve injury, such as chronic compression with or without prior surgical release.
• the deposit site comprises a location within, around, and/or proximate a nerve gap injury repaired with a nerve conduit or graft, such as an allograft, autograft, conduit, or guide.
• the deposit site comprises a location within, around, and/or proximate a targeted muscle reinnervation.
• device 100 can be deployed to promote total muscle reinnervation.
• the deposit site comprises a location within, around, and/or proximate a neurotization.
• device 100 can be deployed to promote direct muscle neurotization, such that device 100 can be deployed within, around, and/or proximate a nerve apposed into and/or onto muscle, such as to promote nerve sprouting into the muscle.
• device 100 can be deployed to promote combined muscle neurotization, such that device 100 can be deployed within, around, and/or proximate a nerve apposed into and/or onto muscle via a nerve conduit, such as to promote nerve sprouting into the muscle.
• the deposit site comprises a location within, around, and/or proximate a nerve end surgically connected to muscle and configured to reactivate the muscle, such as proximate an end-to-end nerve transfer or Oberlin procedure.
• the deposit site comprises a location within, around, and/or proximate a nerve attached to isolated patches of muscles, such as directly via direct neurotization or via existing innervation (e.g. regenerative peripheral nerve interface).
• the deposit site comprises a location within, around, and/or proximate a nerve end surgically connected to muscle and configured to create an electrical activation map on the muscle surface, such as proximate a total muscle reinnervation.
• the electrical activation map can be detected by skin electrodes configured to control electronic prostheses.
• the deposit site comprises a location within, around, and/or proximate a nerve biopsy.
• the deposit site comprises a location within, around, and/or proximate an iatrogenic (e.g. medically, surgically induced) nerve injury. In some embodiments, the deposit site comprises a location within, around, and/or proximate a brachial plexus injury. For example, device 100 can be deployed during an Oberlin or Leechavengvongs procedure. In some embodiments, the deposit site comprises a location within, around, and/or proximate a peripheral nerve injury in which there is no nerve gap. In some embodiments, the deposit site comprises a location within and/or proximate a peripheral nerve injury in which a gap closure is achieved with or without flexion of an extremity.
• an iatrogenic nerve injury e.g. medically, surgically induced
• the deposit site comprises a location within, around, and/or proximate a brachial plexus injury.
• device 100 can be deployed during an Oberlin or Leechavengvongs procedure.
• the deposit site comprises
• the deposit site comprises a location within, around, and/or proximate a tissue bum, such as a superficial bum, a partial thickness burn, and/or a full thickness bum. In some embodiments, the deposit site comprises a location within, around, and/or proximate a truncated nerve following an amputation. For example, device 100 can be deployed at the site of one or more nerves affected by an amputation. In some embodiments, the deposit site comprises a location that contributes to a symptom attributed to a migraine. For example, device 100 can be deployed to treat, or otherwise alleviate, migraine symptoms. In some embodiments, the deposit site comprises a location within, around, and/or proximate muscle tissue.
• the deposit site comprises a location within, around, and/or proximate denervated muscle tissue.
• device 100 can be deployed to promote neuromuscular junction formation.
• the deposit site comprises a location within, around, and/or proximate a nerve end surgically connected to a sensory receptor, such that device 100 can be deployed to promote nerve sprouting into the receptor and/or reestablish sensation.
• the deposit site comprises distributed locations via topical applications onto a surgically created cavity, onto a mucosal surface or cavity, into an organ, and/or onto the skin or injured skin surface.
• device 100 is deployed into a deposit site comprising muscle tissue. In some embodiments, device 100 is deployed into a deposit site comprising tendon, ligament, or cartilage tissue. In some embodiments, device 100 is deployed into a deposit site comprising organ tissue. In some embodiments, device 100 is deployed into a deposit site comprising vascular wall tissue. In some embodiments, device 100 is deployed into a deposit site comprising lymphatic tissue. In some embodiments, device 100 is deployed into a deposit site comprising neural tissue. In some embodiments, device 100 is deployed into and/or through skin tissue (e.g. epidermis, dermis, subcutaneous) proximate the deposit site. In some embodiments, device 100 is deployed topically onto the skin, organ, and/or surgical cavity. In some embodiments, device 100 is deployed into or onto the deposit site percutaneously and/or minimally invasively.
• skin tissue e.g. epidermis, dermis, subcutaneous
• device 100 is deployed into a deposit site comprising oral tissue (e.g. oral mucosa, teeth, tooth pulp, cranial nerve, tongue).
• oral tissue e.g. oral mucosa, teeth, tooth pulp, cranial nerve, tongue.
• device 100 is deployed into the tooth root following a root canal or pulpectomy procedure.
• device 100 is deployed into and/or around the cranial nerves.
• device 100 is deployed into the oral mucosa.
• device 100 is deployed into the tongue or lips.
• device 100 is deployed into a deposit site comprising heart nervous system including both the heart conduction system (e.g. sino-atrial node, atrioventricular node, Purkinje fibers, etc.) and the heart nervous system tissue from the lower cervical to the upper thoracic ganglia (e.g. cardiac plexus, parasympathetic and sympathetic nerve fibers, vagal cardiac nerve, vagus nerve, etc.).
• heart conduction system e.g. sino-atrial node, atrioventricular node, Purkinje fibers, etc.
• the heart nervous system tissue from the lower cervical to the upper thoracic ganglia e.g. cardiac plexus, parasympathetic and sympathetic nerve fibers, vagal cardiac nerve, vagus nerve, etc.
• the treatment site comprises at least a portion of a nerve and device 100 is deployed at a deposit site comprising at least a portion of a neural location selected from the group consisting of: intra-mesoneurium; peri-mesoneurium; intra- epineurium; peri-epineurium; sub-epineurium; intra-fascicular, sub-endoneurium; and combinations of these.
• device 100 is deployed at a deposit site distal to and/or proximal to the treatment site, such as at an ectopic site.
• Device 100 can be deployed into muscle tissue and/or organ tissue distal to the treatment site, such as to promote nerve growth (e.g. neurotization) towards the treatment site, such as to restore sensation and/or motor control.
• Device 100 can be deployed into a deposit site comprising muscle tissue and/or skin tissue (e.g. epidermis, dermis, subcutaneous) distal to the treatment site, such as to recruit, or otherwise promote the growth of, muscle fibers.
• Device 100 can be deployed contemporaneously (e.g. concurrently) with one, two, or more additional treatments provided to the patient (e.g. one or more treatments deployed at the deposit site, the treatment site, and/or another patient location).
• device 100 is deployed contemporaneously with an electrical stimulation.
• device 100 is deployed contemporaneously with a pharmacological treatment.
• device 100 is deployed contemporaneously with a cellular treatment.
• device 100 is deployed contemporaneously with a structural element (e.g. sutures, conduit, wrap, glue).
• device 100 is deployed contemporaneously with physical, occupational therapy and/or electrical, magnetic, and laser stimulation.
• Device 100 can comprise one or more functional elements, functional element 199 shown.
• Functional element 199 can comprise a sensor and/or a transducer.
• functional element 199 comprises a biofeedback element.
• device 100 can further comprise a biofeedback mechanism (e.g. functional element 199) configured to provide an indication of a biological, mechanical, chemical, or electric signal at the deposit site.
• a biofeedback mechanism e.g. functional element 199
• device 100 further comprises one or more pharmacological or other agents, agent 70 shown.
• Agent 70 can be included in (e.g. integrated into) device 100, or it can be provided separately.
• Agent 70 can comprise an antimicrobial agent.
• Agent 70 can comprise a color additive.
• Agent 70 can comprise an adhesive.
• combination of device 100 with an adhesive is configured to reinforce a suture site and/or as a primary means of structural reconstruction of a nerve.
• Agent 70 can comprise an immunosuppressing agent configured to promote, or otherwise support, nerve regeneration, such as FK506.
• Agent 70 can comprise a chemical agent configured to modify, or otherwise effect, an immune response at the deposit site, such as when agent 70 comprises an immunomodulator (e.g. immunosuppressant).
• an immunomodulator e.g. immunosuppressant
• Agent 70 can comprise an antimicrobial agent configured to kill, or otherwise stop or slow down, the growth of microorganisms at the deposit site, such as when agent 70 comprises one, two, or more agents selected from the group consisting of: disinfectant; antiseptic; antibiotic; and combinations of these.
• Agent 70 can comprise a visual additive, such as a visual additive configured to provide visibility to device 100, such as a dye configured to be visualized via an imaging device 30.
• Agent 70 can comprise a lubricating substance configured to minimize friction at the deposit site.
• Agent 70 can comprise a conductive agent configured to increase electrical conductivity at the deposit site.
• Agent 70 can comprise one or more anti-adhesive agents configured to prevent an adhesion at the deposit site, such as to prevent fibrotic adhesions.
• Agent 70 can comprise an anesthetic and/or pain reliever agent configured to induce an insensitivity at the deposit site (e.g. insensitivity to pain and/or sensation).
• Agent 70 can comprise a hemostatic agent configured to promote hemostasis at the deposit site.
• Agent 70 can comprise an antidote configured to counteract a poison and/or toxin at the deposit site, such as an antidote configured to counteract a nerve agent.
• Agent 70 can comprise an anti-inflammatory agent configured to reduce inflammation at the deposit site.
• Agent 70 can comprise a chemoattractant configured to attract motile cells to the deposit site, such as a motile cell selected from the group consisting of: Schwann cells; macrophages; endothelial cells; progenitor cells; and combinations of these.
• Agent 70 can comprise an agent configured to promote the production of angiogenic factors at the deposit 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 70 can comprise an agent configured to promote cell migration, development, and/or maturation at the deposit site, such as a nerve growth factor.
• angiogenin selected from the group consisting of: angiogenin
• growth factors such as fibroblast growth factors, transforming growth factors
• lipids lipids
• Agent 70 can comprise an agent configured to promote cell migration, development, and/or maturation at the deposit site, such as a nerve growth factor.
• device 100 is configured to exhibit cell adhesion properties configured to interact and/or attach to neighboring cells at the deposit 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.
• device 100 is configured to exhibit cell signaling properties configured to communicate and/or coordinate cell actions at the deposit 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.
• device 100 is configured to exhibit pharmacological and/or biological properties configured to support the local microenvironment at the deposit site, such as to promote immunomodulatory action, revascularization, cell chemotaxis, cell development, nerve tissue deposition, ECM 120 or tissue remodeling, nutrient transfer, and/or waste removal.
• device 100 is configured to increase remodeling, vascularization, and/or innervation of a nerve, nerve graft, or nerve conduit.
• Device 100 can be configured to respond to electrical, mechanical, physical, and/or chemical factors, such as factors internal and/or external to device 100.
• device 100 can be configured to respond to temperature, sound waves, electromagnetic waves, and/or light (e.g. coherent light, such as laser).
• device 100 is configured to undergo a physical change upon the application of an ultraviolet light internally and/or externally to ECM 120, such as when device 100 transitions from a fluid to a semifluid and/or solid.
• device 100 is configured to release an agent (e.g. agent 70, such as a chemical, pharmaceutical, and/or other agent) upon the application of an external vibration to device 100.
• agent 70 e.g. agent 70, such as a chemical, pharmaceutical, and/or other agent
• System 10 can further comprise one or more implants, implant 20 shown.
• Implant 20 can comprise a conduit as described herein in reference to Fig. 2.
• Implant 20 can comprise a graft-conduit (e.g. PNM-G 2100) or cap-graft (e.g. PNM-CAP 2200) as described herein.
• System 10 can further comprise one or more imaging devices, device 30 shown, which can be configured to visualize an object (e.g. device 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 nearinfrared visualization; device that provides thermal imaging; 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.
• System 10 can further comprise one or more coating tools, tool 80 shown, which can be configured to coat a surface (e.g. a surface of a deposit site), such as an atomization tool, a brush or brushing tool, and/or a dipping tool, as described herein.
• Tool 80 can comprise a tattoo machine configured to deliver ECM 120 at a defined depth of a surface.
• Tool 80 can comprise a jet injector configured to deliver ECM 120 via high pressure at a defined depth of a surface.
• Tool 80 can comprise a bobbin including a coiled strand or ribbon embedded with ECM 120 configured to wind and/or canvas around a surface.
• Tool 80 can comprise an adhesive or adhesive strip configured to affix ECM 120 to a surface.
• System 10 can further comprise one or more sizing tools, tool 90 shown, which can be configured to measure one or more parameters of implant 20 (e.g. PNM-G 2100, PNM-CAP).
• Sizing tool 90 can be configured to measure the diameter and/or length of implant 20 (e.g. PNM-G 2100, PNM-CAP 2200) as described hereinbelow in reference to Fig. 18.
• Sizing tool 90 can be configured to measure the diameter and/or length of one or more features of device 100 (e.g. the socket 2210 diameter and/or length of PNM-CAP 2200, the lumen diameter and/or length of PNM-G 2100).
• Sizing tool 90 can be configured to measure one or more parameters of the deposit site, such as the length of a nerve gap length or the diameter of a nerve stump.
• System 10 can further comprise one or more support assemblies, assembly 300 shown, which can be configured to receive and/or secure an object (e.g. implant 20, ECM 120, PNM-G 2100, PNM-CAP 2200).
• Support assembly 300 can be constructed and arranged as described herein in reference to Fig. 28.
• System 10 can further comprise one or more environmental chambers, chamber 601 shown.
• Chamber 601 can comprise a temperature-controlled environmental chamber configured to chill and/or freeze an object (e.g. raw material 65) through non-cyclic and/or cyclic refrigeration.
• object e.g. raw material 65
• chamber 601 consists of frozen ice or synthetic ice packs within an insulated container.
• chamber 601 consists of a refrigerator or deli case with or without an incorporated shaker system.
• System 10 can further comprise one or more vessels, vessel 602 shown, which can be configured to store an object (e.g. raw material 65).
• Vessel 602 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 602.
• vessel 602 is configured to store a tissue sample during tissue processing, embedding, and/or sectioning.
• vessel 602 is configured to store one, two, or more tissue samples during decellularization, disinfection, and/or other tissue processing (e.g. permeabilization, digestion, surface treatment, lyophilization, freezing, etc.).
• Vessel 602 can be configured to store and handle one, two, or more tissue samples with minimal manipulation, such as individual nerve segments.
• Vessel 602 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 602 can comprise: silicone; polyolefins, such as polypropylene; other biocompatible materials; and combinations of these.
• System 10 can further comprise one or more mixing devices, device 603 shown, which can be configured to agitate a fluid disposed within a component of mixing device 603.
• Mixing device 603 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 603.
• Mixing device 603 can be configured to agitate a fluid at a speed between approximately 50 rpm and 2,000 rpm.
• Mixing device 603 can be configured to maximize and/or optimize mass transport within the tissue to facilitate desired chemical, physical, and/or mechanical processes.
• mixing device 603 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 603 can be increased and/or cyclic mechanical deformation can be applied to the tissue similarly to improve mass transport.
• Mixing device 603 can include a reaction vessel comprising a 1 liter glass beaker, a 2 liter glass beaker, a spinner flask, a bioreactor bag, or other suitable container.
• mixing device 603 includes a stir plate and agitates a fluid by a rotating magnetic stir bar.
• mixing device 603 includes a fixed overhead stirrer attached to an impeller configured to rotate, thereby agitating a fluid disposed within mixing device 603.
• device 603 consists of an ultrasonic mixing device, such as a device configured to provide mechanical shock waves.
• mixing device 603 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.
• mixing device 603 consists of an orbital shaker platform designed to agitate a fluid contained within mixing device 603 at a specified speed and/or radius.
• mixing device 603 includes a peristaltic pump that defines a set fluid flow rate through mixing device 603.
• mixing device 603 includes a perfusion bioreactor in which a pump, heat exchanger, and/or other components can be used to force a specific steady or periodic flow pattern around or within the tissue samples.
• System 10 can further comprise one or more heating devices 604, device 604 shown.
• Heating device 604 can be configured to warm and/or maintain the temperature of an object (e.g. raw material 65). Heating device 604 can comprise a hotplate comprising electric heating elements. In some embodiments, heating device 604 comprises an agitating hotplate (e.g. 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 604. In some embodiments, heating device 604 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).
• an agitating hotplate e.g. a stirring hotplate
• rotating magnetic field configured to rotate a corresponding magnetic bar that is positioned in fluid proximate a surface of heating device 604.
• heating device 604 can comprise an incubator with or without an incorporated agitation system (e.g. a system configured to shake, rock, mix, stir, and/or
• System 10 can further comprise one or more laboratory instruments, instrument 605 shown, such as an instrument selected from the group consisting of: pipette, such as a serological pipette, a positive displacement pipette; forceps, such as serrated tip forceps, single tooth forceps; scalpel, such as a stainless-steel scalpel; scraper, such as a stainless-steel scraper; blade, such as a stainless-steel blade; a cutting surface, such as a polymeric cutting board; band, such as silicone band; glassware, such as a beaker, a flask, a glass bottle; graduated cylinder; balance; spatula; conical tube; funnel; temperature probe; a measuring device, such as a ruler or caliper; and combinations of these.
• pipette such as a serological pipette, a positive displacement pipette
• forceps such as serrated tip forceps, single tooth forceps
• scalpel such as a stainless-
• System 10 can further comprise one or more lyophilization devices, device 606 shown, such as a device configured to preserve a product (e.g. ECM 120) via a low temperature dehydration process.
• lyophilization device 606 is configured to dehydrate the product residual moisture content of between 0.1% and 10%, such as a residual moisture content between 0.5% and 4%, such as a residual moisture content of less than 4% (e.g. the moisture content as measured via the Karl -Fischer moisture content test).
• lyophilization device 606 is configured to dehydrate the product to a residual moisture content of between 0.2% and 2.5%.
• lyophilization device 606 is configured to create directional porosity in device 100, such as longitudinal porosity, radial porosity, circumferential porosity, and/or combinations of these.
• the low temperature dehydration process executed by lyophilization device 606 can comprise four primary phases: freezing, annealing, primary drying (sublimation), and secondary drying (adsorption).
• the freezing phase can be configured to cool the product within lyophilization device 606 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, etc.
• the annealing phase stabilizes the molecular crystalline structure to minimize disruptions during drying.
• a lower pressure within lyophilization device 606 increase in temperature within lyophilization device 606, can be configured to promote water sublimation until most of the water has been sublimated.
• 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).
• System 10 can further comprise one or more lyophilization receptacles, receptacle 607 shown, which can be configured for use with lyophilization device 606 described herein.
