CN115361912A

CN115361912A - Method and apparatus for in situ formation of a nerve cap with quick release

Info

Publication number: CN115361912A
Application number: CN202180020944.3A
Authority: CN
Inventors: 科琳·布赖特; 任永; 肯恩·马丁; 法哈德·科斯拉维; 阿玛普里特·S·索内
Original assignee: Incept LLC; Tulavi Therapeutics Inc
Current assignee: Incept LLC; Tulavi Therapeutics Inc
Priority date: 2020-01-13
Filing date: 2021-01-13
Publication date: 2022-11-18
Also published as: EP4090261A1; WO2021146330A1; CA3163429A1; AU2021207853A1

Abstract

Methods, devices, and materials for forming implants for treating nerves in situ. The treatment site is located within a cavity defined by the structure. A deformable medium is introduced into the cavity to surround the treatment site. The medium undergoes a transition from a relatively flowable state to a relatively non-flowable state to form a protective barrier around the treatment site. The hydrophilic medium cooperates with the hydrophobic surface of the cavity to facilitate rapid removal of the implant from the cavity after transformation. The implant may be a growth-inhibiting nerve cap to inhibit neuroma formation following planned or traumatic nerve injury, a growth-permitting catheter to facilitate reconnection of a severed nerve, or an anchor to stabilize a pain management electrode relative to a nerve. Access to the nerve treatment site may be open surgery or percutaneous.

Description

Method and apparatus for in situ formation of a nerve cap with quick release

Incorporation by reference of any priority application

This application is a continuation-in-part application of patent cooperation treaty application No. PCT/US2019/040429 filed on 7/2 in 2019, with prior applications claiming rights and benefits from 35u.s.c.119 (e) as non-provisional applications of U.S. provisional application No. 62/692,858 filed on 7/2 in 2018 and U.S. provisional application No. 62/822,881 filed on 3/24 in 2019. This application also claims priority from U.S. provisional patent application No. 62/960,564, filed on 13/1/2020, all of which are hereby incorporated by reference in their entirety.

Background

Neuroma is a benign tumor, originating in neural tissue, consisting of abnormally sprouting axons, schwann cells and connective tissue. Although neuroma may appear after various types of injury, some of the most common and most therapeutically challenging trauma or surgery results from damage or transection of nerve tissue. Amputation requires the severing of one or more sensory or mixed nerves. Chronic neuropathic pain, caused by neuroma formation, occurs after up to 30% of patients, and leads to downstream challenges of wearing prostheses and poor quality of life. In addition to traumatic and amputation-related neuroma, neuroma formation occurs in a variety of clinical indications, such as general surgery (hernia repair, mastectomy, laparoscopic cholecystectomy), gynecological (caesarean section, hysterectomy) and orthopedic (arthroscopy, amputation, knee replacement).

Neuroma is part of the normal repair process following peripheral nerve injury. They form when nerve recovery to the distal nerve end or target organ fails and nerve fibers regenerate incorrectly and irregularly into the surrounding scar tissue. Neuroma includes a disorganized structure of tangled axons, schwann cells, endoneurial cells and perineurial cells in a dense collagen matrix, surrounded by fibroblasts (Mackinnon S E et al 1985. Alternation of neural formation by manipulation of bits micro-environment. Plant Reconstration Surg.76: 345-53). Upregulation of certain channels and receptors during neuroma development can also lead to abnormal sensitivity and spontaneous activity of injured axons (Curtin C and Carroll I.2009.Cutaneous neuroma physiology and its relationship to chronic pain.J.hand Surg am.34: 1334-6). It is known that randomly arranged nerve fibers produce abnormal activity that stimulates central neurons (Wall P D and Gutnick M.1974. Ingoing activity in peripheral nerves; physiology and pharmacology of impulses orientation from exp neuron.43: 580-593). This ongoing abnormal activity can be enhanced by mechanical stimulation (e.g., scarring that is constantly reestablished at the site of injury) (Nordin M et al 1984. Echopic sensory sources and parasthesia in tissues with disorders of cutaneous nerves, dorsal roots and dorsal columns. Pain.20:231-245 scanning J W1981. Development of sexual activity, mechanistic sensitivity, and adenosine sensitivity in transformed cutaneous nerve, exp nerve.73. 345-364).

Neuroma or continuous neuroma at the nerve stump is an inevitable consequence of nerve damage, leading to debilitating pain when the nerve is not or cannot be repaired. It is estimated that approximately 30% of neuromas become painful and problematic. This is particularly likely if the neuroma is present at or near the surface of the skin, as physical stimulation can cause signal transduction in the nerve and thus pain.

In recent years, the number of amputees in the world has increased dramatically, with war injuries and vascular disorders (e.g., diabetes) accounting for approximately 90% of all amputees cases. In the united states alone, there are currently about 170 million amputees, with over 23 million new amputees being discharged from the hospital each year. Furthermore, it is estimated that by 2050, the number of new amputations will increase by 20% per year.

Unfortunately, due to the constant pain of the stump, approximately 25% of amputees are unable to begin rehabilitation, let alone resume daily activities. The cause of this pain may be neuroma. A recent study reported that during the 25 year study, 78% of amputees suffered from mild to severe pain due to neuroma formation, with 63% describing pain as persistent soreness. This pain is also often described as a sharp, shooting or shock-like illusion lasting many years after surgical amputation. In addition, patients palpate the skin overlying the neuroma with tenderness, spontaneous burning, allodynia (allodynia) and hyperalgesia.

While various methods have been used to prevent, minimize or protect neuroma in an attempt to minimize neuropathic pain, the current clinical "gold standard" for treating neuroma is traction denervation, in which the nerve is pulled forward under traction and transects the nerve as far back as possible, with the hope that if a neuroma forms, it will be deep in the tissue. Another accepted method is to bury the proximal nerve ends (which will form neuroma) in a drilled hole in muscle or bone. The nerve is then sutured to the periosteum of the muscle or bone to maintain its position. The rationale is that the surrounding tissue cushions and isolates the neuroma to inhibit stimulation and the resulting pain sensation. However, this procedure can complicate the procedure by requiring a significant amount of additional dissection of otherwise healthy tissue to place the nerve stump. This, coupled with poor and variable treatment, lack of suitable/usable tissue, and the need for additional surgical time, has led to fewer procedures to prevent neuroma formation.

Another approach is to cut back the nerve stump, leaving a section or set of overhanging epineurium. This protruding portion may be ligated to cover the surface of the nerve stump. Alternatively, a segment of the adventitia may be obtained from other nerve tissue, or the corresponding nerve stump may be cut back to form an adventitia cuff that may be used to connect to and cover the other nerve stump.

Another common method is suture ligation, in which a loop of suture is wrapped around the nerve end and tightened. This pressure is believed to mechanically block the exit of the axon and cause the tip to eventually form scar tissue at that location. However, clinical and preclinical evidence suggests that this procedure can lead to the formation of painful neuroma after ligation. Furthermore, ligated nerves are generally not positioned to minimize mechanical irritation of the neuroma, as it is expected that scar tissue will provide adequate protection to the nerve ends.

Other methods used clinically include placing the Nerve stump in a solid implantable silicone or biodegradable polymer tube with an open or more proximally sealed end (e.g., polynanics neuroap) or a stented closed tube (e.g., axogenn Nerve)

) (ii) a The proximal nerve end is then encapsulated with harvested vein or fat grafts in order to provide a physical barrier to abnormal nerve regeneration. The use of biomaterial implant devices and methods requires insertion of sutures in the opening of the device, followed by fixation of the nerve, which can be difficult and further damage to the nerve ends. For example, current procedures for securing NEUROCAP require placement of a suture in the adventitia of the nerve and through the vessel wall, then use of the suture to pull and fill the nerve into the lumen, and placement of multiple sutures to hold the nerve in the device. These methods and devices can also result in mechanical stimulation of the neuroma tissue due to 1) mismatch between tissue conformability and catheter rigidity, and 2) the inability of the cap to prevent the formation of neuroma within the cap due to the potential space, resulting in painful sensations. Although these neurophats degrade over a period of 3 months to 24 months, a substantial degradation-mediated loss of mass occurs within the first 3 to 6 months, resulting in exposure of the temporarily protected neuroma to the surrounding environment And the debris stimulates fibroblast infiltration and scarring around the healing nerve. Thus, the efficacy of these preformed implantable caps is limited by the ability of the cap to conform to the proximal end of the nerve and prevent neuroma formation, followed by their subsequent degradation to expose the neuroma to the surrounding environment and to trigger degradation products of adverse inflammatory reactions. Finally, the time and skill required to fix these implants under surgical magnification (loupes) or surgical microscope limits surgeons from adopting these procedures more extensively, as these procedures require the use of fine sutures (9-0 nylon or 8-0) for suturing.

Unfortunately, current methods for addressing neuroma formation and pain caused by neuroma have not been widely adopted. Thus, in addition to reducing scar formation and perineural adhesions, there is a need for an effective technique or therapy to control or inhibit neuroma formation following inadvertent or planned surgical or traumatic nerve injury.

A variety of biomaterial catheters have been explored in the preclinical field in an attempt to prevent Neuroma Formation, including polylactide/polycaprolactone-based other solid implantable biodegradable polymer catheters (Onode et al (2019) novel clamping with a novel continuous for the Treatment of a fungal Nerve in the Rat diagnostic catheter, J neurosurg.p.1-9, yan et al (2014) Mechanisms of New Capping Technique in advance of Painful neuron Formation, PLOS One,9 (4) p.1-11 Yi et al (2018) Painfu Terminal neuron prediction by Capping PGRD/PDLLA adapter in Rat scientific new adv.Sci, 1-11), atelocollagen (Sakai et al (2005) prediction and Treatment of purification New by an acellulologen Tube in Rat scientific new sources J Biomed. Part B: appl Biomate 73B (355-360)) or Porcine small intestinal submucosa (Tork et al (2018), ePoster: preceding of Neuromas with a Porcine SIS nercap: histopathological Evaluation, http:// meeting. Hand cery.org/files/8/epoters/HSEP106. Pdf) or microcrystalline chitosan (Marcol et al (2011) Reduction of Post-642 classical Neuroma and Epineural scanner Formation in Rat scientific Nerve by Application of Microcrystalline chip. Microware, 31. To date, these approaches have not been successful in preventing neuroma formation, either because solid implants cannot be formed in situ and create potential space to allow nerve growth (outlet) and varying degrees of neuroma formation, or, most importantly, because the persistence of these materials in the body is insufficient to prevent neuroma formation.

Other applications of the techniques directed to wrapping or joining nerves are also described to prevent abnormal axonal growth and promote nerve regeneration. In particular, methods are described for protecting nerves by encapsulating the nerves in an in situ formed hydrogel delivered in a temporary wrap or methods are described for assisting nerve regeneration using a growth supporting/permissive solution or gel, rapidly resorbable thin sheet wrap in combination with an in situ formed inhibitory hydrogel.