• Receptacle 607 can be configured to receive a product (e.g. ECM 120) and can be placed within lyophilization device 606 for the duration of the dehydration process.
• Receptacle 607 can comprise a material selected from the group consisting of aluminum; stainless steel; glass; plastic; and combinations of these. Additionally, receptacle 607 can be depyrogenated, such as to prevent contamination of the product from pathogens on receptacle 607.
• receptacle 607 is inserted into a storage element, such as a selfsealing pouch, prior to its placement within lyophilization device 606. In some embodiments, receptacle 607 insulates one, two, or more sides of the product contained within to promote directional lyophilization. In some embodiments, receptacle 607 compresses one, two, or more regions of the product contained therein in order to create a porosity gradient across the product.
• System 10 can further comprise one or more tubes, tube 608 shown, which can be configured to store an object (e.g. ECM 120).
• Tube 608 can include a top, or other moveable cover.
• System 10 can further comprise one or more batch mills, mill 609 shown, which can be configured to grind soft, fibrous, and/or brittle products (e.g. ECM 120). Mill 609 can be configured to receive tube 608, as described herein, and grind the product within tube 608. In some embodiments, the products are first frozen or maintained in a frozen state through the use of liquid nitrogen and/or dry ice (i.e. cryogrinding).
• System 10 can further comprise one or more containers, bottle 610 shown, which can be configured to store one, two, or more fluids, powders, capsules, and the like.
• Bottle 610 can include a top, or other moveable cover.
• Bottle 610 can comprise a material selected from the group consisting of glass; plastic, such as polypropylene, polyethylene, cyclic olefin copolymer; metal, such as stainless steel; and combinations of these.
• Bottle 610 has a volume of 0.1 L to 5 L, such as 1 L.
• System 10 can further comprise one or more material removal and/or displacement devices, material device 611 shown, which can be configured to shape or otherwise alter a surface of a product.
• Material device 611 can be configured to perform a function selected from the group consisting of drill; grind; rout; plane; bore; cut; ablate; and combinations of these.
• Material device 611 can comprise a drill press, micro drill press, dental pin drilling unit, longitudinal file, cylindrical die, laser cutter, electrosurgery unit, ultrasound cutting, hot wire cutter, and combinations of these.
• System 10 can further comprise one or more tensile tester devices, device 612 shown, which can be configured to determine the tensile or compressive stresses or forces generated by the material (e.g. ECM 120) and/or suture attachment points under displacements or deformations.
• tensile tester devices device 612 shown, which can be configured to determine the tensile or compressive stresses or forces generated by the material (e.g. ECM 120) and/or suture attachment points under displacements or deformations.
• System 10 can further comprise one or more alignment assemblies, assembly 613 shown.
• alignment assembly 613 comprises a Dremel keyless chuck and/or drill bit collet configured to secure an object therein (e.g. implant 20, PNM-G 2100, PNM-CAP 2200, etc.).
• alignment assembly 613 comprises a three-dimensional linear stage configured to allow controlled movement in X,Y,Z directions of an object therein.
• alignment assembly 613 comprises a jig comprising the same dimensions of a device platform (e.g. material device 611, etc.) and is configured to secure an object therein (e.g.
• alignment assembly 613 includes laser alignment at the central axis to ensure concentricity of an object therein in relation to alignment assembly 613, device (material device 611, etc.), and/or other platform.
• System 10 can further comprise one or more material removal assemblies, pin assembly 614 shown.
• pin assembly 614 comprises a Dremel keyless chuck and/or drill bit collet configured to secure a pin, needle, carbide burr, drill bit, and/or other sharpened object therein.
• the pin comprises an outer diameter of 0.7mm. In some embodiments, the pin comprises an outer diameter between 1.5 and 3.0mm, such as an outer diameter of 2.0mm.
• System 10 can further comprise one or more nerve holding and/or processing containers, cassette 620 shown, and as described herein in reference to Fig. 27.
• System 10 can further comprise one or more buffer solutions, solution 701 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).
• solution 701 comprises phosphate buffered solution or phosphate buffered saline (PBS).
• PBS phosphate buffered saline
• solution 701 has a conductivity of between 1 and 30mS/cm, such as 14mS/cm.
• solution 701 comprises a buffer solution in water with the components and ranges of concentration shown hereinbelow in Table 2:
• System 10 can further comprise one or more cooling agents, agent 702 shown, which can be configured to reduce, and/or otherwise regulate, the temperature of a product (e.g. raw material 65).
• Cooling agent 702 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.
• System 10 can further comprise one or more purified waters, water 703 shown, which can comprise water that has been filtered, or otherwise processed, to remove one, two, or more impurities.
• purified water 703 comprises Type I water or water for injection.
• System 10 can further comprise one or more dissociation solutions, solution 704 shown, which can be configured to dissociate adherent cells, cell aggregates, and/or tissues into single-cell suspensions.
• dissociation solution 704 comprises a co-solution comprising 0.02% trypsin and 0.05% ethylenediaminetetraacetic acid (EDTA).
• EDTA ethylenediaminetetraacetic acid
• Dissociation solution 704 can comprise a solution that is warmed to a temperature of approximately 35°C.
• System 10 can further comprise one or more disinfecting solutions, solution 705 shown, which can be configured to destroy one, two, or more microorganisms (e.g. bacteria, virus, fungi).
• disinfecting solution 705 comprises a co-solution comprising 0.1% peracetic acid and 4% ethanol.
• System 10 can further comprise one or more detergent solutions, solution 706 shown, which can be configured to lyse and/or permeabilize cells.
• detergent solution 706 comprises Triton X-100.
• detergent solution 706 comprises a 4% (w/v) sodium deoxycholate solution.
• System 10 can further comprise one or more sucrose solutions, solution 707 shown , which can be configured as an excipient.
• sucrose solution 707 comprises a IM sucrose solution.
• System 10 can further comprise one or more sterile waters, water 708 shown, which can comprise water that has been processed to remove one, two, or more contaminants (e.g. bacteria, virus, fungi).
• sterile water 708 comprises water for injection (WFI).
• System 10 can further comprise one or more digestion solutions, solution 709 shown, which can be configured to break down tissue.
• digestion solution 709 comprises a 0.01 N hydrochloric acid (HC1) solution.
• System 10 can further comprise one or more digestive enzymes, enzyme 710 shown, which can be configured to break down polymeric macromolecules.
• 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 710 is then placed into solution 709 such that the final concentration results in an activity level of between 10 U/mL and 2500 U/mL, such as activity levels of 250 U/mL.
• System 10 can further comprise one or more stabilizing and/or radioprotecting solutions, excipient 711 shown, which can be configured to provide at least one of long-term stabilization, bulking, radioprotection, heat protection, cryoprotection, increase in solubility, increase or decrease in viscosity, optimization of thermal properties for lyophilization and/or other enhancement of a product.
• Excipient 711 can comprise an excipient selected from the group consisting of: sucrose; ascorbic acid, glycerol, glycine, sodium ascorbate; sodium azide; vitamin E; EDTA; mannitol; glycine; Dextran; and combinations of these.
• Excipient 711 can be configured to increase, and/or otherwise improve, the relative solubility of a product (e.g. ECM 120). Excipient 711 can be configured to increase, or otherwise improve, the relative gelation of a product (e.g. ECM 120).
• System 10 can further comprise one or more tools, tool 80 shown.
• Tool 80 can comprise a tool configured to coat a surface (e.g. a surface of a deposit site), such as an atomization tool, a brush or brushing tool, and/or a dipping tool, as described herein.
• Tool 80 can comprise a tool consisting of a tattoo machine for surface delivery of ECM 120 at a defined depth.
• Tool 80 can comprise a jet injector to deliver ECM 120 via high pressure at a defined depth.
• Tool 80 can comprise a bobbin with a coiled strand or ribbon embedded with ECM 120 wound/canvassed around the surface.
• Tool 80 can comprise an adhesive or adhesive strip used to affix ECM 120 to a surface
• Implant 20 comprises a graft-conduit (e.g. artificial, natural) configured to connect, or otherwise provide one, two, or more longitudinal channels, between two or more anatomical elements (e.g. nerve stumps).
• Implant 20 can comprise at least a first end 21 and at least a second end 23, with a lumen 22 therebetween.
• First end 21 can be constructed and arranged to receive at least a portion of a first anatomical element (e.g. first nerve stump) and second end 23 can be constructed and arranged to receive at least a portion of a second anatomical element (e.g. second nerve stump).
• Lumen 22 can be configured to receive, or otherwise comprise, a therapeutic device (e.g. device 100 of the present inventive concepts), such as to maintain the relative positioning of therapeutic device between the two or more anatomical elements.
• first end 21 and/or second end 23 can be configured to receive, or otherwise comprise, a therapeutic device (e.g. device 100 of the present inventive concepts), such that therapeutic device contacts at least a portion of the anatomical elements received by first end 21, second end 23.
• Implant 20 can comprise an inner diameter ID that approximates the outer diameter of at least one of the anatomical elements (e.g. a nerve stump). Implant 20 can comprise an inner diameter (ID) that is smaller than the outer diameter of at least one of the anatomical elements (e.g. a nerve stump). Implant 20 can comprise an outer diameter (OD) that approximates the outer diameter of at least one of the anatomical elements (e.g. a nerve stump). In some embodiments, implant 20 comprises an ID and/or OD that are consistent along its length.
• implant 20 comprises an ID and/or OD that vary along its length, such as an initial IDi and/or ODi to adapt to the diameter of a first anatomical element and a final IDf and/or ODf to adapt to the diameter of a second anatomical element (not shown).
• implant 20 comprises a funnel shape configured to allow for expansion and/or contraction of the ID and/or OD along its length.
• Implant 20 can comprise a length L that approximates the distance between the two or more anatomical elements.
• implant 20 comprises a length L that is greater than the distance between the two or more anatomical elements, such as to overlap at least a portion of at least one of the anatomical elements (as shown).
• implant 20 can comprise an initial length Li that is modified (e.g. trimmed or otherwise reduced) to comprise a final length LF.
• the length of implant 20 can be modified prior to, during, and/or after the procedure in which implant 20 is implanted in the patient.
• Implant 20 can comprise one, two, or more severable portions 24 (not shown) proximate first end 21 and/or second end 23. Severable portions 24 can be removed prior to, during, and/or after the procedure in which implant 20 is implanted in the patient, such as to modify the length of implant 20. In some embodiments, severable portions 24 are configured to be tom away from the remainder of implant 20. In some embodiments, severable portions 24 are configured to be cut away from the remainder of implant 20.
• implant 20 e.g. lumen 22
• implant 20 comprises a volume of device 100 that contracts after implantation within the patient.
• a clinician can remove at least one severable portion 24 comprising a portion of implant 20 that no longer comprises a volume of device 100 (e.g. a portion of implant 20 from which device 100 has contracted away).
• Implant 20 can be configured to accept nerves in multiple orientations, such as a T-shaped implant for an end-to-side repair or a Y-shaped implant for connecting one nerve to two other nerves.
• implant 20 comprises an angled connector configured to receive one, two, or more tissues.
• Implant 20 can comprise one, two, or more materials configured to promote the growth of tissue (e.g. nerve tissue).
• tissue e.g. nerve tissue
• at least a portion of implant 20 comprises a material that is permeable to cells, axons, and/or nutrients (e.g. tissue cells can infiltrate the material of implant 20).
• at least a portion of implant 20 comprises a selective permeability to pre-determined nutrients and/or cell types.
• at least a portion of implant 20 comprises a material that supports, or otherwise promotes, tissue growth (e.g. the material comprises an agent 70 comprising growth factors).
• implant 20 can comprise one, two, or more materials configured to impede the growth of tissue (e.g. nerve tissue).
• At least a portion of implant 20 comprises a material that is impermeable and/or otherwise resistive to cells, axons, and/or nutrients (e.g. tissue cells cannot infiltrate the material of implant 20).
• a first portion of implant 20 can comprise a permeable material and a second portion of implant 20 can comprise an impermeable material.
• Implant 20 can comprise one, two, or more materials configured to provide suture retention strength, such that implant 20 can receive one or more sutures and withstand the respective tractive force.
• Implant 20 can comprise a suture retention strength of no less than 0.02N, such as no less than 0.2N, such as no less 0.5N.
• Implant 20 can be constructed and arranged to withstand tension, flexion, and/or torsion forces exhibited between the two or more anatomical elements (e.g. two or more nerve stumps), such as to withstand motion attributed to characteristic body and/or surrounding tissue movement.
• implant 20 can comprise a longitudinal ultimate tensile strength great than 1 Mpa.
• Implant 20 can be constructed and arranged to prevent, or otherwise resist, the formation of one or more kinks along its length.
• implant 20 is configured to resist kink and/or compressive collapse during a bending of a joint, such as a finger or elbow.
• implant 20 can comprise a radius of kinking curvature less than 2cm.
• Implant 20 can comprise one, two, or more materials configured to degrade at a rate compatible with the rate of tissue regeneration. Nerves can comprise a longitudinal regeneration rate between approximately Imm/day and 5mm/day, and as such, implant 20 can comprise a corresponding degradation rate. For example, implant 20 comprising a length of 2cm can be configured to degrade no earlier than 20 days after implantation.
• implant 20 can comprise a degradation rate that is slower than the longitudinal tissue regeneration rate, such as to allow for sufficient nerve volume regeneration and terminal functional recovery to occur and to prevent fibrotic and/or scar tissue formation.
• a nerve with a gap injury of 1cm can be expected to be crossed by growing axons from proximal to distal nerve stump in no less than 10 days (assuming a longitudinal regeneration rate of Imm/day).
• robust structural tissue formation between the two nerve stumps likely requires more than 20 days and full functional recovery likely requires more than one month. Therefore, the implant 20 can be configured to degrade at a slower rate than that of the nerve healing and/or regeneration process.
• implant 20 can comprise a degradation rate that is faster at the proximal end than at the distal end, corresponding with area or tissue regeneration. For example, robust tissue formation may occur several days earlier at the proximal end of implant 20 than at the distal end. Graftconduit degradation that is too slow may compress and damage a regenerated nerve.
• implant 20 can be configured to degrade proximally before degrading distally, and at a rate matching the progression of tissue formation along the gap injury site.
• Implant 20 can comprise a degradable, artificial conduit configured to break down in situ over one, two, or more periods of time.
• implant 20 comprises a non-degradable artificial conduit, such as a conduit comprising silicone.
• Lumen 22 can comprise device 100 (e.g. ECM 120) comprising a hydrogel configuration, such as a degradable or non-degradable hydrogel.
• Implant 20 can comprise a natural conduit harvested, or otherwise derived, from a tissue source. Implant 20 can further comprise a decellularized, natural conduit. Implant 20 can comprise a decellularized tissue that has been modified to create one, two, or more lumens (e.g. longitudinal tunnel s/channels) of a controlled size. Lumen 22 can comprise device 100 (e.g. ECM 120) comprising a hydrogel configuration, such as a degradable or non-degradable hydrogel. In some embodiments, implant 20 comprises a conduit harvested, or otherwise derived, from porcine tissue (e.g. porcine nerve-tissue).
• porcine tissue e.g. porcine nerve-tissue
• implant 20 comprises a composite of device 100 and one, two, or more other materials, such as a material selected from the group consisting of: polycaprolactone; PLLA; PGA; silicone; polyurethanes; PET; PTFE; decellularized tissue and/or organs; collagen; elastin; GAG; keratin; chitosan; silk; synthetic derived materials; naturally, derived materials; and combinations of these.
• Implant 20 can comprise a conduit at least partially derived, or otherwise fabricated, from device 100 (e.g. ECM 120).
• Implant 20 can comprise a conduit fabricated from raw material 65 comprising a decellularized extracellular matrix (e.g. ECM 120), such as a raw material as described in applicant’s co-pending International PCT Patent Application Serial Number PCT/US2020/053570, entitled “Extracellular Matrix Devices and Methods of Manufacture”, filed September 30, 2020.
• Raw material 65 can be mechanically, physically, and/or chemically modified to create a conduit with a lumen therethrough, such as described herein in reference to Method 1000 of Fig. 6.
• Implant 20 can further comprise one, two, or more cavities, spaces, and/or other reservoirs, reservoirs 25 (not shown).
• Reservoirs 25 can be configured to contain a volume of device 100 (e.g. ECM 120).
• Device 100 can be injected into implant 20 such that lumen 22 and reservoir 25 each receive a volume of device 100.
• the volume of device 100 contracts within lumen 22 (e.g. contracts over time)
• the volume of device 100 within reservoir 25 can be pulled into lumen 22 to provide an additional supply of device 100.
• reservoir 25 is constructed and arranged as a recess within an inner wall of implant 20.
• device 100 can comprise a plurality of reservoirs 25 constructed and arranged along a length of implant 20.
• device 100 can comprise at least one reservoir 25 constructed and arranged proximate first end 21 and/or second end 23.
• Implant 20 can be constructed to comprise device 100 (e.g. ECM 120) and/or can be configured to receive a volume of device 100 (e.g. ECM 120).
• device 100 e.g. ECM 120
• volume of device 100 e.g. ECM 120
• Implant 20 can be constructed and arranged as a nerve graft-conduit, PNM-G 2100, as described hereinbelow.
• Implant can be constructed and arranged as a nerve cap-graft, PNM-CAP 2200, as described hereinbelow.
• the nerve graft-conduit comprises PNM-G 2100 as described herein.
• PNM-G 2100 can comprise a hybrid nerve graft-conduit derived from decellularized peripheral nerves.
• PNM-G 2100 comprises a decellularized porcine peripheral nerve.
• PNM-G 2100 comprises a lumen 2122 surrounded by a luminal wall 2126 and conduit wall 2128, as shown.
• lumen 2122 includes a central neuroinductive septum 2124.
• PNM-G 2100 can comprise a length comprising a proximal bore depth and distal bore depth with a septum length therebetween, as shown.
• PNM-G 2100 can further comprise an outer diameter OD comprising a conduit wall thickness WT and inner diameter ID, as shown.
• the length of PNM-G 2100 can be controlled by one or more of the following factors: native nerve type; native nerve size; animal type from which the native nerve is harvested; animal age from which the native nerve is harvested; cutting tools employed to trim the native nerve; and mechanical stretch applied to elongate the native nerve.
• the proximal and/or distal bore depth of PNM-G 2100 can be controlled by the depth of penetration via a rotating burr or drill bit during manufacturing.
• the proximal and/or distal bore depth of PNM-G 2100 can alternately be controlled by the speed, power, and/or number of passes of laser ablation during manufacturing.
• the proximal and/or distal bore depth of PNM-G 2100 can alternately be controlled by the power setting (J/s), tip size, frequency, power, current, and/or rate and regimen of tip longitudinal travel via electro surgery or ultrasound cutting during manufacturing.
• the outer diameter OD of PNM-G 2100 can be controlled by one or more of the following factors: native nerve type; native nerve size; animal type from which the native nerve is harvested; animal age from which the native nerve is harvested; use of jacketing and circumferential compression ratio; use of mechanical forces to steamroll, massage, and/or stretch the tissue; removal of material around the circumference of the tissue to reduce the OD to a desired size; application of negative hydrostatic pressure to force swelling of the tissue and increase the OD; use of chemical treatment to shrink, remove, and/or swell the tissue and change the OD; use of physical forces, such as heat, to shrink, remove, and/or swell the tissue and change the OD; and addition of material around the tissue to increase the OD, such as a membranous wrap.
• the inner diameter ID of PNM-G 2100 can be controlled by the mechanical, chemical, and/or physical removal of tissue within the native nerve, such as via rotating burrs, luminal filing with abrasive thread, water cutting, ultrasound cutting or drilling, shooting of a micro bullet into the lumen, acid compounds, laser ablation, and electro- surgical methods, such as cutting via electrical current (e.g. cautery).
• electrical current e.g. cautery
• Septum 2124 can be constructed and arranged according to one or more design variables selected from the group consisting of: porosity; pore size; pore interconnectedness; pore alignment; degradation rate; and swell ratio (e.g. all of which can be different along the three main axes).
• septum 2124 is created within PNM-G 2100 by leaving a portion of lumen 2122 undrilled. In some embodiments, septum 2124 is created separately and then inserted into lumen 2122 of PNM-G 2100.
• septum 2124 is created by filling lumen 2122 with a putty, gel, and/or other fluid/semi-solid substance(s) and then solidifying the substance(s) and creating the desired macro and micro properties in situ (e.g. via lyophilization).
• Luminal wall 2126 can be constructed and arranged according to one or more design variables selected from the group consisting of: porosity; pore size; pore geometry; pore interconnectedness; pore alignment; degradation rate; and swell ratio (e.g. all of which can be different along the three main axes).
• Conduit wall 2128 can be constructed and arranged according to one or more design variables selected from the group consisting of: porosity; pore size; pore interconnectedness; pore alignment; degradation rate; and swell ratio (e.g. all of which can be different along the three main axes).
• design variables selected from the group consisting of: porosity; pore size; pore interconnectedness; pore alignment; degradation rate; and swell ratio (e.g. all of which can be different along the three main axes).
• chemical and/or physical treatment are applied to shrink or swell the thickness of conduit wall 2128.
• the pore alignment of conduit wall 2128 can be dependent upon one or more of the following factors: leverage phase separation with defined freezing parameters (e.g. rate, temperature, etc.) to create desired crystals as template for macro porosity; lyophilization with different parameters and/or external aids (e.g.
• porous jacket for water removal and generation of micro porosity; use of mechanical and/or physical forces to generate pores (e.g. laser drilling); use of chemicals to swap water content within conduit wall 2128 with chemicals to induce a controlled phase separation; use of internal or external manifolds with circumferentially distributed pores and the application of high pressure to create radially distributed channels via water cutting; and use of fiber optics with radially distributed outlets to convey laser and create radially oriented pores.
• the swell ratio of conduit wall 2128 can be dependent upon pore distribution, pore size, and/or directionality of pores.
• Swell ratios and the associated directional components can be used to design the desired sizes of PNM-G 2100 upon hydration.
• dry lumen 2122 diameter sizing can be utilized to obtain the hydrated (e.g. operational) lumen size.
• the swelling of PNM-G 2100 upon hydration can be used to create a holding force around a nerve stump, or other mechanical interactions, to anchor, align, secure, position, and/or hold PNM-G 2100 to the nerve stump.
• a dry or partially hydrated PNM-G 2100 can be applied onto the nerve stump prior to hydration, or completion thereof, to secure lumen 2122 onto the nerve and without requiring the use of sutures.
• the application of a dry or partially hydrated PNM- G 2100 can be configured to foster the creation of adhesion forces upon absorbance of the moieties (e.g. via capillarity or hydrophobic/hydrophilic interactions, or other small molecular bond forces) at the deposit site within conduit wall 2128 of PNM-G 2100 and without requiring the use of sutures.
• the nerve graft-conduit comprises PNM-G 2100 as described herein.
• PNM-G 2100 can be configured for implantation at the site of a nerve injury selected from the group consisting of: nerve gap injury, such as to provide end-to-end repair; nerve transection injury, such as to provide short neurorrhaphy repair; nerve transfer, such as to provide end-to-end, end-to-side, or side-to-side repair; and nerve amputation, such as to provide nerve cap-graft and/or nerve extension to expire regenerative potential.
• the deposit site comprises a nerve gap and PNM-G 2100 is configured to connect two nerve stumps.
• An operator can create two proximal and two distal sutures (e.g. 9-0 nylon monofilament suture) to secure PNM-G 2100 to the nerve stumps.
• PNM-G 2100 is configured to have a larger diameter than the nerve stump, thereby creating a hole, or socket, into which an operator can insert the nerve stump, and which can be further secured with two sutures.
• the deposit site comprises an amputated nerve and PNM-G 2100 is attached to an end portion of the amputated nerve.
• An operator can create two proximal sutures (e.g. 9-0 nylon monofilament suture) to secure PNM-G 2100 to the amputated nerve.
• an operator can create a distal suture (e.g. 9-0 nylon monofilament suture) to ligate distal end 2123, as shown in Fig. 4D.
• an operator can create two distal sutures to secure distal end 2123 to surrounding tissue.
• the deposit site comprises a nerve transfer and PNM-G 2100 is configured to connect a healthy nerve to an injured nerve.
• An operator can create two proximal and two distal sutures (e.g. 9-0 nylon monofilament suture) to secure PNM-G 2100 to the healthy nerve and the injured nerve.
• the deposit site comprises an amputated nerve and PNM-G 2100 is attached to an end portion of the amputated nerve.
• An operator can create a centrocentral anastomosis via an end-to-end suture (e.g. 9-0 nylon monofilament suture) between the fascicles of the amputated nerve, with PNM-G 2100 implanted therebetween.
• end-to-end suture e.g. 9-0 nylon monofilament suture
• Implant 20 can comprise a nerve graft-conduit, PNM-G 2100, as described herein.
• PNM-G 2100 can be configured for implantation at the site of a nerve injury selected from the group consisting of: nerve gap injury, such as to provide end-to-end repair; nerve transection injury, such as to provide short neurorrhaphy repair; nerve transfer, such as to provide end-to-end, end-to-side, or side-to-side repair; and nerve amputation, such as to provide nerve cap-graft and/or nerve extension to expire regenerative potential.
• PNM-G 2100 can be configured to connect nerve endings to other tissues and organs to facilitate reinnervation (e.g. cornea, corpus cavernosum, heart, blood vessels, spinal cord, etc.).
• PNM-G 2100 is implanted between muscle tissue and a nerve ending, such that the nerve ending is connected into and/or onto the muscle tissue to foster new functional connections between the nerve axonal sproutings and new and/or existing neuromuscular junctions within the muscle (e.g. direct muscle neurotization).
• PNM-G 2100 is implanted between two nerve endings, one of which is connected to muscle tissue, and such that the muscle tissue is reactivated via the connected nerve endings (e.g. end-to-end nerve transfer, Oberlin Procedure, etc.)
• PNM-G 2100 is implanted between two nerve endings, one of which is connected to muscle tissue, and such that an electrical activation map of the muscle tissue surface is created.
• the activation map can be detected from skin electrodes to control electronic prostheses (e.g. total muscle reinnervation).
• the nerve graft-conduit can comprise PNM-G 2100 as described herein.
• Method 10000 comprises a sequence of submethods, Methods 11000, 12000, 12200, 13000, 14000, 15000, 16000, 17000, 18000, 19000, and 20000 as described herein in reference to Figs. 6-16, respectively.
• Method 11000 comprises a method for harvesting and/or preparing tissue for further manipulation.
• Methods 12000 and 12200 comprise methods for decellularizing the tissue harvested and/or prepared in Method 11000.
• Method 13000 comprises a method for jacketing, or otherwise providing external support to, the decellularized tissue produced in Methods 12000 and 12200.
• Method 14000 comprises a method for lyophilizing the jacketed tissue produced in Method 13000.
• Method 15000 comprises a method for creating one, two, or more desired features (e.g. lumen 2122, socket 2210, anchoring tab 2214, etc.) through the lyophilized tissue produced in Method 14000.
• Method 16000 comprises a method for decellularizing and/or performing other chemical, physical, and/or mechanical treatments of nerve graft-conduit or nerve capgraft produced in Method 15000.
• Method 17000 comprises a method for stabilizing a nerve graft-conduit or nerve cap-graft produced in Method 16000.
• Method 18000 comprises a method for packaging the nerve graft-conduit or nerve cap-graft produced in Method 17000.
• Method 19000 comprises a method for sterilizing the container comprising the nerve graftconduit or nerve cap-graft produced in Method 18000.
• Method 20000 comprises a method for shipping and/or storing the container comprising the nerve graft-conduit or nerve capgraft produced in Method 19000.
• a method of producing a nerve graft-conduit or nerve cap-graft may include and/or omit one, two or more sub-methods as described herein.
• the order of one, two, or more sub-methods can be altered depending on a desired feature of device 100.
• Method 14000 may take place after Method 16000, representing lyophilization of the nerve graft-conduit or nerve cap-graft directly before stabilization and packaging.
• Method 11000 can be configured to harvest and/or prepare raw material 65 from tissue source 60 described herein.
• raw material 65 is selected from a tissue source (e.g. tissue source 60) comprising one, two, or more desired structural and/or functional features.
• tissue source 60 e.g. tissue source 60
• the desired structural and/or functional features are configured to provide a desired structure and/or function to device 100 (e.g. PNM-G 2100, PNM-CAP 2200).
• raw material 65 harvested from a tissue source e.g. tissue source 60
• raw material 65 can be processed to remove connective and/or accessory tissue (e.g. remove non-nerve tissue).
• connective and/or accessory tissue e.g. remove non-nerve tissue
• cleaned raw material 65 can be at least partially immersed in buffer solution 701.
• raw material 65 can be stored in chamber 601 at a temperature between approximately 2°C and 8°C.
• raw material 65 can be rapidly frozen in buffer solution 701.
• raw material 65 is rapidly frozen via cooling agent 702.
• Raw material 65 can be stored and/or transported in chamber 601 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 65 is stored in chamber 601 at a temperature of approximately -80°C for a maximum of six months. In some embodiments, raw material 65 is harvested aseptically and stored at a temperature of between 2°C and 8°C for up to 48 hours in a sealed environment devoid of air (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.
• a preservation solution comprising germostatic or germicidal agents consistent with that used for human organ procurement and transport for transplantation.
• raw material 65 is harvested from a tissue source (e.g. tissue source 60) and preserved for storage and/or transportation. Additionally, raw material 65 can be processed to remove connective and/or accessory tissue (e.g. remove non-nerve tissue). For preservation, raw material 65 can be stored in a preservation solution until proceeding to STEP 11030 described herebelow.
• tissue source e.g. tissue source 60
• connective and/or accessory tissue e.g. remove non-nerve tissue
• frozen and/or preserved raw material 65 is prepared for processing.
• frozen raw material 65 is thawed in chamber 601 at a temperature of between 2°C and 8°C.
• frozen raw material 65 is thawed in chamber 601 for at least 48 hours, such as at least 72 hours.
• preserved raw material 65 is removed from the preservation solution.
• raw material 65 is further processed (e.g. cleaned) to remove additional connective and/or accessory tissue (e.g. remove non-nerve tissue), and as described hereinbelow in reference to Figs. 20A,B and Figs. 21 A, B.
• Raw material 65 can be processed at a temperature of between 2°C and 25°C.
• raw material 65 comprises a portion of nerve tissue comprising one, two, or more side branches.
• the side branches can be removed and processed as described herein.
• raw material 65 is processed manually (e.g. by hand, via surgical instruments such as scalpels).
• raw material 65 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.
• raw material 65 is processed via a chemical treatment (e.g. acid spray plus wash) to partially remove external layers of tissue from the nerve.
• raw material 65 is processed via physical treatment involving heat, cold, radiation, laser, and/or other physical forces to ablate or otherwise remove excess tissue.
• raw material 65 is processed via a combination of manual, mechanical, chemical, and/or physical treatments.
• cleaned raw material 65 is cut, or otherwise divided, into smaller segments, and as described hereinbelow in reference to Figs. 22A, B.
• Cleaned raw material 65 can be cut into segments comprising a length of between 0.5cm and 20cm, such as between 1cm and 10cm.
• Cleaned raw material 65 can comprise segments comprising a diameter of between 0.5mm and 10mm, such as between 1mm and 8mm.
• cleaned raw material 65 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.
• a sizing tool 90 is used to measure the length and diameter of each segment, and as described hereinbelow in reference to Fig. 18.
• each segment is processed manually (e.g. by hand).
• each nerve segment 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.
• each segment is processed via a chemical treatment (e.g. acid spray plus wash) to partially remove external layers of tissue from the nerve.
• each segment is processed via physical treatment involving heat, cold, radiation, laser, and/or other physical forces to ablate or otherwise remove excess tissue.
• each segment is processed via a combination of manual, mechanical, chemical, and/or physical treatments.
• raw material 65 is transferred to one, two, or more vessels 602.
• Cleaned raw material 65 can be transferred at a temperature of between 2°C and 37°C.
• each vessel 602 comprises no more than 25g of cleaned raw material 65 and a mixing device 603 contains no more than six vessels 602.
• each vessel 602 contains a single segment of cleaned raw material 65.
• cleaned raw material 65 is washed with purified water 703.
• Cleaned raw material 65 can be washed at a temperature of between 2°C and 8°C.
• Cleaned raw material 65 is washed with purified water 703 at least two times, such as at least three times, such as at least four times.
• Cleaned raw material 65 and purified water 703 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30.
• vessel 602 is placed into mixing device 603 comprising purified water 703, such as a mixing device comprising at least 3000 mL of purified water 703.
• Mixing device 603 can be combined with heating device 604 configured to agitate purified water 703 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for at least 10 minutes, thereby washing cleaned raw material 65 within vessel 602.
• Purified water 703 is decanted from mixing device 603 and replaced with fresh purified water 703.
• Mixing device 603 is combined with heating device 604 configured to agitate purified water 703 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for at least 10 minutes, thereby washing raw material 65 within vessel 602 a second time.
• Purified water 703 is decanted from mixing device 603.
• cleaned raw material 65 is washed overnight with purified water 703.
• Cleaned raw material 65 can be washed at a temperature of between 2°C and 8°C.
• Cleaned raw material 65 and purified water 703 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30.
• vessel 602 is placed into mixing device 603 comprising purified water 703, such as a mixing device comprising at least 300 mL of purified water 703, such as 3000 mL of purified water 703.
• Mixing device 603 is stored in chamber 601 at a temperature of approximately 5 °C for between 12 hours and 24 hours, such as 16 hours.
• mixing device 603 is combined with heating device 604 configured to agitate purified water 703 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, thereby washing cleaned raw material 65 within vessel 602.
• Methods 12000 and 12200 can each be configured to decellularize cleaned raw material 65 harvested and/or prepared in Method 11000 described herein in reference to Fig. 7.
• Method 12000 comprises STEPs 12010 through 12160, as described hereinbelow.
• dissociation solution 704 and disinfecting solution 705 are prepared.
• Dissociation solution 704 and disinfecting solution 705 can be prepared at a temperature of between 2°C and 25°C.
• cleaned raw material 65 from STEP 12010 is removed from vessel 602 and transferred to one or more cassettes 620, and as described hereinbelow in reference to Fig. 27.
• One or more portions of raw material 65 can be anchored to cassette 620, such as to secure raw material 65 to and/or within cassette 620.
• cleaned raw material 65 from STEP 12010 remains in vessel 602 throughout a decellularization process.
• cleaned raw material 65 is washed with purified water 703.
• purified water 703 is brought in chamber 601 to a temperature of between 2°C and 37°C.
• Cleaned raw material 65 is washed with purified water 703 at least two times.
• Cleaned raw material 65 and purified water 703 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30.
• Purified water 703 is added to mixing device 603. In some embodiments, at least 300 mL of purified water 703 is added to mixing device 603.
• Mixing device 603 is stored in chamber 601 at a temperature of between 2°C and 22°C.
• Mixing device 603 is combined with heating device 604 configured to agitate purified water 703 at 100 ⁇ 10 rpm, for at least 10 minutes, thereby washing cleaned raw material 65 within vessel
• Purified water 703 is decanted from mixing device 603 and replaced with fresh purified water 703.
• Mixing device 603 is combined again with heating device 604 configured to agitate purified water 703 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for at least 10 minutes, thereby washing cleaned raw material 65 within vessel 602 a second time.
• Purified water 703 is decanted from mixing device 603.
• Dissociation solution 704 can comprise a temperature of between 2°C and 37°C, such as 35 ⁇ 2°C. Cleaned raw material 65 and dissociation solution 704 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30. Dissociation solution 704 is added to mixing device 603. In some embodiments, at least 3000 mL of dissociation solution 704 is added to mixing device
• Mixing device 603 is combined with heating device 604 configured to agitate dissociation solution 704 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, thereby washing cleaned raw material 65 within vessel 602.
• Cleaned raw material 65 is washed in chamber 601 at a temperature of between 2°C and 37°C , such as 35 ⁇ 2°C, for between 30 minutes and 180 minutes, such as 60 ⁇ 5 minutes.
• a lipid layer forms on the surface of dissociation solution 704 and is removed using instrument 605.
• Dissociation solution 704 is decanted from mixing device 603.
• cleaned raw material 65 is washed with purified water 703.
• purified water 703 is pre-chilled in chamber 601 to a temperature of between 2°C and 8°C.
• Cleaned raw material 65 is washed with purified water 703 at least six times.
• Cleaned raw material 65 and purified water 703 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30.
• Purified water 703 is added to mixing device 603. In some embodiments, at least 3000 mL of purified water 703 is added to mixing device 603.