Disclosure of Invention

In accordance with one aspect of the present invention, a method of forming a conformable, protective nerve cap in situ is provided to inhibit neuroma formation at severed nerve ends. The method includes the steps of identifying a severed end of a nerve; positioning the severed end in a cavity defined by a structure; introducing a medium into the structure to surround the virtual severed end; and allowing the media to undergo a transition from a first, relatively flowable state to a second, relatively non-flowable state to form a protective, conformable barrier around the severed end. The method may further comprise the step of removing the structure to leave a biocompatible in situ protective nerve cap formed.

The step of identifying a severed end of a nerve may comprise identifying a nerve severed, such as by cutting or ablation or by traumatic means. The structure may include a nerve guide (a neural guide) and the positioning step may include positioning the nerve such that the nerve guide retains the virtual severed end within a cavity spaced from a side wall of the structure. The tip of the severed end may be positioned at least about 0.1mm or 2mm from the sidewall, or more preferably about 1mm from the sidewall. Preferably, the structure is either bioabsorbable or is comprised of a flexible non-degradable material that can be easily removed from the surgical site after in situ nerve cap formation.

The transition from the flowable state to the non-flowable state can occur within about 1 minute, or within about 30 seconds, or within about 10 seconds of the introducing step. The method may further comprise the step of aspirating an amount of axial fluid from the severed nerve prior to the introducing step. The method may alternatively include the step of axonal fusion using a "PEG fusion" protocol described later before placing the nerve within the neural structure.

In one embodiment of the invention, the structure may comprise a first configuration in which the cavity is exposed and a second configuration in which the cavity is partially or fully covered; and further comprising the step of advancing the structure from the first configuration to the second configuration after the step of introducing the nerve. Alternatively, the step of advancing the structure from the first configuration to the second configuration may occur prior to the step of introducing the nerve or mediator. The structure may alternatively comprise an open cell foam, and the cavity comprises a tortuous, interconnected interstitial volume within the foam. In the latter embodiment, the structure will remain in place after integration with and formation of the nerve cap. Alternatively, the structure may comprise a porous lyophilized biomaterial that dissolves within minutes to hours after exposure to a physiological liquid

The step of identifying severed nerves can include the step of severing the target nerve, for example, using scissors, a blade (e.g., a 10 or 11 blade), or a razor blade. This step may additionally include cleanly transecting the nerve at an oblique angle prior to placing the nerve within the structure. Alternatively, the nerve may be repaired with a nerve cutting or trimming device.

The converting step may include a crosslinking reaction or a polymerization reaction, and may use an in situ formed hydrogel that can be embedded with host tissue to form a bond between the hydrogel and the tissue. In a preferred embodiment, the hydrogel is a neutral or negatively charged material with submicron or smaller pores that allow nutrient and protein exchange, but do not allow cellular penetration. In one embodiment, the transformation results in a synthetic crosslinked hydrogel protective barrier through which nerves cannot regenerate around transected nerve stumps. By crosslinking the hydrogel at the distal end of the transected nerve containing the severed axon, the hydrogel provides a physical block to nerve regeneration or neuroma formation and, when it absorbs fluid and equilibrates, draws fluid from the axicon and cellular/cytoplasmic debris from the transected nerve, thereby enhancing the self-sealing ability of the axon membrane.

According to another aspect of the present invention, a method of forming an implant in situ is provided, the implant being quickly releasable from a mold. The method comprises the steps of identifying a nerve; positioning a nerve in a cavity defined by a structure; introducing a medium into the cavity to surround the nerve; and allowing the medium to undergo a transition from a first, relatively flowable state to a second, relatively non-flowable state to form a protective barrier around the nerve; wherein the hydrophilic character of the medium is coordinated with the hydrophobic character of the cavity to facilitate rapid release of the implant from the cavity after transformation.

The positioning step may include positioning a severed end of a nerve into the cavity and allowing the medium to transform to form a protective barrier around the severed end of the nerve. The implant may be removed from the cavity by applying a pulling force of no more than about 10N for no more than about 5 seconds. In some embodiments, the implant can be removed from the cavity by applying a pulling force of no more than about 5N for no more than about 2 seconds, or no more than about 2N. Typically, the implant can be removed from the cavity within 10 seconds, preferably within 5 seconds, preferably within about 2 seconds, without breaking the connection between the implant and the nerve.

The step of introducing the medium may comprise introducing a first volume of medium and, after the first volume is switched, introducing a second volume of medium. In one embodiment, the first layer is delivered first to localize the nerve, followed by the second layer to completely cover the nerve.

Another method of forming an implant in situ includes the steps of determining a site for implant formation; positioning a structure having an implant cavity at the site; introducing a medium into the cavity to form an implant precursor; and allowing the medium to undergo a transition from a first, relatively flowable state to a second, relatively non-flowable state to form the implant; wherein the hydrophilic character of the medium is coordinated with the hydrophobic character of the cavity to facilitate rapid release of the implant from the cavity after transformation.

The implant may be a nerve cap for inhibiting the formation of neuroma around the severed nerve end, a wrap for protecting the nerve from inflammation and scar formation, or may be a nerve conduit for guiding nerve growth. Alternatively, the implant may be a tissue expansion implant or a vaso-occlusive device. Alternatively, the implant may be used as a tendon protector or to promote tendon repair.

A method of forming a nerve cap in situ may include the steps of identifying a severed end of a nerve; positioning the severed end in a cavity defined by the structure; introducing a medium into the cavity to surround the severed end; and allowing the medium to undergo a transition from a first, relatively flowable state to a second, relatively non-flowable state to form a protective barrier around the severed nerve end; wherein the hydrophilic properties of the medium cooperate with the hydrophobic properties of the cavity to facilitate rapid release of the nerve cap from the cavity after the transition.

The method may further comprise the step of removing the structure. The identifying step may include identifying a surgically severed nerve.

The structure may include a nerve guide, and the positioning step may include positioning the nerve such that the nerve guide retains the severed end within the cavity spaced from the structure sidewall. The severed end can be located at least about 1mm from the sidewall. The nerve may be circumferentially covered with a hydrogel layer of at least 0.05mm, preferably at least 0.5mm, more preferably at least 1mm, although greater hydrogel thickness may be acceptable if there is sufficient space.

The transformation may occur within about 30 seconds of the introducing step, preferably within about 10 seconds of the introducing step, preferably within about 5 seconds of the introducing step. The method may further comprise the step of aspirating an amount of axicon from the severed nerve prior to the introducing step.

The structure may comprise a first configuration in which the cavity is exposed and a second configuration in which the cavity is covered. The method may further comprise the step of advancing the structure from the first configuration to the second configuration after the step of introducing the medium. In another embodiment, a nerve is placed within a cavity in a first configuration, the structure is moved to a second configuration in which the nerve is encapsulated in the structure, and then a medium is delivered through a port in the structure. The structure may have a clamshell cover and may have an inlet area for retaining nerves and preventing premature outflow of PEG from the structure. The structure may comprise an open-cell foam, and the cavity may comprise an interstitial volume within the foam. The step of identifying a severed nerve comprises the step of severing a target nerve.

The transformation may comprise crosslinking or polymerization. This transformation can create a synthetic crosslinked hydrogel protective barrier. To prevent the formation of neuroma, the protective barrier may have an in vivo persistence of at least about two months or at least about three months, more preferably 6 months or longer. This transformation can result in swelling of the medium by volume from about 2% to about 60%, alternatively from about 5% to about 30%, preferably 10% to 20%, when the medium is in equilibrium with the surrounding tissue.

The method may further include the step of positioning a structure at the treatment site prior to the step of positioning the severed end. The method may further comprise the step of forming the structure in situ prior to the step of locating the severed ends. The method may further comprise delivering the agent to the periphery of the nerve in two sequential steps. The steps of severing the target nerve and positioning the structure at the treatment site may be accomplished using a single instrument.

The viscosity of the flowable medium can be less than 70,000cps, preferably less than 10,000cps, more preferably less than 500cps. In one embodiment, the viscosity of the flowable medium is similar to water (-1 cps). The flowable medium may have a density of less than 1.1g/cm ³ . The structure may comprise biocompatible silicone. The structure may include a monolithic silicone post for placement of the nerve. The structure may comprise biodegradable for nerve placementA polymer column that remains in place after hydrogel formation. In one embodiment, the polymer posts are lyophilized in place, while in another embodiment, the polymer posts are adhered to the structure with a biocompatible adhesive. In one embodiment, the biodegradable polymer posts remain in place with the hydrogel cap and are separated from the silicone structure when the silicone structure is removed. In another embodiment, the entire structure may include PEG, with or without integral PEG posts for nerve placement. In the latter embodiment, the structure will remain in place and degrade over the time period that the hydrogel degrades.

For use in supporting nerve survival or regeneration. In some embodiments, where the nerve is in a continuous state requiring protection, for example during healing after trauma or compression, there is a need for an in situ formed hydrogel having different properties than hydrogels developed to prevent neuroma formation. In cases where it is desired to protect the nerve from scar tissue and abnormal growth of damaged nerves, such as after nerve release from the nerve, the nerve is separated from the surrounding tissue, preferably a hydrogel that degrades in vivo for a relatively short period of time. Unlike the previously described degradation profile that does not significantly degrade at 3 months to prevent neuroma formation during regeneration, neuroprotection can be achieved in a shorter time with hydrogels that have a degradation profile within 3 months, preferably substantially degrade within 6 weeks, more preferably substantially degrade within 4 weeks, being desirable. Furthermore, unlike hydrogels developed to prevent neuroma, the equilibrium swelling of hydrogels used for protection must be sufficient to accommodate edema on compressed nerves, with a swelling of 20-50% by volume, preferably about 25-40% by volume. When these hydrogels are delivered around an intact nerve, the cavity in which the hydrogel is formed has an entrance and an exit, allowing the nerve to be placed through continuity in an atraumatic manner. In other embodiments following partial or complete nerve transection of a nerve fiber discontinuity, disclosed herein are two-component in situ-forming biomaterial compositions comprising a nerve regeneration-permitting component and a nerve growth-inhibiting component. In some embodiments, the growth-permitting component consists of a thermally-sensitive hydrogel formed in situ and the growth-inhibiting component consists of a chemically cross-linked hydrogel formed in situ. In other embodiments, the growth-permitting component consists of a physically cross-linked hydrogel or viscous solution. Examples of growth permission matrices are provided in pabarri et al 2011.Recent advances in innovative new reduce design. The in vivo duration of the growth-inhibiting component around the partially or completely transected nerve is longer than the time for continuous delivery of the hydrogel around the nerve, because the growth-inhibiting component must bear the load of the nerve until the regenerated proximal nerve has passed through the distal stump, and can begin bearing some load/strain on the nerve again. Preferably, the hydrogel "wrap" around the nerve will carry the load and prevent immunoinfiltration until the nerve is regenerated and then degrades to transfer the natural pressure on the nerve to the regenerated native tissue. In other embodiments, the hydrogel may be suitable for supporting regeneration and functional recovery after allograft and catheter implantation. In one embodiment, the hydrogel is delivered around the allograft to improve maneuverability and facilitate engagement with transected nerves. For example, after selecting cords from autografts to match the length and diameter of the transected nerve, the cords can be bundled together by using the in situ formed hydrogel as an artificial epineurium. Hydrogels can also improve the tailorability and overall treatment of allograft tissue. In a similar approach, the in situ formed hydrogel may be delivered around a catheter (solid, porous, mesh/strut) to adhere the catheter to the proximal and distal transection stumps with or without sutures.