• Mixing device 603 is stored in chamber 601 at a temperature of approximately 5°C.
• Mixing device 603 is combined with heating device 604 configured to agitate purified water 703 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for at least 5 minutes, thereby washing cleaned raw material 65 within vessel 602.
• Purified water 703 is decanted from mixing device 603 and replaced with fresh purified water 703. This process is repeated at least five additional times, thereby washing cleaned raw material 65 within vessel 602 at least six times.
• cleaned raw material 65 is treated (e.g. washed or otherwise treated) with detergent solution 706.
• detergent solution 706 is brought in chamber 601 to a temperature of between 2°C and 37°C.
• Cleaned raw material 65 and detergent solution 706 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30.
• Detergent solution 706 is added to mixing device 603.
• at least 3000 mL of detergent solution 706 is added to mixing device 603.
• Mixing device 603 is stored in chamber 601 at a temperature of between 2°C and 37°C, such as approximately 20°C.
• Mixing device 603 is combined with heating device 604 configured to agitate detergent solution 706 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for between 30 minutes and 180 minutes, such as 60 ⁇ 5 minutes, thereby washing cleaned raw material 65 within vessel 602.
• Detergent solution 706 is decanted from mixing device 603.
• cleaned raw material 65 is washed with purified water 703.
• purified water 703 is brought in chamber 601 to a temperature of between 2°C and 37°C, such as approximately 20°C.
• Cleaned raw material 65 is washed with purified water 703 at least twelve times.
• Cleaned raw material 65 and purified water 703 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30.
• Purified water 703 is added to mixing device 603.
• at least 3000 mL of purified water 703 is added to mixing device 603.
• Mixing device 603 is stored in chamber 601 at a temperature of between 2°C and 37°C, such as approximately 20°C.
• Mixing device 603 is combined with heating device 604 configured to agitate purified water 703 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for at least 5 minutes, thereby washing cleaned raw material 65 within vessel 602.
• Purified water 703 is decanted from mixing device 603 and replaced with fresh purified water 703. This process is repeated at least eleven additional times, thereby washing cleaned raw material 65 within vessel 602 at least twelve times.
• cleaned raw material 65 is treated with sucrose solution 707.
• sucrose solution 707 is pre-chilled in chamber 601 to a temperature of between 2°C and 8°C, such as approximately 4°C.
• Cleaned raw material 65 and sucrose solution 707 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30.
• Sucrose solution 707 is added to mixing device 603.
• at least 3000 mL of sucrose solution 707 is added to mixing device 603.
• Mixing device 603 is stored in chamber 601 at a temperature of between 2°C and 8°C, such as approximately 4°C.
• Mixing device 603 is combined with heating device 604 configured to agitate sucrose solution 707 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for between 5 minutes and 60 minutes, such as 15 ⁇ 5 minutes, thereby washing cleaned raw material 65 within vessel 602.
• Sucrose solution 707 is decanted from mixing device 603.
• cleaned raw material 65 is washed with purified water 703.
• purified water 703 is brought in chamber 601 to a temperature of between 2°C and 8°C, such as approximately 4°C.
• Cleaned raw material 65 is washed with purified water 703 at least six times.
• Cleaned raw material 65 and purified water 703 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30.
• Purified water 703 is added to mixing device 603.
• at least 3000 mL of purified water 703 is added to mixing device 603.
• Mixing device 603 is stored in chamber 601 at a temperature of between 2°C and 8°C, such as approximately 4°C.
• Mixing device 603 is combined with heating device 604 configured to agitate purified water 703 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for at least 5 minutes, thereby washing cleaned raw material 65 within vessel 602.
• Purified water 703 is decanted from mixing device 603 and replaced with fresh purified water 703. This process is repeated at least five additional times, thereby washing cleaned raw material 65 within vessel 602 at least six times.
• cleaned raw material 65 is treated with detergent solution 706.
• detergent solution 706 is brought in chamber 601 to a temperature of between 2°C and 37°C, such as approximately 20°C.
• Cleaned raw material 65 and detergent solution 706 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30.
• Detergent solution 706 is added to mixing device 603.
• at least 3000 mL of detergent solution 706 is added to mixing device 603.
• Mixing device 603 is stored in chamber 601 at a temperature of between 2°C and 37°C, such as approximately 20°C.
• Mixing device 603 is combined with heating device 604 configured to agitate detergent solution 706 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for between 30 minutes and 180 minutes, such as 60 ⁇ 5 minutes, thereby washing cleaned raw material 65 within vessel 602.
• Detergent solution 706 is decanted from mixing device 603.
• cleaned raw material 65 is washed with purified water 703.
• purified water 703 is brought in chamber 601 to a temperature of between 2°C and 37°C, such as approximately 20°C.
• Cleaned raw material 65 is washed with purified water 703 at least 18 times.
• Cleaned raw material 65 and purified water 703 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30.
• Purified water 703 is added to mixing device 603. In some embodiments, at least 3000 mL of purified water 703 is added to mixing device 603.
• Mixing device 603 is stored in chamber 601 at a temperature of between 2°C and 37°C, such as approximately 20°C.
• Mixing device 603 is combined with heating device 604 configured to agitate purified water 703 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for at least 5 minutes, thereby washing cleaned raw material 65 within vessel 602.
• Purified water 703 is decanted from mixing device 603 and replaced with fresh purified water 703. This process is repeated at least seventeen additional times, thereby washing cleaned raw material 65 within vessel 602 at least eighteen times.
• cleaned raw material 65 is treated with disinfecting solution 705.
• disinfecting solution 705 is brought in chamber 601 to a temperature of between 2°C and 37°C, such as approximately 20°C.
• Cleaned raw material 65 and disinfecting solution 705 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30.
• Disinfecting solution 705 is added to mixing device 603.
• at least 3000 mL of disinfecting solution 705 is added to mixing device 603.
• Mixing device 603 is stored in chamber 601 at a temperature of between 2°C and 37°C, such as approximately 20°C.
• Mixing device 603 is combined with heating device 604 configured to agitate disinfecting solution 705 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for between 30 minutes and 240 minutes, such as 120 ⁇ 5 minutes, thereby washing cleaned raw material 65 within vessel 602. Disinfecting solution 705 is decanted from mixing device 603.
• buffer solution 701 is brought in chamber 601 to a temperature of between 2°C and 37°C, such as approximately 20°C.
• Raw material 65 and buffer solution 701 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30.
• Buffer solution 701 is added to mixing device 603.
• at least 3000 mL of buffer solution 701 is added to mixing device 603.
• Mixing device 603 is stored in chamber 601 at a temperature of between 2°C and 37°C, such as approximately 20°C.
• Mixing device 603 is combined with heating device 604 configured to agitate buffer solution 701 at speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for between 5 minutes and 60 minutes, such as 15 ⁇ 5 minutes, thereby washing cleaned raw material 65 within vessel 602.
• Buffer solution 701 is decanted from mixing device 603.
• cleaned raw material 65 is washed with sterile water 708.
• sterile water 708 is brought in chamber 601 to a temperature of between 2°C and 37°C, such as approximately 20°C.
• Cleaned raw material 65 is washed with sterile water 708 at least two times.
• Cleaned raw material 65 and sterile water 708 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30.
• Sterile water 708 is added to mixing device 603.
• at least 3000 mL of sterile water 708 is added to mixing device 603.
• Mixing device 603 is stored in chamber 601 at a temperature of between 2°C and 37°C, such as approximately 20°C.
• Mixing device 603 is combined with heating device 604 configured to agitate sterile water solution at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for between 5 minutes and 60 minutes, such as 15 ⁇ 5 minutes, thereby washing cleaned raw material 65 within vessel 602.
• Sterile water 708 is decanted from mixing device 603 and replaced with fresh sterile water solution.
• Mixing device 603 is combined again with heating device 604 configured to agitate sterile water 708 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for between 5 minutes and 60 minutes, such as 15 ⁇ 5 minutes, thereby washing cleaned raw material 65 within vessel 602 a second time.
• Sterile water 708 is decanted from mixing device 603.
• cleaned raw material 65 is washed with buffer solution.
• buffer solution 701 is brought in chamber 601 to a temperature of between 2°C and 37°C, such as approximately 20°C.
• Cleaned raw material 65 and buffer solution 701 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30.
• Buffer solution 701 is added to mixing device 603.
• at least 3000 mL of buffer solution 701 is added to mixing device 603.
• Mixing device 603 is stored in chamber 601 at a temperature of between 2°C and 8°C, such as approximately 4°C.
• Mixing device 603 is combined with heating device 604 configured to agitate buffer solution 701 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for between 5 minutes and 60 minutes, such as 15 ⁇ 5 minutes, thereby washing cleaned raw material 65 within vessel 602.
• Buffer solution 701 is decanted from mixing device 603.
• cleaned raw material 65 is washed overnight with sterile water.
• sterile water 708 is brought in chamber 601 to a temperature of between 2°C and 37°C, such as approximately 20°C.
• Cleaned raw material 65 and sterile water 708 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30.
• Sterile water 708 is added to mixing device 603.
• at least 3000 mL of sterile water 708 is added to mixing device 603.
• Mixing device 603 is stored in chamber 601 at a temperature of between 2°C and 8°C, such as approximately 4°C, for between 12 hours and 24 hours, such as 16 ⁇ 2 hours.
• mixing device 603 is combined with heating device 604 configured to agitate purified water 703 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, thereby washing cleaned raw material 65 within vessel 602.
• Sterile water 708 is decanted from mixing device 603.
• cleaned raw material 65 comprises a decellularized segmented nerve tissue (referred to as “nerve segment” herein).
• Method 12200 comprises STEPs 12210 through 12290, as described hereinbelow.
• Method 12200 comprises a simplified decellularization process as described hereinabove in reference to Method 12000.
• dissociation solution 704 and disinfecting solution 705 are prepared.
• Dissociation solution 704 and disinfecting solution 705 can be prepared at a temperature of between 2°C and 25°C.
• cleaned raw material 65 from STEP 12010 is removed from vessel 602 and transferred to one or more cassettes 620, and as described hereinbelow in reference to Fig. 27.
• One or more portions of raw material 65 can be anchored to cassette 620, such as to secure raw material 65 to and/or within cassette 620.
• cleaned raw material 65 from STEP 12010 remains in vessel 602 throughout a decellularization process.
• cleaned raw material 65 is washed with purified water 703.
• purified water 703 is brought in chamber 601 to a temperature of between 2°C and 37°C.
• Cleaned raw material 65 is washed with purified water 703 at least two times.
• Cleaned raw material 65 and purified water 703 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30.
• Purified water 703 is added to mixing device 603. In some embodiments, at least 300 mL of purified water 703 is added to mixing device 603.
• Mixing device 603 is stored in chamber 601 at a temperature of between 2°C and 22°C.
• Mixing device 603 is combined with heating device 604 configured to agitate purified water 703 at 100 ⁇ 10 rpm, for at least 10 minutes, thereby washing cleaned raw material 65 within vessel 602.
• Purified water 703 is decanted from mixing device 603 and replaced with fresh purified water 703.
• Mixing device 603 is combined again with heating device 604 configured to agitate purified water 703 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for at least 10 minutes, thereby washing cleaned raw material 65 within vessel 602 a second time.
• Purified water 703 is decanted from mixing device 603.
• cleaned raw material 65 is treated (e.g. washed or otherwise treated) with detergent solution 706.
• detergent solution 706 is brought in chamber 601 to a temperature of between 2°C and 37°C.
• Cleaned raw material 65 and detergent solution 706 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30.
• Detergent solution 706 is added to mixing device 603.
• at least 3000 mL of detergent solution 706 is added to mixing device 603.
• Mixing device 603 is stored in chamber 601 at a temperature of between 2°C and 37°C, such as approximately 20°C.
• Mixing device 603 is combined with heating device 604 configured to agitate detergent solution 706 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for between 30 minutes and 180 minutes, such as 60 ⁇ 5 minutes, thereby washing cleaned raw material 65 within vessel 602.
• Detergent solution 706 is decanted from mixing device 603.
• cleaned raw material 65 is washed with purified water 703.
• purified water 703 is brought in chamber 601 to a temperature of between 2°C and 37°C, such as approximately 20°C.
• Cleaned raw material 65 is washed with purified water 703 at least twelve times.
• Cleaned raw material 65 and purified water 703 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30.
• Purified water 703 is added to mixing device 603.
• at least 3000 mL of purified water 703 is added to mixing device 603.
• Mixing device 603 is stored in chamber 601 at a temperature of between 2°C and 37°C, such as approximately 20°C.
• Mixing device 603 is combined with heating device 604 configured to agitate purified water 703 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for at least 5 minutes, thereby washing cleaned raw material 65 within vessel 602.
• Purified water 703 is decanted from mixing device 603 and replaced with fresh purified water 703. This process is repeated at least eleven additional times, thereby washing cleaned raw material 65 within vessel 602 at least twelve times.
• cleaned raw material 65 is treated with disinfecting solution 705.
• disinfecting solution 705 is brought in chamber 601 to a temperature of between 2°C and 37°C, such as approximately 20°C.
• Cleaned raw material 65 and disinfecting solution 705 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30.
• Disinfecting solution 705 is added to mixing device 603.
• at least 3000 mL of disinfecting solution 705 is added to mixing device 603.
• Mixing device 603 is stored in chamber 601 at a temperature of between 2°C and 37°C, such as approximately 20°C.
• Mixing device 603 is combined with heating device 604 configured to agitate disinfecting solution 705 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for between 30 minutes and 240 minutes, such as 120 ⁇ 5 minutes, thereby washing cleaned raw material 65 within vessel 602. Disinfecting solution 705 is decanted from mixing device 603.
• cleaned raw material 65 is washed with buffer solution 701.
• buffer solution 701 is brought in chamber 601 to a temperature of between 2°C and 37°C, such as approximately 20°C.
• Raw material 65 and buffer solution 701 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30. Buffer solution 701 is added to mixing device 603.
• buffer solution 701 is added to mixing device 603.
• Mixing device 603 is stored in chamber 601 at a temperature of between 2°C and 37°C, such as approximately 20°C.
• Mixing device 603 is combined with heating device 604 configured to agitate buffer solution 701 at speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for between 5 minutes and 60 minutes, such as 15 ⁇ 5 minutes, thereby washing cleaned raw material 65 within vessel 602.
• Buffer solution 701 is decanted from mixing device 603.
• cleaned raw material 65 is washed with sterile water 708.
• sterile water 708 is brought in chamber 601 to a temperature of between 2°C and 37°C, such as approximately 20°C.
• Cleaned raw material 65 is washed with sterile water 708 at least two times.
• Cleaned raw material 65 and sterile water 708 can comprise a ratio between 1 :20 and 1 :50, such as 1 :30.
• Sterile water 708 is added to mixing device 603.
• at least 3000 mL of sterile water 708 is added to mixing device 603.
• Mixing device 603 is stored in chamber 601 at a temperature of between 2°C and 37°C, such as approximately 20°C.
• Mixing device 603 is combined with heating device 604 configured to agitate sterile water solution at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for between 5 minutes and 60 minutes, such as 15 ⁇ 5 minutes, thereby washing cleaned raw material 65 within vessel 602.
• Sterile water 708 is decanted from mixing device 603 and replaced with fresh sterile water solution.
• Mixing device 603 is combined again with heating device 604 configured to agitate sterile water 708 at a speed between 10 rpm and 1000 rpm, such as 100 ⁇ 10 rpm, for between 5 minutes and 60 minutes, such as 15 ⁇ 5 minutes, thereby washing cleaned raw material 65 within vessel 602 a second time.
• Sterile water 708 is decanted from mixing device 603.
• cleaned raw material 65 is treated with one, two, or more excipients 711.
• treatment with excipient 711 comprises sustained immersion in a solution of the excipient for time period that is sufficient to allow for its diffusion into raw material 65 until at least a volume (e.g. 25%, 50%, or 100%) of raw material 65 has been reached.
• treatment with excipient 711 comprises application of the excipient to the surface of raw material 65 via a coating, dipping, and/or spraying process.
• cleaned raw material 65 comprises a decellularized segmented nerve tissue (referred to as “nerve segment” herein).
• Method 13000 can be configured to insert a nerve segment produced in Method 12000 described herein in reference to Fig. 8.
• a suture ligation is created and secured through a first end of the nerve segment.
• a silk suture with a noose is created and secured through the first end of the nerve segment.
• the suture is inserted into lumen 305 of support assembly 300 via proximal end 304, as described herein in reference to Fig. 30.
• the suture is pulled through support assembly 300 such that the nerve segment is advanced into lumen 305.
• the suture is pulled through support assembly 300 until the first end of the nerve segment is proximate distal end 306 of support assembly 300.
• any portion of the nerve segment that is not contained within support assembly 300 can be removed (e.g. cut, or otherwise detached) and discarded.
• support assembly 300 comprises a length of approximately 1cm and any portion of the nerve segment that extends beyond support assembly 300 is removed or otherwise detached (e.g. the nerve segment is cut to comprise a length of approximately 1cm).
• Method 14000 can be configured to lyophilize a nerve segment contained within a support assembly produced in Method 13000 described herein in reference to Fig. 9.
• support assemblies 300 comprising nerve segments can be transferred to, and stored within, chamber 601 comprising heat flow controls and/or methods configured to freeze the nerve segments at a defined rate of freezing and with a defined directionality of the freezing front.
• rate and directionality of freezing can be intended to affect the size and orientation of the water crystals within the nerve segments.
• the water crystal geometries can be configured to affect the geometry of induced macroporosity, pore size, pore orientation, and tissue permeability resulting after lyophilization.
• the nerve segment can be placed into a thermally controlling system placed inside chamber 601.
• Such thermally controlling system can comprise: thermally insulating materials; selective openings to the external environment into chamber 601; internal electrical resistances connected to a controller to provide heating patterns within the geometry of nerve segment; channels for the flow of warmed fluid heated by a heat exchanger connected to a controller and also able to provide heating patterns within nerve segment; and combinations of these.
• Such thermally controlling system can be programmed to control the directionality and speed of the freezing front for the nerve segments.
• Such thermally controlling system can create, for example, 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 a main axis.
• support assemblies 300 comprising nerve segments are stored in chamber 601 overnight.
• one, two, or more support assemblies 300 comprising nerve segments are loaded into lyophilization device 606.
• support assemblies 300 comprising nerve segments are loaded into a preconditioned lyophilization device 606.