In some embodiments, the nerve growth permitting component is delivered first, followed by the nerve growth inhibiting component. The nerve growth permitting component has sufficient mechanical integrity to prevent the growth inhibiting component from entering the desired growth regeneration zone.

In some embodiments, the nerve growth permitting component conforms to the nerve and promotes nerve ingrowth into, through and through the growth permitting biomaterial into the distal stump. In some embodiments, the nerve growth permission component is injected between the proximal and distal ends of the transected nerve. In other embodiments, the nerve growth permitting composition is delivered circumferentially around the nerve at the site of compression, partial transection, gapless full transection, or gapped full transection. In one embodiment, the growth-permitting component is a flowable composition and can diffuse into the area between damaged or transected fibers. In some embodiments, the growth-permissive biomaterial is a temporary filler that prevents the growth-inhibitory biomaterial from accessing the damaged nerve and inhibiting regeneration.

In some embodiments, the nerve growth inhibiting component prevents nerve growth into the material. Preferably, the nerve growth inhibiting composition covers the growth permitting material and the distal and proximal nerve stumps over a distance of at least 2mm, more preferably 10mm, in each direction. In this way, infiltration of immune cells into the regenerated nerve fibers is restricted and the typical constriction of the regenerated nerve cord is prevented.

In some embodiments, the nerve growth inhibiting composition acts as a guide on or along which nerve regeneration may occur. In another embodiment, the nerve growth inhibiting composition is also used as a rod that extends within the growth-permissive zone and extends within the same plane as the nerve to help guide regenerating axons.

In one embodiment, the growth-permitting biomaterial is temporarily placed as a barrier to the growth-inhibiting biomaterial and is removed after the growth-inhibiting hydrogel is formed in situ. The removal of the growth-permissive biomaterial may be performed within 1 hour to 3 months, preferably 1 hour to 2 months, more preferably 1 hour to 3 days after delivery of the growth-inhibiting material. In the latter case, where the two nerves (proximal and distal) are placed in close apposition by suturing or sutureless, the rapid dissolution of the growth-permissive biomaterial eliminates any physical barrier to regenerating neurons.

In some embodiments, the biomaterial component comprises an in situ formed crosslinked gel, an in situ formed thermosensitive hydrogel, an in situ formed thermoreversible hydrogel, a viscous solution of a synthetic or natural polymer, a microparticle, a nanoparticle, a foam, a hydrogel microparticle slurry, or a micelle.

In some embodiments, the growth-permitting component and the growth-inhibiting component both comprise polyethylene glycol (PEG).

In some embodiments, the PEG is a multi-arm PEG. In some embodiments, the PEG is a linear PEG. In some embodiments, the growth inhibiting PEG comprises a multi-arm PEG, and the growth regenerating PEG comprises a linear PEG.

In some embodiments, the PEG consists of a urethane or an amide bond.

In some embodiments, the PEG comprises an ester and/or amine linkage.

In some embodiments, the PEG further comprises a linear end-capped PEG of 5,000 daltons or less, such as PEG 3350.

In some embodiments, the crosslinking is between PEG-NHS ester and PEG-amine or trilysine.

In some embodiments, the growth-permissive gel comprises pores having a size of 1 μm or greater.

In some embodiments, the growth-permissive gel comprises a rod or a filament.

In some embodiments, the growth permissive component comprises chitosan, collagen, laminin, fibrin, fibronectin, HPMC, CMC, or other natural materials. In other embodiments, the components or regions of the components are covalently conjugated to each other. In another embodiment, the components are mixed together.

In some embodiments, the growth-permitting component comprises polylysine, preferably between 0.001wt% and 10wt%, more preferably between 0.01wt% and 0.1 wt%.

In some embodiments, the nerve growth permitting component comprises between 0.001-20%, preferably between 3-6wt% collagen.

In some embodiments, the nerve growth permitting component comprises fibronectin.

In some embodiments, the growth-permissive component comprises poly-l-ornithine.

In some embodiments, the growth-permitting component comprises polylysine, preferably between 0 and 5wt%, more preferably between 0 and 0.1 wt%.

In some embodiments, the swelling of the growth-permitting component is less than 20%, preferably between 0 and 20%. In some embodiments, the viscosity of the growth-permitting component exceeds 5,000cps, preferably between 7,500 and 150,000cps, more preferably between 10,000 and 20,000cps. In some embodiments, the young's modulus of the growth-permitting component is less than 2kPa, preferably less than 1kPa, more preferably less than 600Pa. In one embodiment, the growth-permitting component is a soft gel having a young's modulus between 100 and 300 Pa. In some embodiments, the osmolality of the growth-permissive gel is between 275 and 320mOsm, preferably between 280 and 320 mOsm.

In some embodiments, the swelling of the growth-inhibiting component is less than 50%, preferably from 0 to 30%, more preferably from 5 to 20%. In another embodiment, the swelling of the growth-permitting component is the same as the growth-inhibiting component.

In some embodiments, the swelling of the growth-permitting component is less than or equal to the swelling of the growth-inhibiting component.

In some embodiments, the compressive strength of the growth-inhibiting component is greater than 10kPa, preferably >30kPa.

In some embodiments, the growth-permitting and growth-inhibiting components are different colors.

In some embodiments, the growth-permissive region comprises an agent that supports nerve survival, growth, and regeneration.

In some embodiments, the growth-permissive region allows for infiltration of schwann cells or other supporting cells. In some embodiments, the growth-permissive region may be loaded with cells prior to placement in situ. The growth-inhibitory region may prevent migration of these cells away from the implantation site. In some embodiments, the system comprises a support cell, such as a glial cell, including a schwann cell, oligodendrocyte, or progenitor cell, such as a stem cell.

In some embodiments, the compositions include agents that may include one or more growth factors, anti-inhibitory peptides or antibodies, and/or axon-guiding cues.

In some embodiments, the growth-permissive area may be delivered accurately using a syringe, preferably using an 18 gauge or higher gauge needle, in a volume of 10 microliters to 5 milliliters. Smaller volumes of less than 100 microliters may be delivered to smaller nerves or partially transected nerves, while larger volumes may be delivered to partial or complete lesions in larger caliber nerves.

In some embodiments, the system is delivered to a peripheral nerve, ventral or dorsal root, sympathetic ganglion, cauda equina, spinal cord, or brain. In other embodiments, the system may be delivered to or around the peripheral neurovascular bundle or tendon. In some embodiments, the system is delivered to the nerve without using the structure, although the use of the structure is preferred.

In some embodiments, the growth-permissive and growth-inhibitory region comprises a P2XR receptor antagonist.

In some embodiments, the P2XR receptor antagonist is a P2X7 receptor antagonist, including brilliant blue FCF (BB FCF) or Brilliant Blue G (BBG). In some embodiments, the P2XR antagonist is a P2X3 receptor antagonist, such as AF-219 or gefapixant. In some embodiments, the concentration of the P2XR antagonist is 0.001-0.55% of the hydrogel.

In some embodiments, the hydrogel is delivered circumferentially around the nerve. In other embodiments, the hydrogel is only partially delivered to the nerve surface, for example, between about 25% and 85% of the nerve circumference, preferably between 50% and 80%. In other embodiments, multiple wraps are delivered sequentially to cover a longer length of nerve. For example, in some embodiments, one to four hydrogel wraps are delivered around a nerve when a long length of nerve has been released and moved, such as after displacement of the ulnar nerve. In other embodiments, if the nerve needs protection, but the vessel or nerve bifurcates in the area where encapsulation is desired, the wrap structure can be cut with surgical scissors intraoperatively to leave space for the bifurcated nerve to exit the wrap structure outside of the available exit area of the wrap structure.

In some embodiments, the cap structure or wrap structure is between 10mm and 60mm in length, depending on the length of the nerve. For smaller nerves (small nerve groups) less than 4mm in diameter, it is preferred that the length of the cap structure or wrap structure cavity is between 11mm and 40mm, preferably 15mm to 20mm. For the cap structure, nerves preferably at least 5mm long, more preferably at least 10mm long need to be covered by the hydrogel to secure the nerves in the hydrogel. As a result, to ensure that the hydrogel, preferably 1mm, surrounds the tip of the nerve, the cap structure cavity length needs to be approximately 11mm long, preferably 15mm long for small nerve coverage. For the cap structure and the wrap structure, a longer cavity is preferable for the larger nerve in view of the longer length of these exposed nerves, and thus the cavity length is about 15mm to 60mm, and more preferably 20mm to 50mm.

In some embodiments, kits are disclosed that include two or more in situ-forming hydrogels. The kit includes a dual applicator system explicitly labeled as a growth-permissive applicator and a dual applicator system explicitly labeled as a growth-inhibitory applicator. Each ingredient has a clear color code, including a vial of powder, a reconstitution/dilution solution, and an accelerator (accelerator) solution for a dual applicator system. The kit may also include one or more structures-one structure for receiving a growth-inhibiting hydrogel and another structure for receiving a growth-permitting hydrogel. The kit may include a bioabsorbable structure for receiving a growth-permissive biomaterial, followed by a temporarily non-degradable structure for subsequently receiving a growth-inhibitory hydrogel. In another embodiment, a kit is disclosed comprising an in situ formed hydrogel and a low viscosity gel solution (viscosity between 5,000 and 30,000cps, modulus less than 1 kPa). The kit includes a dual applicator system specifically labeled as a growth-inhibiting applicator and a syringe/applicator system specifically labeled as a growth-permitting applicator. The kit may also include one or more structures-one biodegradable for receiving the growth-permitting gel solution and the other non-degradable for receiving the growth-inhibiting hydrogel. In one embodiment, the biodegradable structure for receiving the growth-permitting gel solution is a biodegradable adhesive sheet that adheres to nerves when wetted and is resorbed within a few days after application. In another embodiment, the biodegradable structure degrades between one to two months and is positively charged to promote neurite extension.

In some embodiments, disclosed herein is a method of delivering a dual gel, comprising forming the hydrogel and the low viscosity gel in situ to treat a condition involving nerves. Nerves may require repair, such as end-to-end anastomosis, repair with allograft or autograft or catheter or wraps, or gap repair. The dual gel system may be delivered to and around the anastomosis site between the proximal nerve and the distal nerve stump, the proximal nerve and the allograft nerve or cord, or as a connector-assisted junction where the connector is provided by an in situ formed hydrogel. The double gel system may be delivered as an adjunct to suture repair, or preferably, without the need to suture nerve tissue.

In some embodiments, the growth-permissive region is delivered between a proximal and a distal nerve stump, between end-to-end anastomosis sites, between a proximal stump and an allograft/autograft, and between an allograft/autograft and a distal stump.

In some embodiments, the growth-permissive zone is injected between the proximal end and the graft and/or the graft and the distal stump, with or without structural assistance. The growth permissive solution may comprise a volume of about 5ul to 3ml, more preferably 10ul to 1cc. Sufficient volume must be delivered to fill the gap between the nerves and also cover 1mm or more of the length of the proximal and distal nerves, preferably 2 to 5mm of the length of the proximal and distal nerves.

In some embodiments, the growth-permissive region is delivered inside a catheter or wrap. In some embodiments, the growth-permissive region is delivered inside a lyophilized PEG catheter or wrap. The lyophilized PEG may comprise cross-linked multi-arm PEG, linear PEG solutions, or combinations thereof, which provide sufficient structural support as a temporary structure during operation to prevent unintended diffusion of growth-permissive substances. After completion of the procedure, the structure will be cleared from the site within 3 to 5 days, preferably not more than 1 to 2 days.

In some embodiments, the growth-permissive region is delivered into a structure that allows the growth-permissive gel to adhere to nerves but not to the structure. In yet another embodiment, the growth-permissive region is delivered to a porous structure, allowing the growth-permissive gel to adhere to nerves and the structure. The growth-permitting regions are inserted into pores of the bioabsorbable structure to provide good adhesion to the structure.

In some embodiments, the growth-suppressing region is delivered after the growth-permissive region. In some embodiments, the growth-inhibiting region is delivered around the growth-permitting region, completely covering the growth-permitting material. In another embodiment, a layer of growth-inhibitory regions is first delivered into the structure, followed by placement of the nerve, delivery of the growth-permissive region, followed by delivery of the last layer of growth-inhibitory regions.

In some embodiments, the growth-inhibitory regions cover the proximal and distal nerves and the growth-permissive regions. In some embodiments, the growth-inhibiting zone extends from the anastomotic orifice down the proximal and distal nerves, at least 2cm in each direction, preferably 1cm in each direction, preferably 5mm covering each nerve stump.

In some embodiments, the kit may include an in situ formed hydrogel. The kit includes a dual applicator system and powder vials, reconstitution/dilution solutions and accelerator solutions for the dual applicator system. The kit may also include a selection of structures within the size and length range for receiving the hydrogel.

In some embodiments, the growth-inhibitory region covers an anastomotic junction or a direct nerve junction.

In some embodiments, the growth-inhibitory region is delivered to cover the junction between the nerve and the catheter or wrap.

In some embodiments, the growth-inhibitory region covers healthy, stressed, or bruised nerves.

In some embodiments, disclosed herein is an in situ formed nerve regeneration construct comprising a growth permissive hydrogel bridge having first and second ends and configured to span the space between the two nerve ends and promote nerve transaxle regeneration; and a growth-inhibiting hydrogel sheath encapsulating the growth-permissive hydrogel bridge and configured to extend beyond the first and second ends to directly contact and adhere the two nerve ends. In some embodiments, the adhesion of the growth-suppressing hydrogel to the nerve provides sufficient strength to maintain the nerve in proximity and support nerve regeneration without the need for suturing. In some embodiments, the nerve can be positioned within the biomaterial in an angled or curved manner to reduce the load on the nerve ends and improve nerve repair.

In some embodiments, disclosed herein is a method of promoting nerve growth between a first nerve end and a second nerve end, comprising placing the first nerve end and the second nerve end in a structural cavity; introducing a growth-permitting medium into the cavity and into contact with the first nerve end and the second nerve end to form a junction; placing the connection in a second structural cavity; and introducing a growth-suppressing medium into the second structural cavity to encapsulate the connection. In some embodiments, disclosed herein is a method of supporting regeneration between a first nerve end and a second nerve end, comprising: the first nerve end and the second nerve end are placed in a bioabsorbable nerve cavity, and a growth-permitting medium is introduced into the cavity and contacted with the first nerve end and the second nerve end to form a connection. The growth-permitting medium/bioabsorbable nerve lumen (e.g., wrap or catheter) can then be handled as a unit with forceps and then transferred into a second structural lumen into which the growth-inhibiting medium is delivered.

In some embodiments disclosed herein, a two-component structure is used to accomplish growth permissive and growth inhibitory biomaterial delivery without the need to handle or otherwise unnecessarily move nerves. For example, the structure comprises an inner bioabsorbable structure (e.g., chitosan, HPMC, CMC film) located inside an outer non-absorbable structure (e.g., silicone). The growth-permissive material is delivered directly to the bioabsorbable structure, while the growth-inhibiting biomaterial is delivered between the bioabsorbable and non-absorbable structures. In one embodiment, the bioabsorbable structure is a sheet that becomes sticky and adherent when wetted. Hydration of the biocompatible biodegradable sheet, which may occur through interaction with naturally moist nerve and/or surrounding tissue fluids or through direct application of an aqueous solution, may crosslink in situ by physical intercalation with the tissue surface to adhere to the tissue. After the hydrogel is formed, the non-resorbable structure is removed.

Use for the prevention of neuronal regeneration and neuroma formation. In some embodiments, disclosed herein is a structure for creating an in situ nerve cap to inhibit neuroma formation, comprising: a recessed wall defining a cavity, the wall having a top opening for accessing the cavity, the top opening lying on a first plane and having an area smaller than a second plane that conforms to an interior dimension of the cavity and is spaced apart from the cavity and parallel to the first plane; and a concave nerve guide carried by the wall and providing lateral access to the cavity.

For neuroprotective and regenerative applications. In some embodiments, disclosed herein is a structure for forming an in situ wrap around a nerve-to-nerve connection comprising: a recessed wall defining a cavity, the wall having a top opening for accessing the cavity, the top opening lying on a first plane and having an area smaller than a second plane conforming to an interior dimension of the cavity and spaced from and parallel to the first plane; a first concave nerve guide carried by the wall and providing a first lateral access for positioning a first nerve end in the cavity; and a second concave nerve guide carried by the wall and providing a second lateral access for positioning a second nerve end in the cavity.

In some embodiments, disclosed herein are Cap compositions for forming growth-inhibiting hydrogels in situ, having: a compressive strength of greater than 10kDa for more than 3 months, an in vivo persistence (including less than 15% mass loss) for at least 3 months, and/or a swelling of less than 30% for more than 3 months. In some embodiments, disclosed herein is a composition for forming a growth-inhibiting hydrogel in situ, having: a compressive strength of greater than 20kDa for more than 6 months, an in vivo persistence (including less than 15% mass loss) for at least 6 months, and/or an in vitro swelling of less than 20% for more than 6 months. In a preferred embodiment, the growth-inhibiting hydrogel for preventing neuroma formation has a compressive strength of greater than 30kDa, an in vivo durability (including less than 15% mass loss) of greater than 4 months, and/or a swelling of less than 20% for greater than 4 months. In a preferred embodiment, the growth-inhibiting hydrogel swells radially outward upon degradation, preventing nerve compression. Thus, swelling of the hydrogel occurs simultaneously with loss of the hydrogel's tensile strength, preventing compression of the encapsulated nerve and adjacent structures. In some embodiments, disclosed herein are wrap compositions for in situ formed growth-inhibiting hydrogels having a compressive strength of greater than 10kDa for greater than 2 weeks, having in vivo persistence for at least 4 weeks, and/or less than 60% swelling for greater than 3 months. In this embodiment, at least 10% swelling is required to accommodate any swelling of the post-traumatic nerve. In a preferred embodiment, the hydrogel swells at least 20%, more preferably greater than 30% after equilibration, but does not swell more than 60% at any point in time prior to clearance. In this embodiment, the hydrogel swells sufficiently to allow any nerves to swell, then remains on the nerves to prevent infiltration of immune cells, and secondly, as degradation and loss of tensile strength occurs, additional expansion in the radially outward direction allows the nerves to slide within the hydrogel. In another embodiment, a wrap that degrades in a shorter time is needed to prevent nerve compression in the first week after surgery. These inclusion hydrogels remained in place in the body for at least 2 weeks, but cleared rapidly from the site, and essentially cleared from the site within 4 weeks. These more rapidly degrading encapsulate hydrogels allow for slippage during the hydrogel degradation phase, during which the hydrogel forms a viscous solution around the nerve, through which the nerve can freely move.

In some embodiments, the composition comprises one or more of: poly (ethylene glycol) succinimide carbonate, P2XR receptor antagonists, such as P2X7 receptor antagonists.

In some embodiments, the P2X7 receptor antagonist is brilliant blue FCF (BB FCF) or Brilliant Blue G (BBG).

In some embodiments, a method of forming a nerve wrap in situ, comprising identifying a portion of a nerve; positioning a nerve in a cavity defined by a structure; introducing a medium into the cavity of the structure to encapsulate the nerve; and allowing the medium to undergo a transition from a first, relatively flowable state to a second, relatively non-flowable state to form a protective barrier around the nerve.

In some embodiments, the nerve is healthy, stressed, contused, partially or fully transected. In some embodiments, the nerve is transected during surgery, while in other embodiments, damage to the nerve, such as neuroma formation, has occurred 3 months to about 10 years ago and was excised prior to application of the hydrogel. In other embodiments, the nerve is repaired within a few minutes to three months after injury, typically within one day to 14 days after injury. In further embodiments, the surgeon may choose not to operate until 2 months to 6 months after initial nerve trauma, for example, after trauma, when the extent of nerve damage or the ability of nerves to regenerate is unclear, and the surgeon is inclined to assess whether nerve function can be restored without surgical intervention.

In some embodiments, the nerve is first repaired by direct anastomosis, repaired with an allograft or autograft, or repaired with a catheter prior to delivery of the growth-permissive and growth-inhibitory biomaterial.

In some embodiments, the method comprises removing the structure.

In some embodiments, the structure comprises a nerve guide, and positioning comprises positioning the nerve such that the nerve guide maintains the nerve spaced apart from a sidewall of the structure. In one embodiment, the hydrogel is formed around a nerve placed in a cylindrical tube, which is subsequently removed, after which the neurohydrogel is spun and placed in a second cylindrical tube, and a second administration of the growth-inhibiting hydrogel is performed. By appropriate placement of the gradually larger diameter tube, nerves can be delivered to the center of the formed hydrogel. This same approach can be used for growth-permissive or growth-inhibitory gel applications.

In some embodiments, the method of forming a nerve wrap in situ comprises covering a nerve with a protective barrier around the nerve that is at least 0.1mm thick of a biomaterial, preferably 0.5mm to 10mm thick, more preferably 0.5mm to 5mm thick.

In some embodiments of delivering the in situ-forming hydrogel into a cap or wrap structure, the transformation occurs within about 10 seconds of the introducing step, preferably within 5 seconds of the introducing step. In some embodiments where the hydrogel is delivered into a wrap/cap structure, the transition preferably occurs within 15 seconds of the introduction step, or takes longer because of the wrapping of the longer nerve segment. To accommodate different gel times, the kit will be designed for small nerves (nerves less than 4 mm) and large nerves (nerves greater than 4 mm). The kit may also contain different needle gauges to accommodate delivery of different volumes and gel times to the nerve cap/wrap. For example, a small nerve kit may contain 22 gauge needles and a large nerve kit may contain 20 or 18 gauge needles.