• support assemblies 300 comprising nerve segments are lyophilized via lyophilization device 606. In some embodiments, support assemblies 300 comprising nerve segments are lyophilized for 72 hours.
• support assemblies 300 comprising nerve segments are removed from lyophilization device 606.
• support assemblies 300 comprising nerve segments are transferred to, and stored within, chamber 601 comprising a temperature between 2°C and 8°C.
• the nerve segments comprise lyophilized nerve segments (referred to as “lyophilized nerve segment” herein).
• Method 15000 can be configured to create one, two, or more features (e.g. lumen 2122, socket 2210, anchoring tab 2241, etc.) through lyophilized nerve segments produced in Method 14000 described herein in reference to Fig. 10.
• alternate methods such as laser ablation, electrosurgery, or ultrasound cutting, are utilized to create the one, two or more features, such as described in reference to Fig. 41.
• Method 15000 can be configured to increase and/or decrease permeability of the nerve segment radially, longitudinally, circumferentially, or combinations of these.
• one of proximal end 304 and distal end 306 of support assembly 300 comprising the lyophilized nerve segment is inserted and secured within alignment assembly 613, as described hereinbelow in reference to Fig. 26.
• Alignment assembly 613 can be positioned below pin assembly 614 comprising a pin therein.
• one, two, or more features are created within the lyophilized nerve segment.
• pin assembly 614 is lowered towards alignment assembly 613 comprising support assembly 300 such that the pin penetrates at least a portion of the lyophilized nerve segment.
• the pin can be configured to create a channel extending through at least a portion of the lyophilized nerve segment. In some embodiments, the pin creates a channel that extends the full length of the lyophilized nerve.
• Pin assembly 614 can be raised away from alignment assembly 613 such that the pin is removed from the lyophilized nerve segment. Pin assembly 614 can be repositioned above alignment assembly 613 comprising support assembly 300. In some embodiments, alignment assembly 613 can be used to reposition support assembly 300 below pin assembly 614.
• support assembly 300 comprising the lyophilized nerve segment is released and removed from alignment assembly 613.
• support assembly 300 includes one or more components that can be inserted into the channel(s) created to maintain the channel(s) during rehydration of the lyophilized nerve segment.
• the lyophilized nerve segment comprises a nerve graft-conduit (referred to as “PNM-G 2100” herein) or nerve cap-graft (referred to as “PNM-CAP 2200” herein).
• PPM-G 2100 nerve graft-conduit
• PNM-CAP 2200 nerve cap-graft
• Method 16000 can be configured to decellularize PNM-G 2100 or PNM-CAP 2200 prepared in Method 15000 described herein in reference to Fig. 11.
• PNM-G 2100 or PNM-CAP 2200 is treated (e.g. washed) with detergent solution 706.
• PNM-G 2100 or PNM-CAP 2200 is treated with detergent solution 706 for a duration of approximately 8 hours.
• PNM-G 2100 or PNM-CAP 2200 is washed with purified water 703.
• PNM-G 2100 or PNM-CAP 2200 is washed with purified water 703 for a duration of at least 5 minutes at least six times.
• PNM-G 2100 or PNM-CAP 2200 is washed with purified water 703.
• PNM-G 2100 or PNM-CAP 2200 is washed with purified water 703 for a duration of at least 24 hours.
• PNM-G 2100 or PNM-CAP 2200 is washed with purified water 703.
• PNM-G 2100 or PNM-CAP 2200 is washed with purified water 703 for a duration of at least 5 minutes at least six times.
• Method 17000 can be configured to stabilize PNM-G 2100 or PNM-CAP 2200 prepared in Method 16000 described herein in reference to Fig. 12.
• a suture ligation is created and secured through a first end of the PNM-G 2100.
• a silk suture with a noose is created and secured through the first end of PNM-G 2100 or PNM-CAP 2200.
• the suture is inserted into lumen 305 of support assembly 300 via proximal end 304, as described herein in reference to Fig. 30.
• the suture is pulled through support assembly 300 such that PNM-G 2100 is advanced into lumen 305. In some embodiments, the suture is pulled through support assembly 300 until the first end of PNM-G 2100 or PNM-CAP 2200 is proximate distal end 306 of support assembly 300. [298] In STEP 17050, comprising an optional step, any portion of PNM-G 2100 or PNM-CAP 2200 that is not contained within support assembly 300 can be removed (e.g. cut, or otherwise detached) and discarded.
• support assembly 300 comprises a length of approximately 1cm and any portion of PNM-G 2100 or PNM-CAP 2200 that extends beyond support assembly 300 is removed or otherwise detached (e.g. PNM-G 2100 or PNM-CAP 2200 is cut to comprise a length of approximately 1cm).
• support assemblies 300 comprising PNM-G 2100 or PNM-CAP 2200 can be transferred to, and stored within, chamber 601 comprising a temperature of - 80°C. In some embodiments, support assemblies 300 comprising PNM-G 2100 or PNM- CAP 2200 are stored in chamber 601 for a few hours.
• one, two, or more support assemblies 300 comprising PNM-G 2100 or PNM-CAP 2200 are loaded into lyophilization device 606.
• support assemblies 300 comprising PNM-G 2100 or PNM-CAP 2200 are loaded into a preconditioned lyophilization device 606.
• support assemblies 300 comprising PNM-G 2100 or PNM-CAP 2200 are lyophilized via lyophilization device 606. In some embodiments, support assemblies 300 comprising PNM-G 2100 or PNM-CAP 2200 are lyophilized for 72 hours.
• PNM-G 2100 or PNM-CAP 2200 are removed from support assembly 300, and stored at a temperature of between 15°C and 25°C in a very dry and sterile environment prior to packaging.
• Method 18000 can be configured to package PNM-G 2100 or PNM-CAP 2200 produced in Method 17000 described herein in reference to Fig. 13.
• PNM-G 2100 or PNM-CAP 2200 can be packaged for bulk storage and/or sterilization.
• Method 18000 is configured to be performed within an environment suitable for aseptic processing, such that that materials, devices, and components utilized in Method 18000 are transferred to and/or contained within an environment suitable for aseptic processing.
• Method 18000 is performed utilizing a sterile work area and/or sterile handling, such as to prevent or otherwise reduce contamination from microorganisms (e.g. bacteria, fungi, virus, etc.).
• a sterile work area and/or sterile handling such as to prevent or otherwise reduce contamination from microorganisms (e.g. bacteria, fungi, virus, etc.).
• microorganisms e.g. bacteria, fungi, virus, etc.
• Method 19000 can be configured to sterilize the packaged PNM-G 2100 or PNM-CAP 2200 produced in Method 18000 described herein in reference to Fig. 14.
• the containers comprising PNM-G 2100 or PNM-CAP 2200 can be sterilized via gamma irradiation, such that the containers are exposed to gamma radiation (e.g. Cobalt 60).
• gamma radiation e.g. Cobalt 60
• the containers comprising PNM-G 2100 or PNM-CAP 2200 can be sterilized via electron-beam irradiation (“e-beam irradiation” herein), such that the containers are exposed to a stream of electrons.
• e-beam irradiation electron-beam irradiation
• Method 20000 can be configured to ship and/or store the sterilized PNM-G 2100 or PNM-CAP 2200 produced in Method 19000 described herein in reference to Fig. 15.
• the containers comprising PNM-G 2100 or PNM-CAP 2200 can be stored at a temperature of between 15 °C and 25 °C, such as at a temperature of approximately 20°C.
• Method 30000 can be configured to deploy a nerve graft-conduit comprising PNM-G 2100 or never cap-graft PNM-CAP 2200 as described herein.
• the deposit site comprises one, two, or more injured nerves.
• the container comprising PNM-G 2100 or PNM-CAP 2200 is retrieved from a non-sterile field (e.g. storage) by a first, non-sterile operator (e.g. circulating nurse, etc.) and is transported to the room within which the clinical procedure is to be performed (e.g. treatment room, operating room).
• a non-sterile field e.g. storage
• a first, non-sterile operator e.g. circulating nurse, etc.
• the first operator opens the container in proximity to the sterile field where a second sterile operator (e.g. scrub nurse, surgeon, etc.) assists with the transfer of all internal components (e.g. PNM-G 2100 or PNM-CAP 2200) to the sterile field using aseptic techniques.
• a second sterile operator e.g. scrub nurse, surgeon, etc.
• all internal components e.g. PNM-G 2100 or PNM-CAP 2200
• the second operator rehydrates PNM-G 2100 or PNM-CAP 2200.
• PNM-G 2100 or PNM-CAP 2200 is immersed in IX PBS for a duration of between 1 minute and 10 minutes.
• PNM-G 2100 or PNM- CAP 2200 is configured to increase in size logarithmically.
• the second operator transfers the rehydrated PNM-G 2100 or PNM-CAP 2200 to a third, sterile operator (e.g. physician, surgeon, etc.) proximate to a surgical field.
• a third, sterile operator e.g. physician, surgeon, etc.
• the third operator deploys, or otherwise delivers, PNM-G 2100 or PNM-CAP 2200 at the deposit site.
• the third operator alters the size of PNM-G 2100 or PNM-CAP 2200 to a desired length prior to deploying PNM-G 2100 or PNM-CAP 2200 at the deposit site, such as by trimming or otherwise reducing the length of PNM-G 2100 or PNM-CAP 2200.
• the deposit site comprises a nerve gap and PNM-G 2100 is configured to connect two nerve stumps.
• the third operator can create two proximal and two distal sutures (e.g. 9-0 nylon monofilament suture) to secure PNM-G 2100 to the nerve stumps.
• the deposit site comprises an amputated nerve and PNM-G 2100 or PNM-CAP 2200 is attached to an end portion amputated nerve.
• the third operator can create two proximal sutures (e.g. 9-0 nylon monofilament suture) to secure PNM-G 2100 or PNM-CAP 2200 to the amputated nerve.
• the deposit site comprises a nerve transfer and PNM-G 2100 is configured to connect a healthy nerve to an injured nerve.
• the third operator can create two proximal and two distal sutures (e.g. 9-0 nylon monofilament suture) to secure PNM-G 2100 to the healthy nerve and the injured nerve.
• Sizing tool 90 can be constructed and arranged to measure and/or alter the one or more dimensions (e.g. length, diameter, etc.) of PNM-G 2100 or PNM-CAP 2200 and/or one or more dimensions (e.g. length, diameter, etc.) of the lumen 2122, socket 2210, and/or anchoring tab 2214 prior to implantation.
• Sizing tool 90 can comprise elements with which the diameter of PNM-G 2100 or PNM-CAP 2200 and/or the diameter of the deposit site comprising a nerve stump is measured.
• sizing tool 90 comprises two, three, or more circular openings, holes 92a-c as shown.
• Each hole 92 can comprise a unique outer diameter OD.
• hole 92a can comprise an outer diameter smaller than hole 92b, which comprises an outer diameter smaller than hole 92c.
• An operator e.g. physician, surgeon, etc.
• PNM-G 2100 or PNM-CAP 2200 can insert through each hole 92 to measure or otherwise confirm the device’s diameter.
• the operator can trim or otherwise alter the diameter of PNM-G 2100 or PNM-CAP 2200 to achieve a desired diameter prior to implantation.
• An operator e.g. physician, surgeon, etc.
• Sizing tool 90 can further comprise elements with which the length of PNM-G 2100 or PNM-CAP 2200 is measured.
• sizing tool 90 comprises two, three, or more protrusions, protrusions 94a-d as shown.
• Each protrusion 94 can comprise a unique width W.
• protrusion 94a comprises a width smaller than protrusion 94b, which comprises a width smaller than protrusion 94c, which comprises a width smaller than protrusion 94d.
• An operator e.g. physician, surgeon, etc.
• the operator can trim or otherwise alter the length of PNM-G 2100 or PNM-CAP 2200 to achieve the desired length prior to implantation.
• Sizing tool 90 can further comprise one, two, or more elements with which the diameter of the lumen 2122 of PNM-G 2100 or socket 2214 of PNM-CAP 2200 is measured.
• sizing tool 90 comprises two, three, or more cylindrical protrusions, protrusions 94a-d as shown.
• Each protrusion 94 can comprise a unique outer diameter OD.
• protrusion 94a can comprise a diameter that is smaller than the diameter of protrusion 94b, which comprises a diameter that is smaller than the diameter of protrusion 94c, which comprises a diameter that is smaller than the diameter of protrusion 94d.
• Sizing tool 90 can further comprise a length measurement element, ruler 96, comprising two or more markings at regular intervals configured to measure a distance between two reference points. In some embodiments, ruler 96 is utilized to measure a length of PNM-G 2100 or PNM-CAP 2200.
• the nerve graft-conduit can comprise PNM-G 2100 and the nerve cap-graft can comprise PNM-CAP 2200 as described herein.
• the compressed, lyophilized PNM- G 2100 or PNM-CAP 2200 increases in size logarithmically, as shown.
• PNM-G 2100 or PNM-CAP 2200 is rehydrated via immersion in IX PBS for a duration of between 1 minute and 10 minutes.
• Unprocessed nerve tissue comprises excess connective tissue, as shown in Fig. 20A, that is manually, mechanically, chemically, and/or physically removed to result in processed nerve tissue, as shown in Fig. 20B.
• Unprocessed nerve tissue comprises excess connective tissue, as shown in Fig. 21 A, that is manually, mechanically, chemically, and/or physically removed to result in processed nerve tissue, as shown in Fig. 21B.
• Untrimmed nerve segments comprise a native nerve branch system, as shown in Fig. 22A.
• the native nerve branch system is trimmed or otherwise cut into two or more nerve segments according to one, two, or more features (e.g. nerve size, location, fascicular complexity, etc.), such as shown in Fig. 22B.
• the nerve segments can be trimmed or otherwise cut to comprise various diameters and lengths.
• Figs. 23A and B photographs of a nerve segment prepared for insertion into a nerve jacket are illustrated, consistent with the present inventive concepts.
• the nerve jacket can be constructed and arranged similar to that of support assembly 300 described herein.
• a single suture is created and secured to a first end of the nerve segment.
• the suture comprises a silk suture comprising a noose configured to choke the first end of the nerve segment.
• FIGs. 24A and B photographs of a nerve segment inserted into a nerve jacket are illustrated, consistent with the present inventive concepts.
• the nerve jacket can be constructed and arranged similar to that of support assembly 300 described herein.
• the suture created in Figs. 23A and B can be threaded through lumen 305 of support assembly 300, such that the nerve segment is pulled and compressed within lumen 305.
• support assembly 300 comprising the nerve segment therein is trimmed or otherwise cut to a length of 1cm.
• FIG. 25A and B photographs of a nerve graft-conduit implanted at a nerve injury site are illustrated, consistent with the present inventive concepts.
• PNM-G 2100 can be placed proximate a nerve injury site, such as between two nerve stumps. At least two proximal and at least two distal sutures can be created, thereby securing the interface between PNM-G 2100 with each nerve stump
• FIG. 26 a photograph of an alignment assembly positioned below a pin assembly is illustrated, consistent with the present inventive concepts.
• Alignment assembly 613 can comprise a Dremel keyless chuck configured to secure support assembly 300 comprising a nerve segment, as shown. Alignment assembly 613 can be positioned directly below pin assembly 614. Pin assembly 614 can be lowered towards alignment assembly 613 to create one, two, or more channels within the nerve segment, as described hereinabove in reference to Fig. 11. Alignment assembly 613 can also comprise a three-dimensional linear stage that allows movement of the support assembly 300 in X,Y,Z directions. Alignment assembly 613 can be raised towards pin assembly 614 using the linear stage to create one, two, or more channels within the nerve segment.
• Nerve cassette 620 can comprise a housing 621 comprising one, two, or more channels 625 each configured to receive a nerve segment, such as raw material 65 described herein. Housing 621 can further comprise one or more dividers 623 configured to provide a barrier between adjacent channels 625. Housing 621 can comprise one or more fluid-permeable materials configured to allow for the passage of a fluid between cassette 620 (e.g. channels 625 therein) and an external environment.
• Channels 625 can be configured to prevent or otherwise reduce bunching of the nerve segments.
• Each channel 625 can comprise a straight and/or spiral shape.
• each nerve segment can be removably attached to housing 621 via one or more anchoring elements 627. In some embodiments, both ends of the nerve segment are secured via an anchoring element 627.
• Support assembly 300 can comprise a housing 301 constructed and arranged as a cylindrical tube with a lumen 305 therethrough. Housing 301 comprises a proximal end 304 and a distal end 306. Support assembly 300 can be configured to receive raw material 65, ECM 120, and/or PNM-G 2100 (collectively “contents” herein).
• Support assembly 300 can comprise a plurality of pores 309. Each pore 309 can have diameter of between 0.1 micron and 500 microns. Pores 309 can be distributed uniformly along the longitudinal and/or circumferential directions of housing 301. In some embodiments, pores 309 are configured to allow for the passage of a fluid or vapor between support assembly 300 (e.g. contents therein) and an external environment. In some embodiments, a vacuum source is applied to support assembly 300 and pores 309 are configured to apply a uniform vacuum to the contents within support assembly 300. The vacuum source can be configured to maintain uniform contact between an outer surface of the contents and an inner surface of support assembly 300.
• Support assembly 300 can comprise an insulating material to prevent the passage of a fluid, vapor, and/or heat between the tissue contained within support assembly 300 and an external environment (except for in a select axis).
• Support assembly 300 can comprise a material selected from the group consisting of: expanded and non-expanded poly fluoro tetraethylene; poly sulfones; cellulose acetate; polyamide; polyvinylidene fluoride; polysulfone; polyethersulfone; polyvinyl chloride; polyimide; polyacrylonitrile; polyethylene glycol; polyvinyl alcohol; poly(methacrylic acid); poly(arylene ether ketone); poly(ether imide); and polyaniline nanoparticles; stainless steel, such as 316L stainless steel; aluminum; cobalt alloy; titanium alloy; and combinations of these.
• support assembly 300 comprises a hydrophobic, expanded poly fluoro tetraethylene (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 1mm and 10mm.
• ePTFE expanded poly fluoro tetraethylene
• Support assembly 300 can further comprise one, two, or more elements configured to impart structural modifications to the contents therein.
• support assembly 300 can be configured to impart a plurality of channels (e.g. lumens 2122) to raw material 65, ECM 120, and/or PNM-G 2100 via ablation and/or sublimation processes using an excimer laser.