In some embodiments, the converting comprises crosslinking or polymerizing. In some embodiments, the transition comprises gelation following a temperature change from room temperature to body temperature.

In some embodiments, the transformation creates a synthetic crosslinked hydrogel protective barrier. In some embodiments, the transition results in a natural crosslinked hydrogel protective barrier, such as may be obtained with fibrin sealant (Tisseel, baxter).

In some embodiments, the protective barrier has an in vivo persistence of at least about two months.

In some embodiments, the protective barrier has an in vivo persistence of at least about three months.

In some embodiments, the protective barrier has an in vivo persistence of at least about six months.

In some embodiments, the protective barrier does not degrade in vivo.

In some embodiments, the transformation results in swelling of the media volume in the range of about 2% to about 60%.

In some embodiments, the transformation results in swelling of the media volume in the range of about 50% to about 40%.

In some embodiments, the method comprises forming the structure in situ prior to positioning the severed ends; and/or delivering the medium around the nerve in two sequential steps.

In some embodiments, the steps of severing the target nerve and positioning the structure at the treatment site are accomplished by a single instrument.

In some embodiments, the flowable hydrogel precursor medium has a viscosity of less than 70,000cps, preferably less than 10,000cps.

In some embodiments, the flowable medium has a density of less than about 1.2g/cm ³ Or about 1g/cm ³ The density of water.

In some embodiments, the structure is comprised of biocompatible medical grade silicone.

In some embodiments, the structure comprises an integral post for placement of a longer length nerve.

In some embodiments, the cap or wrap structure consists of PEG.

In some embodiments, the structure has a clamshell cover and a separate port or inlet for delivery of the hydrogel.

In some embodiments, the P2XR receptor antagonist is a P2X7 receptor antagonist, including brilliant blue FCF or Brilliant Blue G (BBG). In some embodiments, the P2XR antagonist is present in the hydrogel at a concentration of between 0.001-0.55%.

In other embodiments, the growth-permitting region and/or the growth-inhibiting region comprises the antioxidant methylene blue. In other embodiments, the growth permissive and/or growth inhibitory composition comprises FD & C No. 1 alone or in combination with FD & C No. 5 to produce a blue, blue-green/blue-green, and different shade green hydrogel. In other embodiments, the growth-permissive zone and/or the growth-inhibitory zone comprises a linear-capped PEG (3.35 kDa or 5kDa, or mixtures thereof) having a solids content of from 1wt% to 50wt%, more preferably 10-20 wt%.

In some embodiments, in situ forming a hydrogel as a cap is disclosed herein. In some embodiments, the nerve cap is not preformed.

Some embodiments disclosed herein include in situ formed hydrogel scaffolds or hydrogel scaffolds that can be formed/encapsulated in situ around nerves. In some embodiments, these hydrogels are delivered without a preformed wrap or catheter.

In some embodiments, neuroblastoma formation of nerves is prevented by delivery of an in situ formed "no site bridging" catheter, which is an in situ formed hydrogel structure with an open lumen that allows nerves to regenerate along and over the hydrogel until their regenerative capacity ceases. In some embodiments, the channel within the hydrogel is 1cm or longer in length, preferably 2cm or longer in length. In some embodiments, the channel is comprised of a rapidly resorbing biomaterial, such as low molecular weight PEG, collagen, hyaluronic acid, or hydroxymethylcellulose, or a combination thereof. The hydrogel forms around the nerve and rapidly absorbed biomaterial pathway. The biomaterial cylinder retains its three-dimensional structure for a sufficient time to provide a scaffold on which growth-inhibiting cylinders can be formed. To achieve this configuration, the nerves are placed in a bioabsorbable wrap structure, and the structure is filled with a rapidly resorbing material. The wrap structure is wrapped circumferentially around the nerve and rapidly resorbs the biomaterial, after which the growth-inhibiting hydrogel is delivered around the nerve and wrapped in a second, larger wrap structure. The larger wrap structure may be a biodegradable or removable non-degradable structure.

In some embodiments, disclosed herein are systems and methods for delivering a hydrogel circumferentially around a nerve in a properly designed structure. In some embodiments, the systems and methods may include using a structure or specific design of PEG hydrogel to prevent neuroma formation, such as circumferential delivery and coverage of nerve endings, sufficient in vivo persistence beyond the time (3 months or more) that the nerve can regenerate, minimal swelling to prevent nerve compression or loss of adhesion between the nerve and hydrogel, sufficient tensile strength to prevent nerve growth into the hydrogel, and delivery of them into a removable structure.

According to another aspect of the present invention, there is provided a kit for forming an implant in situ for guiding nerve regeneration between two nerve endings. The kit includes a first component for producing a first growth-permissive hydrogel; a second component for producing a second growth-inhibiting hydrogel; at least one structure having concavity; a first applicator for delivering a growth-permissive hydrogel into the cavity; and a second applicator for delivering the growth-inhibiting hydrogel into the cavity.

The first component may include a powdered growth-permitting hydrogel precursor, a reconstituting solution, and an accelerator solution. The powdered growth-permitting hydrogel precursor may contain an agent that stimulates nerve regeneration. The second component may include a powdered growth inhibiting hydrogel precursor, a reconstituting solution, and an accelerator solution. The first component includes a powdered growth-permitting gel precursor and a reconstituting solution. The second component may comprise a pre-filled syringe containing a growth-permitting gel.

The kit may further include a first structure having a first configuration for receiving the growth-inhibitory hydrogel, and a second structure having a second, different configuration for receiving the growth-permissive hydrogel. The concave shape may have a surface of hydrophobic character. At least one of the growth-permitting hydrogel and the growth-inhibiting hydrogel may have hydrophilic properties.

The second structure may comprise a biocompatible biodegradable sheet material having a thickness of less than about 60 microns or less than about 40 microns.

Also provided is a kit for forming a hydrogel nerve cap in situ. The kit may include a dual applicator system; a vial containing a powdered hydrogel precursor; reconstituting the solution; an accelerator solution; and at least one nerve cap structure.

In situ formed nerve regeneration constructs are also provided. The construct comprises a growth-permissive hydrogel bridge having first and second ends and configured to span the space between the two nerve ends and promote nerve transaxle regeneration; and a growth-inhibiting hydrogel sheath encapsulating the growth-permissive hydrogel bridge and configured to extend beyond the first and second ends to directly contact both nerve ends.

Also provided is a structure for forming an in situ nerve cap to inhibit neuroma formation. The structure includes a recessed wall defining a cavity, the wall having a top opening for accessing the cavity, the top opening lying on a first plane and having an area less than an area of a second plane conforming to an interior dimension of the cavity and spaced apart in the cavity and parallel to the first plane; and a concave nerve guide carried by the wall and providing lateral access to the cavity; wherein the recessed walls have hydrophobic characteristics.

The structure is configured to receive a second biodegradable structure containing a nerve end to be repaired. The former may promote nerve regeneration across the nerve-nerve connection. The structure can promote hydrogel formation to prevent nerve compression and scar tissue formation around the nerve.

Also provided is a structure for creating an in situ nerve conduit for promoting regeneration of a nerve-to-nerve junction. The structure includes a recessed wall defining a cavity, the wall having a top opening for accessing the cavity, the top opening lying on a first plane and having an area smaller than a second plane conforming to an interior dimension of the cavity and spaced apart to access the cavity and parallel to the first plane; a first concave nerve guide carried by the wall and providing a first lateral access for positioning a first nerve end in the cavity; and a second concave nerve guide carried by the wall and providing a second lateral access for positioning a second nerve end in the cavity.

Also provided is a composition for forming a growth-inhibiting hydrogel in situ. The hydrogel exhibits a compressive strength of greater than 10kDa for more than 3 months; in vivo persistence for at least 3 months, including less than 15% mass loss, and less than 30% swelling for more than 3 months. Degradation of the hydrogel may result in outward radial swelling of the hydrogel.

The composition may comprise one or more poly (ethylene glycols) having biodegradable amide or urethane linkages; poly (ethylene glycol) succinimide carbonate; a P2XR receptor antagonist; a P2X7 receptor antagonist. The P2X7 receptor antagonist may be brilliant blue FCF (BB FCF) or Brilliant Blue G (BBG).

The composition may further comprise one or more poly (ethylene glycol) s having biodegradable ester linkages; poly (ethylene glycol) succinimide adipate; a multi-arm PEG with arm lengths between 1 and 10 kDa; and at least one multi-arm PEG with an arm length of 5 kDa.

Also provided is a composition for forming a growth-inhibiting hydrogel in situ. The composition comprises a hydrogel that exhibits a compressive strength of greater than about 10 kPa; in vivo persistence for at least about 2 weeks; and an initial swelling of greater than about 20% but less than about 100%

Degradation of the hydrogel can result in outward radial swelling of the hydrogel with a swelling volume of less than about 160%. Degradation of the hydrogel may occur in less than about 16 weeks. The growth-inhibiting hydrogel may be configured to encapsulate a growth-permissive gel having a Young's modulus of less than about 10kPa and a viscosity of greater than about 5,000cP.

The composition may include one or more poly (ethylene glycol) s having biodegradable ester linkages; poly (ethylene glycol) succinimide adipate; PEG with an arm length between 1 and 10 kDa; or at least one PEG with an arm length of 5 kDa.

An absorbable, in situ-formed electrode anchor is also provided, including a mass of hydrogel polymerized in situ around an electrode and configured to maintain the electrode in electrical communication with a nerve. The hydrogel may be conductive.

Also provided is a shaped in situ implant comprising an amount of hydrogel that transitions from a relatively flowable state to a relatively non-flowable state upon contact with a structure and is removed from the structure by a tensile force of no more than about 5N, wherein the hydrophilic properties of the hydrogel and the hydrophobic properties of the structure facilitate removal. The formed in situ implant may include a nerve cap, a nerve conduit for guiding nerve regeneration, or a nerve wrap for preventing scar formation and nerve constraint.

Some embodiments disclosed herein have been shown to prevent the formation of neuromas in the preclinical setting and 1) eliminate the need to suture, pull or stuff nerves within a catheter, 2) conform to nerve stumps, provide a physical barrier for nerve regeneration, and 3) provide mechanical strength to prevent nerve regeneration for a period of growth regeneration following nerve injury, for two months, preferably three months or more, to prevent nerve growth. The in situ forming implants described herein are conformable to surrounding tissue, adhere to but do not compress underlying nerve tissue, are flexible such that they can move over areas of tissue involving joints or areas where nerves slide relative to other tissue, and prevent the formation of scar tissue and adhesions around nerves. Finally, some of these in situ forming implants can be delivered without advanced surgical training. In other cases, there remains a need for a technique to prevent nerve growth into the surrounding tissue and to direct the growth of transected or compressed nerves into the distal nerve stump or allograft/autograft. Thus, in some aspects, a seamless thread technique is described that can guide nerve regeneration from a proximal nerve stump into a distal nerve stump either directly (by direct engagement/anastomosis with the distal nerve stump) or indirectly (by nerve conduit, guide channel, allograft, autograft) or through a growth-permissive matrix. In addition, in some aspects, techniques are described that allow compression of the anastomotic site to promote better nerve regeneration. By delivering the hydrogel circumferentially around the nerve, the tension can be distributed circumferentially and at a distance on the nerve to evenly distribute the tension on the nerve surface. In this way, tension is transferred to the hydrogel, rather than being created at the foci of three or four sutures at the anastomosis site. As is done in catheters, additional detensioning can be achieved by creating a relaxation in the nerve (placed in a curve) prior to delivery of the growth-inhibiting hydrogel, such that the tension at the anastomosis site is minimized. Finally, in some aspects, there is a need for a general technique that can be quickly and widely applied to nerves to prevent inadvertent damage to adjacent nerves during various surgical procedures.

Also provided is a kit for forming an implant in situ for guiding nerve regeneration between two nerve stumps. The kit includes a first component for producing a first growth-permissive hydrogel; a second component for producing a second growth-inhibiting hydrogel; at least one structure having concavity; a first applicator for delivering a growth-permissive hydrogel into the cavity; and a second applicator for delivering the growth-inhibiting hydrogel into the cavity.

The first component may include a powdered growth-permitting hydrogel precursor, a reconstituting solution, and an accelerator solution. The second component may include a powdered growth inhibiting hydrogel precursor, a reconstituting solution, and an accelerator solution. It is also possible to provide a first structure having a first configuration for receiving a growth-inhibiting hydrogel and a second structure having a second, different configuration for receiving a growth-permitting hydrogel. The concave shape may have a surface of hydrophobic character. At least the growth-permitting hydrogel may have hydrophilic properties.

Kits for forming hydrogel nerve caps in situ are also provided. The kit includes a dual applicator system; a vial containing a powdered hydrogel precursor; reconstituting the solution; an accelerator solution; and at least one nerve cap structure.

Also provided is an in situ formed nerve regeneration construct comprising a growth-permissive hydrogel bridge having first and second ends and configured to span the space between the two nerve ends and promote nerve transaxle regeneration; and a growth-inhibiting hydrogel sheath encapsulating the growth-permissive hydrogel bridge and configured to extend beyond the first and second ends to directly contact both nerve ends.

Also provided is a structure for forming an in situ nerve cap to inhibit neuroma formation, comprising a recessed wall defining a cavity, the wall having a top opening for accessing the cavity, the top opening lying on a first plane and having an area less than an area of a second plane conforming to an interior dimension of the cavity and spaced apart in the cavity and parallel to the first plane; and a concave nerve guide carried by the wall and providing lateral access to the cavity. At least the surface of the recess wall may have hydrophobic properties.

There is also provided a structure for creating an in situ nerve conduit to promote regeneration of a nerve-to-nerve connection, the structure comprising a concave wall defining a cavity, the wall having a top opening for accessing the cavity, the top opening lying on a first plane and having an area smaller than a second plane conforming to an internal dimension of the cavity and spaced apart into the cavity and parallel to the first plane; a first concave nerve guide carried by the wall and providing a first lateral access for positioning a first nerve end in the cavity; and a second concave nerve guide carried by the wall and providing a second lateral access for positioning a second nerve end in the cavity.

Also provided is a composition for forming a growth-inhibiting hydrogel in situ, the composition having a compressive strength greater than 10kDa for more than 3 months; in vivo persistence for at least 3 months, including less than 15% mass loss; and less than 30% swelling for more than 3 months. The composition may comprise poly (ethylene glycol) succinimide carbonate. The hydrogel may contain a P2XR receptor antagonist and/or a P2X7 receptor antagonist. The P2X7 receptor antagonist may be brilliant blue FCF (BB FCF) or Brilliant Blue G (BBG).

Also provided is a shaped in situ implant comprising an amount of hydrogel that transitions from a relatively flowable state to a relatively non-flowable state within a structural cavity and is removed from the cavity by a pulling force of no more than about 5N, wherein the hydrophilic properties of the hydrogel and the hydrophobic properties of the cavity facilitate removal. The implant may be a nerve cap or a nerve conduit for guiding nerve regeneration.

Some embodiments disclosed herein have been shown to prevent the formation of neuromas in the preclinical setting and 1) eliminate the need to suture, pull or stuff nerves within a catheter, 2) conform to nerve stumps, provide a physical barrier for nerve regeneration, and 3) provide mechanical strength to prevent nerve regeneration during the growth regeneration phase following nerve injury, for two months, preferably three months or more, to prevent nerve growth. The in situ forming implants described herein are conformable to surrounding tissue, adhere to but do not compress underlying nerve tissue, are flexible such that they can move over areas of tissue involving joints or areas where nerves slide relative to other tissue, and prevent the formation of scar tissue and adhesions around nerves. Finally, some of these in situ forming implants can be delivered without advanced surgical training. In other cases, there remains a need for a technique to prevent nerve growth into the surrounding tissue and to direct the growth of transected or compressed nerves into the distal nerve stump or allograft/autograft. Thus, in some aspects, a seamless thread technique is described that can guide nerve regeneration from the proximal nerve stump into the distal nerve stump either directly (via direct engagement/anastomosis with the distal nerve stump) or indirectly (via nerve conduit, guide channel, allograft, autograft) or via growth-permitting matrix. In addition, in some aspects, techniques are described that allow compression of the anastomotic site to promote better nerve regeneration. By delivering the hydrogel circumferentially around the nerve, the tension can be distributed circumferentially and at a distance on the nerve to evenly distribute the tension on the nerve surface. In this way, tension is transferred to the hydrogel, rather than being created at the foci of three or four sutures at the anastomosis site. As is done in catheters, additional detensioning can be achieved by creating relaxation in the nerves (placed in a curve) prior to delivery of the growth-inhibiting hydrogel, such that the tension at the anastomosis site is minimized. Finally, in some aspects, there is a need for a general technique that can be quickly and widely applied to nerves to prevent inadvertent damage to adjacent nerves during various surgical procedures.

Drawings

Fig. 1A is a perspective schematic view of a nerve end positioned within a structural cavity. Allowing nerves to be directed into the entry area of the structure. The length of the structure provides sufficient surface area on which the hydrogel forms and adheres to neural tissue.

FIG. 1B is a side elevational view of a section through the FIG. 1A construction.

FIG. 1C is a top view of the FIG. 1A construction.

FIG. 1D is an end view of the FIG. 1A construction.

FIG. 1E is a cross-sectional view taken along line 1E-1E in FIG. 1B.

FIG. 1F is a top view of another embodiment of the FIG. 1A construction.

Fig. 2 is a schematic view of a forming barrier formed according to some embodiments of the present invention.

Fig. 3A is a perspective view of a structure having a stabilizing feature.

Fig. 3B is a perspective view of a structure for forming a wrap around a nerve or a growth-permitting region in a gap between nerve ends. Thus, depending on the application, the wrap structure may comprise a growth-permissive or growth-inhibitory hydrogel.

Figure 4A shows electrical stimulation of nerve ends through growth-permitting media in open surgery.

Fig. 4B shows anchoring of the electrodes near the nerve to deliver pain control stimulation in a percutaneous procedure.

Figures 5A-5M show a series of steps for creating a growth-permitting hydrogel attachment encapsulated by a growth-inhibiting hydrogel barrier.

Fig. 6 is a perspective view of a clamshell structure.

Figures 7-10C show embodiments of tools for transecting nerves and/or creating hydrogel connections.

Fig. 11A-11E show views of a structure and method of use thereof.

Fig. 12 is a perspective view of a structure with a stabilizing rod.

Figures 13A-13D are perspective views of a cap structure with a partial covering and an inner rod to support a nerve.

Fig. 14A-14C are perspective views of a cap structure having a partial clamshell.

Figures 15A-15C are perspective views of tearable nerve cap structures.

Figures 16A-16E show perspective views and photographs of hydrogels formed in situ around nerves (growth inhibition and growth allowance), including cap structures and wrap structures.

Figures 17A-17B show preclinical data demonstrating neuroma formation after delivery of a hydrogel having sufficient initial mechanical strength but insufficient in vivo durability (relative to a hydrogel having longer duration mechanical strength and durability).

Figures 18A-18B illustrate mixing element designs for increasing hydrogel consistency when delivering low volume precursor solutions.

Fig. 18C and 18D show the configuration of the distal mixing tip.

FIG. 18E is a section taken along line 18E-18E of FIG. 18D

Fig. 19 schematically illustrates a dual chamber syringe system.

Fig. 20 schematically illustrates a dual chamber syringe and mixer system.

Detailed Description

Some aspects of the invention relate to the in situ formation of a protective barrier around nerve endings using injectable or surgically introducible mediators, which may be gels/hydrogels or gel precursors, to block nerve regeneration and/or neuroma formation as well as inflammation and adhesions, etc. around/in contact with nerves. Access may be by open surgery or percutaneous (needle, intravascular/transvascular) methods. The nerve endings or stumps may be formed by transection (cutting), traumatic injury, or by ablation including Radiofrequency (RF), cryotherapy, ultrasound, chemistry, heat, microwave, or other means known in the art.

The hydrogel may "adhere" to the end of the nerve, providing a suitable, consistent, cushioning barrier around the end of the nerve, rather than a cap with voids (the presence of inflammatory cells/fluid cysts that support neuroma formation). The hydrogel is transparent for visualization, low swelling, conformable, and delivered into the structure to produce a hydrogel cap with a volume of 0.05 to 10ml, preferably 0.1 to 5ml, more preferably 0.1 to 2.8 ml. The barrier may inhibit the formation of neuroma completely by mechanically blocking nerve regeneration. The medium may additionally comprise any of a variety of drugs, for example, drugs for inhibiting nerve regeneration, as discussed in further detail herein.

The diameter of the target nerve can vary widely, with spherical or non-spherical outer configurations, and the cutting or severing angle and precision can also vary. Although it will be appreciated that all nerves will benefit from this hydrogel technique, nerves of approximately 0.2mm to 15mm, preferably 1mm to 5mm, more preferably 1mm to 2mm, can be treated in this way. According to some embodiments of the invention, capping is best achieved by forming a soft, cushioned, and conformable protective barrier in situ. A flowable medium or medium precursor can be introduced to surround and conform to the configuration of the nerve tip and then converted to a non-flowable state to form a protective plug that closely conforms to and bonds with the nerve tip. To include the mediator prior to and during transformation (e.g., cross-linking), the mediator may be introduced into a structure in which the nerve ends have previously or will be placed. Filling the medium into the structure allows the medium to surround the nerve ends and turn into a solid state while contained in a predetermined volume and configuration to consistently produce a protective, conformable nerve cap regardless of the diameter and configuration of the nerve stump. The structure prevents the medium from contacting the surgical site and forms a smooth shape around the nerve, allowing the nerve and structure to slide in the surrounding tissue.