• channels e.g. lumens 2122
• Support assembly 300 can be constructed and arranged to slidingly receive contents therein, such as described hereinabove in reference to Figs. 23 A, B and Figs. 24A,B.
• a nerve segment can be pulled into assembly 300 (e.g. lumen 305) using a suture ligation anchored at one end of the nerve segment.
• the suture ligation can be pulled through assembly 300 (e.g. lumen 305) until a desired portion of the nerve segment is positioned within the assembly.
• Support assembly 300 can be constructed and arranged as a compressive, porous, tubular structure configured to constrict or restrict the contents therein.
• Support assembly 300 can be configured to promote the homogenous and/or unidirectional lyophilization of the contents therein.
• Support assembly 300 can be configured to homogenize the diameter of the contents therein.
• Support assembly 300 can be configured to reduce the size of the contents therein according to a diameter conversion factor.
• Support assembly 300 can be configured to alter the length of the contents therein according to a conversion factor.
• support assembly 300 further includes a component to be inserted into a socket or lumen of the contents therein. This component can maintain (e.g. reinforce) the socket or lumen during rehydration, disinfection, and/or storage of the object (e.g. implant 20, ECM 120, PNM-G 2100, PNM-CAP 2200).
• Support assembly 300 and the related methods can be configured to manufacture at least a portion of implant 20, ECM 120, PNM-G 2100, and/or PNM-CAP 2200 as described herein.
• a nerve graft-conduit for nerve gap injury repair (PNM-G 2100, herein) and a neuro-inhibitory nerve cap-graft (PNM-CAP 2200 herein), which is manufactured using methodologies equivalent to those described for PNM-G 2100, but with some modifications.
• PNM-G 2100 to address the need for solutions for nerve gap repair, which requires a physical bridge between nerve endings.
• the resulting PNM-G 2100 is a graft-conduit device, which applicant has demonstrated to be fully decellularized, biocompatible, and non-cytotoxic, and with shape, size, and surgical handling characteristics suitable for nerve gap repair applications.
• PNM-G 2100 implantation in a rodent nerve gap injury model applicant observed that PNM-G 2100 functioned as a potent and effective inhibitor, rather than inducer of nerve growth. This unexpected outcome is highly desired, yet seldom achieved with current approaches for the prevention of painful neuroma.
• Applicant has determined that the neuro-inhibitory activity of PNM-G 2100 resulted from both the collapse of the device lumen and the excessive compression of the annular matrix surrounding it.
• PNM-CAP 2200 a neuro-inhibitory nerve cap-graft configured to the formation of neuroma following nerve damage sustained during limb amputations.
• PNM-CAP 2200 comprises a decellularized nerve graft-conduit similar to PNM-G 2100, but with the distal end of the material either compressed to form an anchoring tab 2214 or ligated to compress the matrix at the distal end to create a progressive restriction of nerve growth.
• Applicant’s findings demonstrate that PNM-CAP 2200 has potential for use in the field of neuroma prevention and/or treatment.
• FIG. 29A-E histological images of native nerve tissue versus decellularized nerve tissue and assay results for the decellularized nerve tissue are illustrated, consistent with the present inventive concepts.
• PNM-G 2100 comprises an off-the-shelf graft-conduit that is fully decellularized and non-cytotoxic, with shape, size, and handling characteristics suitable for surgical use. Applicant sought to ensure the device materials were fully decellularized and free of myelin, a nerve tissue lipoprotein that can cause a significant inflammatory response. Applicant began by testing the baseline decellularization protocol used for the peripheral nerve matrix (PNM), which is obtained from small, morselized, short segments of porcine sciatic nerve. The nerve segments were then exposed to a series of chemical and enzymatic treatments designed to remove cellular content while maintaining tissue structure and composition.
• PPM peripheral nerve matrix
• Figs. 30A-F a generalized nerve jacketing process is illustrated, consistent with the present inventive concepts.
• Human nerve repair applications require the use of grafts and conduits with a controllable range of diameters and lengths to address the spectrum of anatomical and surgical scenarios.
• Applicant has developed a process that utilizes microporous tubular ‘jackets’ to control the graft-conduit geometry by: restricting the outer diameter of the graft-conduit to the desired size; eliminating the natural taper/reduction in diameter of the nerve from proximal to distal ends; imparting a straight, aligned, and round cross-sectional geometry to the nerve graft-conduit; and creating a temporary external mechanical support for the nerve segment to support additional features, such as the creation of a lumen.
• Nerve jackets may also enable future features including preferential sublimation of water during lyophilization along the longitudinal direction of the decellularized nerve segment to create aligned micro-porosity, and partial sublimation along the radial direction of the nerve segment to improve tissue permeability.
• Fig. 30A shows a nerve segment following a first water bath.
• Fig. 3 OB shows the nerve segment ligated distally.
• Fig. 30C shows the free end of a snare that has been threaded through the nerve jacketed channel.
• Fig. 30D shows the nerve jacket slid along the snare thread to meet the nerve segment.
• Fig. 30E shows the ligated end of the nerve segment pulled through the nerve jacket.
• Fig. 30F shows the resulting jacketed nerve segment.
• FIGs. 31A-D scanning electron microscopy (SEM) images for morphological assessment of nerve graft-conduits following lyophilization in tubing with various porosities are illustrated, consistent with the present inventive concepts.
• Applicant has conducted tests on four different nerve jackets; three comprising expanded polytetrafluoroethylene (ePTFE) tubing with varying porosities (e.g. low porosity of 56% as shown in Fig. 3 IB, intermediate porosity of 67% as shown in Fig. 31C, and high porosity of 79% as shown in Fig. 3 ID) and the other comprising nonporous silicon tubing as shown in Fig. 31 A.
• ePTFE expanded polytetrafluoroethylene
• Results demonstrate that ePTFE jacketed groups, when compared to the nonporous tubing group, have a more porous structure on examination under SEM. ePTFE jackets appear to improve matrix porosity compared to those made with nonporous nerve jackets. Therefore, applicant has tested PNM-G 2100 made with varying porous nerve jackets in a short-term pilot rat sciatic gap model. Results from the gap model demonstrate positive feedback regarding the texture, suturability, and handling characteristics of the rehydrated PNM-G 2100; particularly those formed using the lower porosity ePTFE jacket material. However, applicant observed a significant diameter mismatch between the nerve and PNM-G 2100.
• FIGs. 32A-C photographs of a nerve graft-conduit lumen formation are illustrated, consistent with the present inventive concepts.
• applicant has attempted multiple lumen creation strategies.
• Fig. 32A one method entailed holding a decellularized, jacketed, and lyophilized nerve segment within a dedicated support system, and creating a lumen using a needle inserted longitudinally throughout the full length of the graft.
• FIGs. 33A-G in-vitro and histological images of a nerve graftconduit are illustrated, consistent with the present inventive concepts.
• Applicant has conducted an 8 week animal study that included a 1cm sciatic nerve gap injury model in rats using PNM-G 2100 that were produced using the optimized decellularization method, the correct source tissue diameter, small pore ePTFE jacketing, and the lumen creation method described hereinabove.
• the study consisted of four experimental groups: I) PNM-G 2100 without the lumen; II) PNM-G 2100 with the lumen; III) PNM-G 2100 with lumen filled with ECM 120; and IV) inverted autograft as a positive control and standard of care.
• the inverted autograft control was used as it is known to support nerve regeneration across nerve gap injuries.
• assessment of nerve regeneration motor electrophysiology demonstrated an absence of any appreciable recovery in nerve function in any of the PNM-G 2100 groups, while the nerve autograft was found to recover approximately 20% of the native nerve motor electrophysiologic function. This indicated to applicant that PNM-G 2100 did not support nerve regeneration as initially expected, but rather inhibited it.
• FIGs. 34A and B photographs of a nerve graft-conduit and a nerve cap-graft following implantation at a sciatic nerve injury are illustrated, respectively, consistent with the present inventive concepts.
• Applicant has performed a study to assess the use of PNM-CAP 2200.
• PNM-CAP 2200 were implanted into a model of neuroma formation consisting of the removal of a 1cm portion of the sciatic nerve, and either treatment of its free proximal end with PNM-CAP 2200 or leaving it untreated, which is known to lead to neuroma (e.g. neuroma control).
• the distal nerve stump is left disconnected as shown in Fig. 34B.
• Treatment utilizing PNM-G 2100 is shown in Fig. 34A.
• FIGs. 35A-H histological images of a neuroma control, a nerve graft-conduit, and a nerve cap-graft at 8 weeks post-injury in a rodent model are illustrated, consistent with the present inventive concepts.
• Applicant has conducted studies that demonstrate a progressive loss of myelin starting at the proximal end of PNM-G 2100, PNM- CAP 2200 with near total loss at the distal end. This loss of myelination was accompanied by a progressive decrease in axon number from the proximal to distal end of PNM-G 2100, PNM-CAP 2200.
• Applicant observed the progressive dissipation of axonal growth was accompanied by increasing replacement of the test article by dense connective tissue, with few, if any, axons observable within these areas. Despite the observed progressive loss of myelin, decrease in axonal number, and replacement of PNM-G 2100, PNM-CAP 2200 with connective tissue, applicant observed the linear orientation and organization of the remaining axons in the native nerve and proximal portion of PNM-G 2100, PNM-CAP 2200 was largely maintained, while lacking the presence of aberrant axonal growth producing neuroma-like lesions. Applicant maintains these results are in direct contrast to the histopathologic findings for the transections without repair (i.e.
• neuroma positive control group which was characterized by a well-organized axonal structure at the proximal aspect of the nerve, transitioning into a tangled and randomly-oriented array of axons distally from the level of the transection. This irregular, tangled mass of axons is consistent with neuroma formation.
• Figs. 35A-D Bielschowsky’s silver stain, thereby staining axons brown, was performed on the neuroma control, PNM-G 2100, and PNM-CAP 2200.
• Figs. 35E-H hematoxylin and eosin with Luxol fast blue, thereby staining myelin blue and connective tissue pink, was performed on the neuroma control, PNM-G 2100, and PNM-CAP 2200. Applicant observed the neuroma control is characterized by significant axonal outgrowth from the proximal to distal end as shown in Figs.
• Figs. 36A-T results demonstrating nerve tissue maintaining multiple structure and functional components following decellularization are illustrated, consistent with the present inventive concepts.
• Applicant has developed a technology platform based on decellularized porcine nerve tissue to address peripheral nerve injuries and other conditions. While each of the products derived from this platform have different features, intended use, and stage of development, these products share two common characteristics: the same source of nerve material (i.e. young and healthy porcine sciatic nerve); and a decellularization process, which consists of a series of chemical and enzymatic treatments.
• the decellularized tissue includes a multi-fascicular internal nerve structure, structural and functional proteins like collagen I (as shown in Figs. 36E and F), collagen III (as shown in Figs. 36G and H), collagen IV (as shown in Figs. 361 and J), and laminin (as shown in Figs. 36K and L), as well as neurotrophic factors including BDNF (as shown in Fig. 36Q), CNTF (as shown in Fig. 36R), NT-3 (as shown in Fig.
• Figs. 37A and B a photograph and graphical representation of a gel derived from porcine sciatic nerve promoting Schwann cell (SC) proliferation and axon regrowth at the site of a nerve injury are illustrated, respectively, consistent with the present inventive concepts.
• SC Schwann cell
• ECM 120 is derived from a decellularized matrix via solubilization and other processes as described hereinabove.
• ECM 120 can be delivered at the site of nerve injury and is intended to act both as a scaffold and modulator for native nerve regeneration processes.
• ECM 120 can have a critical impact on the nerve repair and functional recovery following multiple types of acute nerve injuries.
• ECM 120 has been found to support Schwann cell (SC) migration, axon elongation, and a transition from pro- to antiinflammatory macrophage phenotype in a manner which has been shown to promote effective nerve healing.
• SC Schwann cell
• axon elongation a transition from pro- to antiinflammatory macrophage phenotype in a manner which has been shown to promote effective nerve healing.
• delivery of ECM 120 to the site of an injured peripheral nerve can promote SC proliferation and axon regrowth.
• ECM 120 can be formed into a nerve graft-conduit, PNM-G 2100, configured to support nerve regrowth at the site of a nerve injury.
• PNM-G 2100 is configured to serve as a physical bridge for nerve gap injuries.
• PNM-G 2100 can comprise an internal central lumen running along its full length, and the lumen can be surrounded by a neuro-inductive matrix with longitudinally aligned pores; these features are critically important in the initial phases of nerve regeneration as they allow a low resistance path for budding nerve growth and directional cues (i.e. neuro-conductive), which combined with neurotrophic factors, can lead to faster and enhanced regeneration of the tissue (i.e. neuro-inductive).
• PNM-G 2100 utilizes a series of mechanical and physical treatments resulting in a regular tubular geometry with an open lumen spanning the full length of the graft, with a neuro-inductive matrix surrounding the lumen. Therefore, PNM-G 2100 can foster nerve regeneration between two nerve endings by providing a template for axonal regeneration via the internal lumen and neuro-inductive matrix.
• ECM 120 can be formed into a cap with an anchoring tab, such as PNM-CAP 2200 including anchoring tab 2214, configured to inhibit neuroma formation.
• PNM-CAP 2200 is configured to prevent the development of painful neuroma, a common condition following limb amputation caused by an aberrant nerve tissue growth at the severed free nerve ending.
• Inhibition of neuroma formation can be obtained by creating a socket 2210 within PNM-CAP 2200 to receive a free nerve ending and a surrounding the nerve ending with a neuro-inhibitory matrix 2212, permitting the longitudinally aligned growth of the nerve, but progressively slowing the axon growth to a stop without formation of neuroma.
• the inhibition is achieved by exhausting the regenerative potential of the sprouting axons while extending their growth into progressively more restrictive, longitudinally-oriented pores.
• FIG. 40 a table containing feature development parameters of a nerve cap-graft is illustrated, consistent with the present inventive concepts. Applicant has identified six PNM-CAP 2200 design features: outer diameter; socket 2210 diameter; socket 2210 length; neuro-inhibitory matrix 2212 length; neuro-inhibitory matrix 2212 morphology; and anchoring tab 2214.
• the outer diameter of PNM-CAP 2200 can be configured to provide sufficient suture retention strength and separation from surrounding tissues without compromising permeability to nutrients or creating excessive bulk.
• PNM-CAP 2200 comprises an outer diameter of between 2.5mm and 4mm, such as approximately 3mm, when hydrated.
• Applicant can control the nerve outer diameter by modifying the nerve tissue harvest process, such as by stratifying and selecting segments from well-defined anatomical locations to provide several predictable sizes.
• Nerve tissue can be harvested at the main trunk of the porcine sciatic nerve, representing one of the largest diameter nerves available. The sciatic nerve branches into the tibial, common peroneal, and sural nerves.
• Each nerve branch can be harvested at a specified length from the branching point, or at secondary branching points, to achieve control in nerve diameter and fascicular complexity. Additionally, different size jacketing of the nerve can be utilized to provide a consistent outer diameter to the end product.
• the outer diameter of PNM-CAP 2200 can be measured via a vernier caliper and image-based technique for the dry and rehydrated states, respectively.
• the socket 2210 diameter of PNM-CAP 2200 can be configured to allow a clinician (e.g. surgeon) to insert the severed proximal free nerve ending into the product and secure it via standard sutures.
• PNM-CAP 2200 comprises a socket 2210 diameter of between 1mm and 2mm when hydrated.
• the diameter of socket 2210 can be controlled by removing tissue from the nerve in a frozen or lyophilized state to form a central bore via the use of carbide burrs or drill bits mounted in the drill press setup as described hereinabove, and whereby different burr shapes and sizes can be employed.
• the diameter of socket 2210 can be controlled by removing tissue from the nerve in a frozen or lyophilized state to form a central bore via the use of laser ablation, in which the specific size and shape of socket 2210 created is controlled by a user-defined dimension vector graphic.
• the diameter of socket 2210 can be controlled by removing tissue from the nerve in a frozen or lyophilized state to form a central bore via the use of electrosurgery or ultrasound cutting, whereby a different tip size can be employed.
• the diameter of socket 2210 can be measured using a set of pin gauges when PNM-CAP 2200 is in a dry state. Additional visualization of the diameter of socket 2210 can be obtained by tissue surface marking dye and resin casting of the socket. Additional examination under SEM and Micro Computed Tomography (Micro CT) can be performed to further characterize the final geometry of socket 2210 and the morphology of the surface of neuro-inhibitory matrix 2212.
• the length of socket 2210 of PNM-CAP 2200 can be configured to allow sufficient overlap with the proximal free nerve ending to secure the nerve in position, as well as protect it from surrounding tissue, while leaving sufficient space for sutures.
• socket 2210 comprises a length of between 2mm and 5mm, such as approximately 3mm, when hydrated.
• the length of socket 2210 can be controlled by the depth of burr penetration into PNM-CAP 2200 using the same drill press setup as described hereinabove.
• the length of socket 2210 can be controlled using laser ablation by altering settings of: wavelength; power; pulse width; speed; number of passes; vertical distance between tissue and head of laser beam; and combinations of these.
• the length of socket 2210 can be controlled using electrosurgery or ultrasound cutting by altering: power; frequency/waveform, current; rate and regimen of tip longitudinal travel; and combinations of these.
• the length of socket 2210 can be measured using a depth micrometer while the PNM- CAP 2200 is in a dry state. Additional visualization of the diameter of socket 2210 can be obtained by tissue surface marking dye and resin casting of socket 2210.
• the length of neuro-inhibitory matrix 2212 of PNM-CAP 2200 can be configured to act as a substrate for progressive inhibition of nerve outgrowth in a well -organized tissue architecture.
• PNM-CAP 2200 comprises a neuro-inhibitory matrix 2212 length of between 5mm and 20mm, such as approximately 10mm, when hydrated.
• the length of neuro-inhibitory matrix 2212 can be controlled by providing a decellularized and lyophilized tissue of known length, with standard socket 2210 depth, with the remainder of the length of the product representing neuro-inhibitory matrix 2212 and anchoring tab 2214.
• graft-conduit length e.g. 5cm
• axonal exhaustion begins at the proximal end of the graft-conduit with axonal growth termination in ⁇ 10mm length.
• the porosity of neuro-inhibitory matrix 2212 and pore size of PNM-CAP 2200 can be configured to allow a limited number of axons to grow within each pore and travel distally through the inhibitory matrix.
• PNM-CAP 2200 comprises an average pore size of between 10pm and 80pm, such as between 25 pm and 50pm, when hydrated.
• PNM-CAP 2200 comprises a porosity of between 20% and 50%, such as approximately 30%, when hydrated.
• the porosity and pore size of neuro- inhibitory matrix 2212 can be controlled by controlling ECM 120 freezing conditions (e.g.
• the porosity of neuro-inhibitory matrix 2212 can be modified along the length of the PNM-CAP 2200 by providing varying levels of mechanical compression to the inhibitory matrix throughout the manufacturing process (e.g. tapered jacketing, conical or cylindrical compression mold), thereby creating an increasingly dense structure from the proximal to distal end of the PNM-CAP 2200.
• pore size, geometry, alignment, and interconnectivity can be investigated using a modified vascular corrosion casting process.
• the casting process can include perfusing the tissue using a commercially available radiopaque methylmethacrylate-based casting material kit.
• the tissue-polymer composites are treated with a caustic solution to remove all tissue prior to imaging. Imaging can be performed under SEM, or using Micro CT with 3D reconstruction as necessary to fully visualize and quantify the resultant structures.
• Anchoring tab 2214 of PNM-CAP 2200 can be configured to allow firm surgical fixation to surrounding tissue to maintain desired alignment.
• the anchoring tab 2214 can be produced by applying mechanical compression with a bench press to one end of the PNM- CAP 2200, trimming anchoring tab 2214 to the desired shape and size via a punch and die, and punching two holes on either end of the tab to allow anchoring.
• Anchoring tab 2214 is configured to remain intact (i.e. compressed) during hydration to allow it to serve its function at the time of implantation.
• Anchoring tab 2214 is further configured to possess sufficient suture retention strength for its intended application.
• anchoring tab 2214 is crosslinked using physical (e.g. heat) or chemical (e.g. carbodiimide, glutaraldehyde) processes to prevent swelling during hydration, or to increase suture retention strength.
• FIG. 41 a table containing feature development parameters of a nerve graft-conduit is illustrated, consistent with the present inventive concepts. Applicant has identified five PNM-G 2100 design features: outer diameter; lumen diameter; overall length; neuro-inductive matrix morphology; and device degradation rate.
• PNM-G 2100 comprises an outer diameter of between 2mm and 3mm when hydrated.
• the nerve outer diameter can be controlled by modifying the nerve tissue harvest process, such as by stratifying and selecting segments from well-defined anatomical locations to provide several predictable sizes.
• Nerve tissue can be harvested at the main trunk of the porcine sciatic nerve, representing one of the largest diameter nerves available.
• the sciatic nerve branches into the tibial, common peroneal, and sural nerves. Nerve tissue can be harvested from a specific one of these branches at a specified length from the branching point, or at secondary branching points, to achieve control in features such as nerve diameter and fascicular complexity. Additionally, different size jacketing of the nerve can be utilized to provide a consistent outer diameter to the end product.
• the outer diameter of PNM-G 2100 can be measured via a vernier caliper and image-based technique for the dry and rehydrated states, respectively.
• PNM-G 2100 comprises a lumen diameter of between 1mm and 2mm when hydrated.
• the diameter of lumen 2122 can be controlled by removing tissue from the nerve in a frozen or lyophilized state to form a central bore via the use of carbide burrs or drill bits mounted in the drill press setup as described hereinabove, and whereby different burr shapes and sizes can be employed.
• the diameter of lumen 2122 can be controlled by removing tissue from the nerve in a frozen or lyophilized state to form a central bore via the use of laser ablation, in which the specific size and shape of the lumen created is controlled by a user-defined dimension vector graphic.
• the diameter of lumen 2122 diameter can be controlled by removing tissue from the nerve in a frozen or lyophilized state to form a central bore via the use of electrosurgery or ultrasound cutting, whereby different tip size can be employed.
• the diameter of lumen 2122 can be measured using a set of pin gauges when PNM-G 2100 is in a dry state. Additional visualization of the diameter of lumen 2122 can be obtained by tissue surface marking dye. Additional examination under SEM and Micro CT can be performed to further characterize the lumen final geometry and the morphology of the lumen-matrix surface.
• PNM-G 2100 comprises an overall length of between 15mm and 30mm when hydrated.
• the overall length can be controlled by modifying the initial tissue segment length.
• the overall length can be controlled by trimming PNM-G 2100 prior to implantation.
• PNM-G 2100 comprises a neuro-inductive matrix 2112 comprising an average pore size of between 80pm and 250pm, such as between 100pm and 200pm, when hydrated.
• PNM-G 2100 comprises a neuro-inductive matrix 2112 comprising a porosity of between 50% and 80%, such as approximately 60%, when hydrated.
• the porosity and pore size of the neuro-inductive matrix 2112 can be controlled by modifying: one, two, or more freeze casting parameters; one, two, or more laser ablation parameters; and/or one, two, or more porogen leaching parameters.
• PNM-G 2100 comprises a degradation rate of between 30 days and 90 days when hydrated.
• Histological images indicate PNM-G 2100 maintains a continuous structure 30 days post-implantation.
• the degradation rate can be controlled by modifying PNM-G 2100 to comprise a higher density morphology, chemical cross-linking, and/or physical cross-linking.
• Fig. 42 a table containing nerve graft-conduit lumen formation strategies is illustrated, consistent with the present inventive concepts.
• the collapse of the lumen resulted from the use of methods which primarily displaced, rather than removed, the matrix.
• the radially-displaced matrix expanded inward toward the center of the lumen causing closure.
• the lumen should be sized to allow rapid creation and propagation of the nerve growth cone from the proximal stump and support the formation of a tissue bridge across the device in vivo. Also, the lumen should be designed such that the wall of the PNM- G 2100 retains sufficient suture strength for use in nerve gap repair applications, but also retains sufficient kink resistance to prevent the blockage of nerve growth through the lumen.
• Fig. 42 applicant intends to evaluate a diverse set of material removal strategies including mechanical (e.g. burr drilling, etc.), physical (e.g. laser ablation, electrosurgery, ultrasound cutting, etc.), and chemical (e.g. focal corrosion, etc.). Applicant will pair these removal strategies with tools to hold the PNM-G 2100 in place during the lumen formation process, and to center, size, and orient the lumen’s longitudinal axis within the volume of the PNM-G.
• mechanical e.g. burr drilling, etc.
• physical e.g. laser ablation, electrosurgery, ultrasound cutting, etc.
• chemical e.g. focal corrosion, etc.
• lumen 2122 is formed within PNM-G 2100 via drilling, such as burr drilling. Material can be removed by rotational filing or cutting of the tissue therein.
• Control variables can comprise one or more of the following: bit type; bit size; bit geometry; rotational speed; rate and regiment of bit longitudinal travel.
• lumen 2122 is formed within PNM-G 2100 via longitudinal filing.
• Material can be removed by longitudinal filing of the tissue therein.
• Control variables can comprise one or more of the following: file size; rate and regimen of file longitudinal travel.
• lumen 2122 is formed within PNM-G 2100 via a die, such as cylindrical die. Material can be removed via coring the tissue therein via a die. Control variables can comprise one or more of the following: die size; rate and regimen of die longitudinal travel; die rotational speed.
• lumen 2122 is formed within PNM-G 2100 via ablation, such as laser ablation.
• Material can be removed via ablation/sublimation of the tissue therein using laser energy.
• Control variables can comprise one or more of the following: laser type (e.g. carbon dioxide, argon ion, Er-YAG (erbium-doped yttrium aluminum garnet), Nd-YAG (neodymium-doped yttrium aluminum garnet); wavelength; power; pulse width; speed; number of passes; and/or vertical distance between tissue and head of laser beam.
• lumen 2122 is formed within PNM-G 2100 via electrical surgery cutting technique (e.g. cautery). Material can be removed via ablation/sublimation of the tissue therein using electrical current energy.
• Control variables can comprise one or more of the following: tip size; current; rate and regimen of tip longitudinal travel.
• lumen 2122 is formed within PNM-G 2100 via hot wire cutting. Material can be removed via combusting the tissue therein using thermal energy.
• Control variables can comprise one or more of the following: wire size; temperature; rate and regimen of tip longitudinal travel.
• lumen 2122 is formed within PNM-G 2100 via crosslinking.
• Material can be removed via mild chemical treatment to stabilize the luminal surface.
• Control variables can comprise one or more of the following: chemical type; concentration; duration.
• lumen 2122 is formed within PNM-G 2100 via corrosion.
• Material can be removed via focal delivery of a strong acid or base to corrode the tissue therein.
• Control variables can comprise one or more of the following: chemical type; concentration; chemical flow rate; rate of longitudinal motion of capillary tip.
• support assembly 300 includes a protrusion to be inserted into a newly formed lumen 2122 before rehydration, which can increase the stability of lumen 2122.
• lumen 2122 is filled with a material (e.g. PNM) to enhance stability of lumen 2122, mechanical properties of PNM-G 2100, and neuroinductive properties of PNM-G 2100.
• the devices will undergo hydration protocols that simulate use in the operating theater pre-implantation (e.g. 10 minutes in cold sterile saline), and extended in vivo hydration (e.g. 24 hours in sterile saline at 37°C) to assess the stability of the lumen.
• Pre- and post-hydration lumen diameters at different locations along the length of the PNM-G 2100 will be assessed by fixing the rehydrated PNM-G 2100 in 10% formalin and embedding in paraffin, and serial 20pm sections will be cut via a microtome and stained with hematoxylin and eosin.
• Image analysis applied to the histologic images will be used to measure geometrical features of the lumen.
• SEM and Micro CT may be used to assess lumen geometry at multiple levels of the graft-conduit as needed, and as described above.
• FIG. 43 a table containing nerve graft-conduit matrix morphology control strategies is illustrated, consistent with the present inventive concepts.
• the matrix of the PNM-G 2100 did support and induce axonal growth (i.e. neuro-inductive).
• the matrix was found to be neuro-inhibitory. Therefore, in addition to lumen 2122 as described herein, the annular matrix surrounding it represents a second functional element critical to the success of PNM-G 2100.
• the matrix In order to convert the existing matrix from neuro-inhibitory to neuro-inductive, it is believed the matrix must have suitable porosity, pore size distribution and interconnectedness, longitudinal pore alignment, and cell adhesion properties to freely allow and support the infiltration of SC and neurites during the nerve regeneration process from the proximal toward the distal portion of the device.
• Fig. 43 in order to create such a neuro-inductive annular structure, applicant intends to evaluate a diverse set of morphology control strategies including physical (e.g. freeze casting, laser ablation, etc.), and chemical (e.g. porogen leaching, etc.) to affect both the size, density, and alignment of the pores within PNM-G 2100 matrix.
• physical e.g. freeze casting, laser ablation, etc.
• chemical e.g. porogen leaching, etc.
• PNM-G 2100 matrix morphology is controlled via freeze casting. Water freezing, crystal formation, and subsequent water formation can be used to create porosity and other desired morphological features within the matrix.
• Control variables can comprise one or more of the following: freezing parameters; lyophilization parameters; and jacketing compression ratio.
• PNM-G 2100 matrix morphology is controlled via ablation, such as laser ablation.
• Micropore formation can be obtained via ablation/sublimation of the tissue therein using laser energy in conjunction with a metal mesh mask and a raster motion of the laser beam.
• Control variables can comprise one or more of the following: laser type; wavelength; power; pulse width; speed; number of passes; vertical distance between tissue and head of laser beam; and mesh mask geometry.
• PNM-G 2100 matrix morphology is controlled via porogen leaching.
• Organic and/or inorganic porogens e.g. NaCl, other salts
• Control variables can comprise one or more of the following: salt type; salt solution concentration; impregnation parameters; rate of crystallization.
• PNM-G 2100 comprises an initial nominal average pore size of between 100pm and 200 pm, and a porosity of approximately 60%.
• Fig. 44 a table containing nerve graft-conduit clinical requirements is illustrated, consistent with the present inventive concepts.
• applicant has identified features of PNM-G 2100 internal lumen (e.g. lumen 2122) and matrix (e.g. matrix 2112) morphology to obtain desired neuro-inductive properties for use in a rodent model.
• matrix e.g. matrix 2112
• the PNM-G 2100 is available in various diameters and lengths.
• PNM-G 2100 can comprise an outer diameter of between 1mm and 7mm.
• the PNM-G 2100 comprises an outer diameter configured to accommodate a 2mm nerve (e.g. digital nerves).
• PNM-G 2100 comprises an outer diameter configured to accommodate a 4mm nerve (e.g. distal ulnar or radial nerve).
• PNM-G 2100 can comprise an overall length of between 3mm and 10mm.
• PNM-G 2100 comprises an overall device length of at least 3cm to allow trimming and application across a reasonable range of clinically relevant nerve injuries.
• PNM-G 2100 is configured to allow for growth of nerve tissue within PNM-G 2100.
• PNM-G 2100 can be permeable and supportive to the nerve growth cone, thereby allowing cells to move longitudinally across the whole length of the device to ultimately bridge the two nerve stumps. This can be achieved with a hollow lumen 2122 or by filling such lumen with ECM 120.
• PNM-G 2100 is configured with sufficient strength to sustain the tensions, flexions, and torsions between two nerve stumps as a result of physiological motion.
• PNM-G 2100 comprises sufficient suture retention strength. In some embodiments, PNM-G 2100 comprises a suture retention strength > IN/cm. In some embodiments, PNM-G 2100 is compatible for use with nylon monofilament sutures.
• PNM-G 2100 can comprise one, two, or more features to support surgical handling.
• PNM-G 2100 is configured for rapid rehydration, such as rehydration in less than 10 mins.
• PNM-G 2100 comprises a non-tacky surface.
• PNM-G 2100 is easily trimmable by a clinician (e.g. surgeon).
• PNM-G 2100 comprises degradation rates compatible with the speed of nerve growth, such as Imm/day. For example, for a 2cm device, the material will need to remain in place for a minimum of 20 days. In some embodiments, PNM-G 2100 is configured to degrade between 30 days and 60 days. The degradation rate is slower than the speed of nerve regeneration in order to maintain physical guidance between the proximal and distal nerve stumps.
• PNM-G 2100 comprises a kink resistance to prevent closure of lumen 2122 during flexion, thereby inhibiting and/or disrupting nerve regeneration and growth.
• PNM-G 2100 comprises a radius of curvature ⁇ 2cm.
• PNM-G 2100 comprises neuro-inductive properties to enhance its ability to support nerve remodeling and regeneration (as compared with collagen and/or synthetic nerve conduits). In some embodiments, PNM-G 2100 comprises > 20% greater functional recovery than collagen nerve conduits.
• FIG. 45 through Fig. 53 various graphical representations, photographs, histological images, and graphs regarding the development of an acellular nerve cap-graft xenograft for neuroma prevention are illustrated, consistent with the present inventive concepts.
• Neuroma formation following limb amputation is a prevalent and debilitating condition that can deeply affect quality of life and productivity.
• Several approaches exist to prevent or treat neuromas however, no approach is either consistently reliable or surgically facile, with high rates of neuroma occurrence and/or recurrence.
• the present study describes the development and testing of a xenogeneic nerve cap-graft graft made from decellularized porcine nerve.
• the grafts were tested in vitro for cellular removal, cytotoxicity, mechanical properties, and morphological characteristics. The grafts were then tested in rat sciatic nerve gap reconstruction and nerve amputation models for 8 weeks. Gross morphology, electrophysiology, and histopathology assessments were performed to determine the ability of the grafts to limit pathologic nerve regrowth. In vitro testing showed well decellularized and demyelinated nerve cap-graft graft structures without any cytotoxicity from residual reagents. The grafts had a proximal socket for the proximal nerve stump and longitudinally oriented internal pores. Mechanical and surgical handling properties suggested suitability for implantation as a nerve graft.
• the grafts were well integrated with the proximal and distal nerve segments without evidence of fibrotic adhesions to the surrounding tissues or bulbous outgrowth of the nerve. Electrophysiology revealed absence of nerve conduction within the remodeled nerve cap-graft grafts and significant downstream muscle atrophy. Histologic evaluation showed well organized, but limited axonal regrowth within the grafts without fibrous overgrowth and neuromatous hypercellularity. These results provide proof of concept for a novel xenograft-based approach to neuroma prevention.
• Neuromas are pathologic, non-neoplastic, nerve lesions resulting from aberrant and disorganized axonal outgrowth and cellular proliferation caused by nerve injury. These lesions originate from a physiologic outgrowth to restore continuity of the affected nerve; however, when the regenerating bridge fails to reach the distal target, a tangled bulbous mass forms, with axonal trapping and leading to neuropathic pain. This condition is frequently chronic, physically debilitating, and results in reduction of quality of life. Painful neuromas reduce the mobility of those affected, and may lead to psycho-social conditions such as depression and withdrawal.
• neuromas Despite the availability of multiple surgical approaches, there is no clinical ‘gold-standard’ for treatment of neuromas, and success depends on both the nature and location as well as careful patient selection.
• up to 20-30% of neuromas are refractory to these treatments regardless of the type of surgical intervention, and reoperation rates for neuroma are as high as 65%.
• Additional approaches to treat and prevent neuromas include the application of a device (cap or allograft) to the end of the transected nerve.
• a device can be made of synthetic or biologic materials and appear to attenuate painful neuroma formation, potentially by isolating free sensitized axons. While the cap devices have shown promising results in preclinical studies, they have yet to be widely adopted, as the limited clinical evidence available suggests that nerve cap-grafting remains inferior to existing surgical approaches. Recent studies have demonstrated that application of an acellular allograft provides a template for termination of the axonal growth potential within the length of the device; while this approach has shown promise, the length (i.e. 5 cm) of allograft required to exhaust the regenerative potential of the injured nerve creates the need for a more extensive surgical site.
• the resulting decellularized nerve matrix was quantitatively assessed for cytotoxicity to demonstrate the removal of potentially cytotoxic reagents used for the decellularization process. Additional processing of the decellularized nerve tissues was performed to create axon growth -restrictive longitudinal porosity within the graft and an interface for connecting the graft to the nerve end. The resulting grafts were then implanted in a 10 mm rat sciatic nerve gap model for 8 weeks to explore whether such a cap graft approach could be used to inhibit nerve growth, which we believe is an important step towards the prevention of neuroma. Additional pilot studies in a model of nerve amputation were then performed, further demonstrating the utility of the grafts for neuroma prevention.
• the nerve cap-graft graft developed in this study is a cylindrical tissue graft derived from porcine sciatic nerve extracellular matrix.