Referring to fig. 1A to 1D, a nerve cap structure 10 is shown. The structure 10 extends between a proximal end 12 and a distal end 14 and includes a sidewall 16 extending therebetween. The side walls 16 are concave to create a structural cavity 18 therein. The structural cavity 18 is exposed to the exterior of the structure through a window 20.

The proximal end 12 of the structure 10 is provided with a nerve guide 22 to facilitate passage of a nerve 24 to position a nerve end 26 within the structural cavity 18. The nerve guide 22 may include a window or opening in the proximal end wall 12 of the structure 10 and is configured to support a nerve at a level that positions the nerve end 26 within the structural cavity 18. In the illustrated embodiment, the nerve guide 22 includes a support surface 28 on the upwardly concave housing to create a nerve guide channel 30. See fig. 1D.

FIG. 1E is a cross-sectional view taken along line 1E-1E in FIG. 1B. The width of the window 20 in the circumferential direction is smaller than the inner diameter of the cavity. This results in the recessed wall sections 29 on either side of the window 20 having edges lying on a plane 33, which plane 33 is parallel to a central vertical line (not shown) passing through the centre of the window 20. The edge plane 33 is parallel to and spaced from the tangent 31 to the inner surface of the side wall 16. The distance between the edge plane 33 and the tangent 31 is in the range of about 2% to about 30%, typically in the range of about 5% to about 20% of the inner diameter of the cavity.

Referring to fig. 1B and 1C, nerve ends 26 are positioned such that nerve ends 26 are separated from the inner surface of the sidewall of template 10 by at least 1mm, preferably 2mm or more, in any direction. This allows the medium 27 to flow into the structural cavity and around the nerve end 26 to provide a protective barrier in all directions.

Generally, a sufficient volume of hydrogel precursor is introduced into the cavity to produce a continuous protective coating around the nerve ends and at least about 1mm or 2mm or 5mm or more from nerve end to end. In some embodiments, the axial length of the cavity will be up to 10mm or 12mm up to about 2cm or less, and the ID width may be less than about 2.5mm or 2mm or less. The flowable precursor is typically at least about 100 microliters or 200 microliters or more in volume, but does not exceed about 500 microliters for small nerves (e.g., nerves up to about 4mm in diameter). Structural cavities for larger nerves (e.g., nerves having a diameter of about 4mm to about 10 mm) can accommodate at least about 1ml or 1.5ml, but typically less than about 4ml or 3ml of flowable precursor. As discussed further elsewhere herein, the precursor may be introduced into the cavity as two or three or more separate layers that adhere together to form the final cap. For example, the first substrate layer may be introduced into the cavity before or after the nerve is positioned in the cavity. During or after the transformation of the first layer, a second layer can be introduced to bond to the first layer and encapsulate the nerve, forming a protective nerve cap. The first layer will preferably contact at least the inferior surface of the nerve and may partially surround the nerve while exposing at least the superior portion of the nerve surface. Within about 5 minutes, preferably within about 1 minute or within about 30 seconds after the delivery of the first layer is completed, a second top layer is applied in contact with the exposed portion of the nerve and the exposed surface of the top layer to encase the nerve and form the final cap configuration. In another preferred embodiment, the nerve is fully or partially embedded in the precursor solution until it forms a gel. The mixer/blunt needle on the applicator tip is removed and a new mixer/blunt needle tip is attached so that additional material can be delivered in the second layer to cover the nerve.

Referring to fig. 1F, the inner surface of the cavity may be provided with one or more surface structures 35 to facilitate mixing and/or filling of the cavity. In the illustrated embodiment, the surface structure 35 includes a flow guide in the form of a radially inwardly extending protrusion structure for facilitating flow, such as a helical thread. The spacing and depth of the wires may be optimized with the viscosity of the flowable medium to promote filling and complete circumferential coverage of the nerve roots when the flowable hydrogel precursor medium is injected into the cavity.

After the media is transitioned from the relatively flowable state to the relatively non-flowable state, the structure 10 may be left in place, or may be peeled away to leave a barrier 60 of the plug structure formed, as schematically illustrated in FIG. 2.

To stabilize the structure 10 after placement and during the filling and transition stages, at least one stabilization feature 32 may be added. See fig. 3A. The stabilizing feature 32 may be at least one or two or four or more ridges, flanges or feet that provide lateral support surfaces 34 for contacting adjacent tissue and stabilizing the structure against movement. The lateral support surfaces 34 may extend along or parallel to a tangent to the sidewalls of the structure 10.

In one embodiment of the present invention, an aqueous bi-hydrogel construct is provided having connectivity at the junction between two nerve endings by creating a growth-permissive hydrogel junction between the two opposing nerve endings, and then encapsulating the junction with a growth-inhibitory hydrogel. The use of an in situ cross-linked hydrogel as a growth-permitting medium produces a joint of sufficient mechanical integrity and adhesion that can be picked up as a unit as if it were an intact nerve, which is then placed in a second structure to form an outer growth-inhibiting hydrogel capsule.

In another embodiment, an in situ formed thermosensitive hydrogel (such as a PEG-PCL-PEG triblock copolymer) is selected as the growth-permissive hydrogel. The hydrogel formed at the junction is soft enough that nerves can grow unimpeded through the hydrogel, but is sufficiently viscous to prevent the inhibitory hydrogel from flowing out to the junction between the two nerves. In another embodiment, the growth-permissive biomaterial provides a temporary barrier to the egress of the growth-inhibitory hydrogel, for example by using a viscous hyaluronic acid, pluronic, PEG, fibrin, or collagen solution.

In another embodiment, the injectable growth-permissive biomaterial is delivered in a bioabsorbable wrap structure, such wrap consisting of PEG-, pullulan-collagen or HPMC-based dry sheet. These films can be cast in the form of caps or wraps and dried by solvent casting with organic or aqueous solvents, and the films dried by evaporation at room temperature or in a lyophilizer. For example, these materials may be formed into a hat-like shape, similar to pullulan or gelatinThe process of making capsules (Capsugel Plantcaps) in which the biomaterial is melted, compressed and formed into the desired structure. Plasticizers include sorbitol and glycerin. General instructions on soft gelatin capsule formation can be found at the SaintyCo site (https://www.saintytec.com/soft-gelatin-capsules-manufacturing-process/) With small or large automated packaging equipment, the structure is adapted to the appropriate size and shape for use as a cap and wrap around the nerve.

Other sheets of interest include fibrinogen and thrombin sheets (US 10,485,894), hydroxypropylcellulose (JP 2009/183649A) or hydrophobic polymers (US 2012/0095418). The film has a sufficient thickness to support delivery of the hydrogel precursor solution, and desirably has a thickness of 10 to 100 microns, preferably 50 to 200 microns, more preferably 10 to 150 microns. The membrane preferably swells minimally after hydration to a thickness of less than 50%. In one embodiment, the thin film bioerodible cap and wrap structure dissolves into the growth-permitting molecules or polymers after 5 minutes. In another embodiment, the membrane remains in place and is cleared within 1 day to 6 months, preferably 1 day to 3 months.

In another embodiment, the injectable growth permissive biomaterial is delivered in a more traditional wrap sheet configuration, similar in size and configuration to the commercially available product (Axoguard neuroprotector), from about 1 to 4cm in length and from about 0.5cm to 4cm in width (similar in thickness and size to the oral tablet Listeria POCKETPAKs). When wrapped around nerve and growth-permitting biomaterials, the wraps of these structures are about 2mm x 40mm long (2 mm, 3.5mm, 5mm, 7mm, 10mm, and 20mm to 40mm long) in diameter. The wrap structure or catheter structure comprising the biodegradable, biocompatible material may or may not be pre-assembled into a second larger wrap structure. If the latter is the case, for example, after the growth-permitting biomaterial is placed around the nerve or nerve stump in the configuration of a wrap, the physician can deliver the growth-inhibiting biomaterial directly around the growth-permitting material without the need to handle the biomaterial wrap. By delivering the growth-permissive biomaterial in a biocompatible, biodegradable wrap, a softer or lower viscosity solution, gel or slurry can be delivered to the nerve in a close-fitting manner without dripping from the site. Finally, the growth-inhibiting biomaterial is delivered into a second larger diameter wrap structure around the nerve (typically 1-4mm larger in diameter than the wrap structure diameter, and if the second wrap structure is not biodegradable, it can be removed and discarded.

Referring to fig. 3B, the structure 10 includes curved sidewalls 66 that define a structure cavity 68. First and second nerve guides 70 and 72 communicate with cavity 68 and are sized and oriented to allow positioning of the first and second nerve ends in cavity 68 at a location where they will face each other and be surrounded by flowable medium introduced into cavity 68.

Referring to fig. 5A-5E, a series of steps for forming a bi-hydrogel conducting nerve connection between two nerve ends is shown. The first structure 50 includes elongated side walls that are curved to form a concave, e.g., semi-cylindrical, structure with an inner diameter that is larger than the diameter of the target nerve. The structure 50 has a first end 52, a second end 54, and an elongate channel 56 extending therebetween. A first nerve end 58 is positioned within the channel 56 from the first end 52. A second nerve end 60 extends from the second end 54 into the channel 56. The result is a structural cavity 62 formed between the first and second nerve ends and the side wall of the structure 50.

As shown in fig. 5B, a transformable growth-permitting hydrogel precursor is introduced into the structural cavity 62 to adhere to the nerve ends and polymerize in situ to form a conductive bridge 64 between the first nerve end 58 and the second nerve end 60. After the gel transitions to a less fluid state, the structure 50 is removed as shown, leaving connections comprising nerve ends connected by conductive bridges 64 of the polymerized growth-permitting gel 62. See fig. 5C.

Thereafter, the polymeric linkage is placed within the second structure 66, the second structure 66 having a central chamber 68 separating first and second

neural supports

70, 72, as shown in fig. 3B. A second growth-inhibiting hydrogel precursor is introduced into the central chamber 68 to surround and cover the formation of the conductive bridges 64 and nerve endings to produce a final configuration in which the first growth-permitting polymer bridges 62 are encapsulated by a second growth-inhibiting polymer capsule 70. See fig. 5E.

The nerve capping or nerve regeneration structures of some embodiments of the present invention may be provided in a clamshell configuration, as shown in fig. 6. The first housing 80 defines a first cavity 82 and the second housing 84 defines a second complementary cavity 86. The first and second housings are connected by a hinge 88 (e.g., a flexible living hinge made of thin polymer film). The first and

second housings

80, 84 may be rotated toward each other about a hinge 88 to form an enclosed chamber structure.