• the grafts have longitudinal pores with a diameter that is sufficient to allow initial axonal ingrowth, but too restrictive to allow nerve regeneration throughout the graft.
• the grafts are intended to be secured to the proximal stump of a transected nerve using standard surgical techniques.
• the nerve cap-grafts were tested both in vitro and in vivo. In vitro testing was used to assess the critical properties required for implantation as a nerve cap-graft graft.
• the degree of tissue decellularization, the potential for cytotoxicity from residual reagents used during decellularization, and the structural and mechanical properties of the grafts were assessed.
• the grafts were then assessed in vivo in a rat model of sciatic nerve transection to explore the ability of the nerve cap-graft graft to limit nerve growth in a controlled fashion without eliciting neuroma.
• the decellularized nerve segments were then pulled through the lumen of pre-cut polytetrafluoroethylene (PTFE) tubing with inner diameters smaller than those of the nerve segments (2.1 mm internal diameter, and 1 cm in length) until the length of each nerve segment was centered within the PTFE tube.
• the excess tissue protruding from each end of the tubing helped securing the nerve tissue to the tubing during the lyophilization process.
• the PTFE tubing containing segments of decellularized nerve were then frozen at -20°C, and lyophilized in a benchtop lyophilizer (Labconco Freezone 2.5 plus) for 72 hours.
• a total of 2 PBS and 2 water washes were performed to remove the disinfecting solution from the tissue and to neutralize the pH of the grafts.
• the nerve cap-graft grafts were then stored in sterile PBS at 4°C until use.
• a schematic outline of the decellularization process, nerve cap-graft graft structure and features, and representative images of the nerve cap-graft graft are shown in Figure 45.
• Figure 46 Neve cap-graft Graft decellularization analysis. A. Semi quantitative scale for myelin quantification using Woelcke and Eosin stains at lOx magnification.
• NRU Neutral Red Uptake
• Schwann cells (Rat, RT4-D6P2T, ATCC CRL-2768) were separately plated at a density of 10,000 cells/well in a 96-well plate and incubated in DMEM (10013CV, Corning) with 10% FBS (35016CV, Coming) and 1% Penicillin/Streptomycin (15140122, Gibco) at 37°C, 5% CO2 overnight. Cell culture media was then aspirated from the wells with cells and replaced with either extraction media, positive control media (cell culture media with 0.05 mM Doxorubicin), negative control media (fresh cell culture media), or vehicle control media (cell culture media incubated for 48h).
• DMEM 10013CV, Corning
• FBS 35016CV, Coming
• Penicillin/Streptomycin 15140122, Gibco
• JEOL JSM- 6510LV/LGS scanning electron microscope
• Nerve cap-graft graft suture retention force was assessed consistent with the methods described in ASTM F3225-17. Briefly, a tensile force test was performed on a uniaxial testing system (Mark-10, ESM303) mounted with a 5 lb. load cell (Mark-10, M5-5 Force Gauge). All nerve cap-graft grafts were tested in a hydrated state at room temperature. Each graft received a single 7-0 nylon suture placed between the outer surface of the graft and the socket lumen. The suture was located 2 mm from the end of the graft through the luminal wall. A loop of 1 cm in diameter was then created with the suture thread and secured with multiple knots to prevent slippage.
• the distal portion of the graft was secured in vertical position to the lower stage/grip of the uniaxial tensile system and a folded rectangle of 220 grit sandpaper was used to prevent slippage of the sample from the grip.
• the upper stage of the uniaxial tensile system was mounted with a hook connected to the load cell and then lowered to engage the suture loop tethered to the edge of the graft.
• the test was performed by pulling the upper stage at the constant rate of 75 mm/min until graft or suture failure.
• the maximum force (in gf) recorded during the test was considered the suture retention force of the graft.
• the nerve cap-graft was attached to the distal portion in these experiments to evaluate if the nerve cap-graft arrested axon growth despite the presence of the distal nerve to receive any regenerating axons across the nerve cap-graft.
• the nerve cap-graft group in the nerve amputation model was anchored to surrounding muscle tissue adjacent to the distal end of the graft with a 4-0 nylon suture. The muscle and skin were then closed using standard techniques. All animals were survived for 8 weeks following implantation.
• the rat sciatic nerves were exposed and electrophysiology was performed on the proximal nerve stump under anesthesia as described below. Animals were then sacrificed and the sciatic nerve and remodeled nerve cap-graft graft were explanted for gross morphologic examination and histologic assessment.
• Electrophysiology Electrophysiologic function was assessed by measurement of compound motor action potential (CMAP) at the gastrocnemius muscle. Briefly, prior to euthanasia and under anesthesia, the sciatic nerve was isolated and two insulated steel electrodes (26g) were placed around the sciatic nerve proximal to the injury and a bipolar concentric recording electrode (Neuroline Concentric 25 x 0.30 mm, Ambu Inc. MD 21045 Columbia, United States) inserted into the mid body of the gastrocnemius muscle. Supramaximal stimulation was determined by progressively increasing the stimulation current with 0.5mA increments (100 psec pulse duration) until a plateau was reached. Supramaximal CMAP was then recorded in triplicate and the average calculated for statistical assessment.
• CMAP compound motor action potential
• samples were processed and embedded in paraffin blocks and cross-sections of 5 pm in thickness were cut at 2 mm intervals along the longitudinal direction of each sample (3-5 cross-sections per graft), placed on a single slide, and stained with H&E and Luxol Fast Blue (for cellular content and myelin) or Bielschowsky’s silver stain (for assessment of axonal morphology).
• the samples were then evaluated by a board-certified veterinary pathologist with expertise in neuropathology for evidence of nerve regeneration, fibrotic tissue deposition, axonal morphology, and neuroma formation.
• Figure 49 Sudden retention analysis.
• B. Distribution of peak force values reached prior to Nerve cap-graft Graft wall failure. n 10 samples, presented as box and whisker plot).
• the nerve cap-graft graft graft grafts were successfully attached to both the proximal and distal ends of the sciatic nerve at the time of implantation, with the proximal end of the nerve inserted into socket 2210 of the nerve cap-graft .
• the diameter of the graft was observed to be greater than that of the peripheral nerve trunk, and the trunk fit well within the socket of the graft.
• the lumen of the graft was closed around the end of the proximal segment of the sciatic nerve.
• significant remodeling of the graft was observed, with few, if any, remnants of the originally implanted material visible within the site of implantation on macroscopic evaluation.
• Figure 51 Electrode results.
• A. CMAP results (n 4) compared to contralateral side.
• B. Gastrocnemius muscle weight (n 5) compared to contralateral side).
• the nerve cap-graft graft (Figure 52B) was characterized by well organized, but rapidly dissipating axonal structures within the proximal end of the graft when used to repair a nerve gap injury.
• the axons were well aligned, confined to the boundaries of the nerve cap-graft graft, and no axonal escape or evidence of neuroma was noted.
• Myelin labeling demonstrated well organized, myelinated axons within the proximal stump with little to no myelination at mid graft.
• the nerve capgraft grafts were well integrated with the proximal and distal nerve stumps with no evidence of foreign body reaction, rejection, or excessive fibrosis noted on histologic examination and the remodeling grafts were replaced by connective tissue. A prominent angiogenic response was noted along the periphery of the nerve cap-graft grafts. Despite the observed loss of myelin, decrease in axonal number, and replacement of the graft with connective tissue along the longitudinal direction, the linear orientation and organization of the remaining axons in the native nerve and proximal portion of the nerve cap-graft graft was largely maintained. Notably lacking was the presence of aberrant axonal growth producing neuroma-like lesions. These results are consistent with degradation and remodeling of the test article into collagenous connective tissue without significant neuronal outgrowth or neuroma formation.
• Figure 52 Histologic assessment of autograft and nerve cap-graft graft in gap injury model.
• A. The reverse autograft is characterized by well -organized axonal outgrowth from the proximal to distal ends of the graft as shown in Bielschowsky’s silver stain (top). Similarly, myelination is observed throughout the length of the graft as shown in Luxol fast blue/hematoxylin and eosin stained samples (bottom).
• B. In contrast, the nerve cap-graft graft is characterized by rapid axonal exhaustion, with few axons shown at mid graft (top).
• Figure 53 Histologic assessment of untreated and nerve cap-graft graft treated nerve amputation.
• the untreated nerve amputation group is characterized by well -organized axons within the proximal aspect of the amputated nerve, transitioning to an unprotected, disorganized mass of axons at the distal end of the sample. Multiple paths of uncontrolled axonal growth can be observed (top). Similarly, well organized myelination is observed within the proximal aspect of the amputated nerve, with disorganized tissue at the distal aspect of the amputated nerve.
• B Histologic assessment of untreated and nerve cap-graft graft treated nerve amputation.
• the goal of this study was to develop and test a novel nerve cap-grafting approach entailing an axonal growth restrictive matrix composed of a xenogeneic decellularized nerve graft segment.
• the nerve cap-graft graft was approximately 1 cm in length, had a defined interface (e.g. socket 2210) for the proximal nerve stump, and longitudinally oriented pores/channels. Decellularization of the nerve cap-graft graft was confirmed via histologic analysis, which showed that the graft was largely devoid of both nuclei and myelin. Cytotoxic assessment of the nerve cap-graft graft showed absence of cytotoxic residual reagents.
• the nerve cap-graft graft was also assessed for surgical handling and determined to be compatible for use in nerve repair applications. Following implantation in both a nerve gap injury and nerve amputation models, the nerve cap-graft graft appeared to integrate well with the surrounding nerve and muscle tissue without evidence of fibrotic adhesions. Electrophysiology following nerve cap-graft graft implantation after 8 weeks showed complete absence of nerve conduction, indicating the nerve cap-graft graft effectively inhibited nerve axonal regrowth across the nerve injury models used.
• axonal sprouting lacks appropriate directional guidance and signaling cues, leading to a disorganized and unregulated growth (neuroma formation). Histologically, neuroma formation is identifiable as early as at 28 days after injury in rodents.
• the present exploratory study utilized a porcine decellularized nerve extracellular matrix structure applied to the proximal stump of the rat sciatic nerve following transection in both gap and nerve amputation models.
• the device was designed with a socket to host the proximal nerve stump and prevent axonal escape at the nerve-device interface.
• the main working hypothesis of the present study was that the application of a longitudinally-oriented, but complex and restrictive, guiding structure would allow limited and controlled axonal outgrowth, effectively inhibiting the formation of neuroma. This approach could offer multiple advantages over existing clinical methods for neuroma prevention.
• transposition of the nerve stump into muscle, bone, or a vein is often preferred.
• This approach provides a dedicated space where nerve regeneration can occur; however, the lack of a directional structural template still exposes the nerve to the risk of disorganized axonal outgrowth or aberrant reinnervation.
• more complex surgical approaches have been used to create new functional interfaces between nerve and muscle tissue in amputees (e.g. targeted muscle reinnervation, regenerative peripheral nerve interface), reducing the rate of neuroma and enabling control of muscles and external prostheses; however, these approaches are highly specialized microsurgical procedures and require use of different surgical expertise, when, ideally, neuromas should be prevented by the vascular or trauma surgeons performing the amputation.
• nerve cap-grafting devices made of synthetic or biological degradable materials are intended to provide protection from the tissue injury inflammatory environment. While these devices can provide a macrostructural template to contain axonal regeneration, they can also create nerve ‘compartment syndrome’ as they can become a ‘barrier/trap’ for growing/ sprouting axons. Furthermore, the degradation products of the synthetic material used for one of these technologies (PLLA-PCA) has been associated with 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 human data.
• the preliminary approach described here may improve upon the aforementioned limitations of existing approaches by using a degradable ECM material that has been shown to modulate an anti-inflammatory, constructive remodeling profile when used for nerve applications.
• This nerve cap-graft graft offers a protected and directional path for organized nerve regeneration, with the addition of intrinsically resistive elements for axonal sprouting.
• Such a combination of features can become an effective approach for neuroma prevention by allowing the development and exhaustion of the nerve regenerative potential, but without requiring extensive lengths, as previously described for the use of acellular nerve allografts.
• porcine sciatic nerves have an average of 11-20 fascicles, including small branches (2 mm in diameter), whereas rat sciatic nerves have an average of 3 fascicles.
• the device constitutes a neuroinductive matrix contained within a nerve graft structure (PNM-G 2100).
• PNM-G 2100 can be sutured to a peripheral nerve stump at one end and a distal nerve stump at the opposite end.
• PNM-G 2100 can facilitate the growth of axons from the peripheral nerve stump through to the distal nerve stump.
• Fig 54A illustrates a representative front of axon progression through PNM-G 2100 to the distal nerve stump at 1, 2, 4, and 8 weeks.
• Axons grow at a relatively equal rate, resulting in growth cone presence at similar depth for each time point. Additionally, myelin is present around axons at a uniform depth difference from the growth cone, consistent with the myelination of healthy growing axons.
• Representative cross-sections at the 4-week timepoint further illustrate the relative positioning of growth cones, myelinated axons, and unmyelinated axons within PNM-G 2100.
• a graphical model of the representative axon growth in Fig. 54A through PNM-G 2100 is illustrated in Fig 54B.
• Axon growth cones move through the device in a wave front of uniform distribution.
• Axon density at the neurorrhaphy plane approaches a constant over time, which is consistent with the axonal density of the peripheral nerve stump. With increasing depth, the axon density decreases, and the slope of this decline becomes less steep over time as more axons infiltrate further into the device.
• the axon density approaches a plateau of constant density across the device, which represents the time at which there are no growth cones within PNM-G 2100 and the axons are fully formed across the gap injury into the distal stump.
• the myelin density follows the trend of axon density with a time shift to account for the region of unmyelinated axons immediately following the growth cone.
• the device constitutes a neuroinhibitory matrix contained within a cap structure (PNM-CAP 2200), which is sutured to a peripheral nerve stump in order to prevent neuroma formation.
• PNM-CAP 2200 can slow and eventually stop the growth of axons from the peripheral nerve stump before the end of the neuroinhibitory matrix.
• Fig 54C illustrates a representative front of axon progression through PNM-CAP 2200 at 1, 2, 4, and 8 weeks. As depth into PNM-CAP 2200 decreases, the front becomes less uniform, indicating that some axons growth has been slowed or stopped.
• myelination of axons decreases as axons progress into the matrix, resulting in a greater length of unmyelinated axons at later timepoints.
• Representative cross-sections at the 4-week timepoint further illustrate the relative positioning of growth cones, myelinated axons, and unmyelinated axons within PNM-CAP 2200.
• a graphical model of the representative axon growth in Fig. 54C through PNM-CAP 2200 is illustrated in Fig 54D. Growth cones move through the device in a wave front.
• the neuroinhibitory nature of the PNM-CAP 2200 matrix causes both the axon density of each front and the distance between fronts to decrease over time as axon growth slows.
• Axon density at the neurorrhaphy plane approaches a constant over time.
• the axon density decreases rapidly, although the slope of this decline becomes less steep over time as more axons infiltrate further into the device.
• Axon density does not extend the entire length of the device.
• the myelin density follows the trend of axon density; however, the myelin is a consistently lower density, and does not penetrate to the same depth into the device as the axon growth.
• Method 40000 can be configured to customize the choice of raw material 65.
• Method 40000 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 graftconduit (e.g. PNM-G 2100) or nerve cap-graft (e.g. PNM-CAP 2200).
• a nerve segment comprising a larger diameter requires longer incubation period in detergent solution to obtain the same level of decellularization as a nerve segment comprising a small diameter.
• fascicular complexity can impact the degree of decellularization.
• a nerve graft-conduit e.g. PNM-G 2100
• nerve cap-graft e.g. PNM- CAP 2200
• the decellularization process can be tailored to each individual nerve segment to produce equally decellularized grafts with different features (e.g. external diameter and fascicular complexity).
• a graft (e.g. PNM-G 2100, PNM-CAP 2200) can comprise a nerve segment from the main sciatic trunk comprising a large diameter and high fascicular number. This nerve segment can require a longer incubation in detergent solution, such as an incubation period of 10 hours.
• a graft (e.g. PNM-G 2100, PNM-CAP 2200) can comprise a nerve segment from the sural branch of the sciatic nerve comprising a smaller diameter and low fascicular number. This nerve segment can undergo a shorter incubation in detergent solution, such as an incubation period of 6 hours.
• the cellular content in the detergent solution may be monitored through one, two, or more features, such as turbidity, absorbance, conductivity, and pH.
• one, two, or more features of a desired nerve segment are defined.
• the external diameter, length, and/or structure/fascicular complexity of the desired nerve segment can be defined based on the intended use. For example, if a graft (e.g. PNM- CAP 2200) is intended to cap a nerve stump comprising a diameter of 3 mm in diameter with the goal of preventing neuroma formation, the graft would be derived from a nerve segment sourced from anatomic locations of similar external diameter.
• the intended use as PNM- CAP 2200 can also inform the structure/fascicular complexity of the nerve segment source. In some embodiments, it is desirable to select a nerve segment from an anatomic location characterized by high fascicular complexity.
• a nerve segment having one, two, or more the desired features as defined in STEP 40010 is selected.
• the source nerve segment would be selected from a nerve branch and location known to possess at least one of the desired features.
• the nerve segment as selected in STEP 40020 is further selected to begin at a known distance from a branching point (e.g. a proximal branching point).
• the nerve segment location is selected to comprise at least one desired feature and/or to allow sufficient a graft length.
• a desired degree of tissue processing and/or decellularization of the nerve segment for a target application is defined.
• the nerve segment can be processed and/or decellularized to a degree (e.g. high degree) configured to completely remove adhesive molecules, such as laminin, which is known to support nerve regeneration.
• the nerve segment can be processed and/or decellularized to a degree (e.g. low degree) configured to preserve adhesive molecules, such as laminin.
• tissue processing and/or decellularization parameters include the incubation time, the temperature, the concentration of the reagent use, the degree of washing following treatment with the reagents, etc.
• the degree of tissue processing and/or decellularization of the nerve segment can be analyzed during and/or following the tissue processing and/or decellularization to confirm that the desired features have been obtained.
• the degree of tissue processing can be measured and/or otherwise tracked during the process by measuring the absorbance to visible light through the solution used for decellularization and/or the solution used for washing the tissue following tissue processing.
• the degree of tissue processing and/or decellularization and the structural and fascicular complexity of the nerve segment can be also confirmed by performing histological analysis of a sample of the nerve segment. The diameter and length of the nerve segment can be confirmed with standard tools after completion of the process.

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