Fig. 7 shows a perspective view of a gripping tool 700, the gripping tool 700 configured to cut nerve tissue and to receive a structure for forming a hydrogel nerve connection after severing a nerve (as disclosed elsewhere herein). The tool 700 may include a plurality of proximal movable handles 702, each connected to a shaft 706 connected at a pivot 704, and may have an unlocked configuration as shown, movable to a locked configuration using a locking mechanism 705 (e.g., a series of interlocking teeth). The distal end 707 of the shaft 706 may include an end effector 708, the end effector 708 may include a sidewall 710, the sidewall 710 may have a curved geometry as shown, and a complementary cutting element 712 operably connected to the curved sidewall. In some embodiments, the structure 10 may be attached to the side wall 710 after cutting the nerve. In other embodiments, the structure may include an integrally formed cutting element. In some embodiments, the cutting element may be separated or otherwise removed after cutting, leaving the structure in place.

Fig. 8 is a close-up view of the end effector 708 of fig. 7, further illustrating that the end effector 708 may also carry the structure 10. Fig. 9 is a side close-up view of the distal end of one embodiment of a tool, showing that each end effector can include a cutting element and/or structure.

Figures 10A-10C illustrate various stages of a method of severing a nerve while removing axial fluid from the nerve endings to improve the snug fit between the nerve endings and the hydrogel. In some embodiments, the opposing end effector 708 may include a blade 712, which blade 712 may have equal or unequal lengths. As shown in some embodiments, the blades 712 on each end effector 708 may be generally opposed, but offset from each other. Activation of the end effector 708 may cause the blade to transect the nerve 24, creating a nerve end 26. The blade may be in a configuration as previously described. An absorbent material 780 (e.g., a swab) can be attached to one or more end effectors 708 (e.g., within a structure) and in close proximity (e.g., directly adjacent to one or more blades 712) to absorb any shaft pulp after nerve transection. The tip of the swab may for example be less than 5mm, more preferably less than 2mm, so that it can fit properly within the structure and support the nerve when the hydrogel is delivered.

Referring to fig. 11A-11E, in some embodiments, a delivery needle 1102 is advanced into an opening 1104 of a cap structure 1100 to deliver a hydrogel precursor into and around a nerve 1124. The opening 1104 may communicate with the cavity 18 through the sidewall at about the level of or below the support surface 28 to facilitate introduction of media below the nerve to form a first layer on the inferior side and partially wrap around the nerve. Also shown is a nerve guide 1122, which may be as described elsewhere herein. See fig. 11A.

The hydrogel can be delivered in two or more consecutive applications to partially (e.g., half) fill the structure and form a hydrogel layer 1150, as shown in fig. 11B. A second volume of precursor may then be introduced to completely fill the structure as shown in fig. 11C and form a hydrogel cap that wraps around the nerve end, after which the structure is removed. As shown in fig. 11D, the hydrogel can be delivered in a small bolus 1152 to surround the nerve endings, then the remainder of the cap is subsequently filled to form a hydrogel cap as shown in fig. 11E, after which the structure is removed. Thus, multiple layers (two or three or four or more) of hydrogel caps may be formed to encapsulate the nerve ends.

Referring to fig. 12, in some embodiments, a support rod 1215 is placed near and in contact with a portion of a nerve 1224. The wand 1215 provides additional strength to the nerve 1224 and naturally adheres to the nerve 1224 such that the nerve 1224 adheres to the wand 1215 regardless of the position of the wand. The hydrogel solution is then delivered onto or around the nerve 1224 and biodegradable rod 1215 to form an enhanced nerve cap. The rod 1215 can be biodegradable.

Referring to fig. 13A-13D, in some embodiments, one, two or more holes 1310 are provided on the sides of the cap or wrap structure 1300 to guide the needles to deliver the precursor solution to the correct location. The holes 1310 may be located at one of many locations around the structure as needed to deliver the precursor solution. A post 1330 may be included at the bottom of the cap or wrap structure to provide additional support to the nerve. The nerve length is placed on top of the post 1330, taking care that the nerve endings are not in contact with the post 1330. The post 1330 may be integral with the cap or wrap structure and subsequently removed when the structure is removed. Alternatively, the posts 1330 may comprise biodegradable posts that remain integral with the hydrogel cap. See fig. 13B and 13C. Alternatively, a first layer of hydrogel may be formed at the bottom of the cavity prior to introducing the nerve ends into the cavity. The nerve ends may then be placed on top of the first substrate layer. The precursor may then be introduced to encapsulate the nerve and bind to the first substrate layer. The first substrate may be formed during the clinical procedure or preformed during manufacture of the structure.

In some embodiments, the cap structure may include a partial cover 1320, as shown in fig. 13A. The structure is tilted so that the precursor material will flow to and fill the distal cap first, around the proximal nerve stump, and then subsequently fill the remainder of the nerve cap. As shown in fig. 13D, the cap or wrap structure may also include a raised tab (tabs) 1333 to tilt the longitudinal axis of the structure. By slightly tilting the cap structure, spillage of precursor material from the nerve entry region can be minimized.

Figures 14A-14C illustrate various views of an embodiment of a neural cap structure 1400 similar to that shown in figures 13A-13D, in which a partial cover 1420 is connected to an insert 1440 by a hinge 1428 to help center the cover 1420 on the window of the cap structure 1400. Also shown is a nerve guide 1405, which may be as described elsewhere herein.

Fig. 15A-15C show various views of a tearable cap structure 1500, which cap structure 1500 can include a peelable sheath 1560, which sheath 1560 includes a sidewall 1561 in which a nerve (nerve channel 1562) is placed. The precursor solution is delivered into first nerve channel 1562 and around first nerve channel 1562, and peelable sheath 1560 is subsequently peeled away from nerve 1524, for example using tearable tab 1564 as shown in figure 15A. The neurohydrogel 1570 is then rotated approximately 90 degrees and placed in the second larger diameter peelable cap structure 1501. A precursor solution is then applied into the nerve channel to surround the nerve and the first cap structure. The peelable sheath is then torn from the second tearable cap structure 1501. The final cylindrical cap structure contains the centered nerve. The nerve 1524 may then be rotated back to the normal physiological position, as shown in fig. 15B. Fig. 15C shows an alternative tearable cap structure design, which may include multiple pull tabs.

Figures 16A-16E show hydrogel filling the cap structure and surrounding the nerves in the cap structure. Fig. 16B shows a photograph of the hydrogel formed inside the cap structure. Fig. 16C shows a high resolution image of the cap. Figure 16D shows an example of a cap and wrap around the sciatic nerve of a pig. An example of a growth-permissive hydrogel (pink) that surrounds the nerve in a wrap structure and then is embedded in a second (blue) growth-inhibitory hydrogel wrap. See also fig. 5A-5E. The hydrogel was cut into cross-sections to see the growth-permissive (pink) hydrogel embedded in the growth-inhibitory (blue) hydrogel, as shown in fig. 16E.

Figure 17A shows neuroma formation after delivery of DuraSeal in a cap configuration around transected rat sciatic nerve. Figure 17B illustrates the absence of neuroma formation following delivery of the formulation of the invention around transected rat sciatic nerves. The hydrogel cap retains mechanical strength and in vivo durability for at least about 3 months, more preferably about 6 months

Figures 18A-18B schematically illustrate embodiments of mixing elements for mixing a two-part hydrogel system. In some embodiments, one static mixer 1800 delivers the hydrogel precursor solution into the central chamber, allowing the initial material out of the mixer to flow back and recirculate. The second static mixer captures the well-mixed solution and delivers it through the needle tip. Also shown are a fluid inlet 1802 (from a dual lumen applicator) and a fluid outlet 1804 (to a blunt needle).

Referring to fig. 18A and 18B, a mixer 1800 for mixing the two-part precursor components of the gels of the present invention is shown. The mixer at 1800 includes a housing 1802 having an inlet port 1804 and an outlet port 1806 in fluid communication via a flow path 1808. The inflow port 1804 and the outflow port 1806 can include luer connectors or other standard connection structures. The media introduced through the inflow ports 1804 passes along a flow path 1808 through at least a first static primary mixing column 1810. The mixing column 1810 includes an inner column of mixing elements in the structure of a tubular housing 1812 and a baffle 1814.

The media exiting the first static mixing column 1810 enters the secondary mixing chamber 1816. In the illustrated embodiment, the secondary mixing chamber 1816 passes media along a flow path 1808 into an optional second static mixing column 1818. The media exiting the second mixing column 1818 is directed out of the outflow port 1806.

The total volume of mixing medium delivered is typically less than about 5ml, typically no more than about 2ml, and in some applications less than 1ml. The first component entering the primary mixing column 1810 will generally be the first component to exit the primary mixing column 1810. The secondary mixing chamber 1816 is used to achieve different types of fold mixing to mix the effluent from the primary mixing column 1810 with itself and achieve excellent uniformity. The addition of the third mixing function by the addition of the optional second static mixing column 1818 further ensures uniformity of mixing of the hydrogel components such that the first 0.5ml or 1ml of hydrogel exits the outflow port 1806.

The mixing column 1810 preferably includes at least 4 mixing elements 1814, typically about 6 to 12 baffles 1814, typically no more than about 32 baffles 1814. The baffle may have an outer diameter of no more than about 1/8 inch, and in some embodiments no more than about 1/16 or 1/32 inch. The length of the static primary mixing column 1010 is typically less than about 4 inches, typically less than about 2 inches or less than about 1.5 inches. In one embodiment, the length is in a range from about 0.4 to 1.0 inches, more specifically about 0.5 to 0.7 inches.

Referring to fig. 18C-E, a combination dual chamber dispenser and mixing assembly 1830 is illustrated. Referring to fig. 18C, the dispensing and mixing assembly 1830 includes a housing 1832, the housing 1832 enclosing a first chamber 1834 and a second chamber 1836 for containing and holding separate first and second components. The components begin to mix at the merge point 1838 of the flow path from the chambers, for example, in response to advancing a plunger (not shown) into the proximal ends of the first and second chambers. The combined media streams are then propelled through a primary mixer.

In fig. 18D, a combined dual chamber dispenser and mixing assembly 1830 is shown with a single primary mixing post 1810 and secondary mixing chamber 1816. The media exiting the static mixing column 1810 passes along the flow path 1808 through the second mixing cavity 1816 and eventually through the aperture 1820 into the exit cavity 1822. A baffle 1824 may be provided to direct effluent from the static mixing column 1810 into the second mixing chamber 1816, which second mixing chamber 1816 will be substantially full before exiting through the aperture 1820.

Referring to fig. 19, a two component syringe for use with the mixer of fig. 18A is shown. The syringe includes a housing 1842 (the housing 1842 encloses a first chamber 1844 having a first plunger 1846) and a second chamber 1848 (the second chamber 1848 encloses a second plunger 1850). The first and second plungers are connected by a bridge 1852 to prevent dispensing from either chamber before the other.

An adapter 1854 may be provided for removable coupling to the housing 1842, and for removable coupling to the first chamber 1858 and the second chamber 1864 of the adapter 1854. The adapter includes a first connector 1856 for removably coupling to a first chamber 1858 containing a first medium 1860. Second connector 1862 may be removably coupled to second container 1864, and second container 1864 may contain second medium 1866.

Proximal retraction of the first and second plungers draws first medium 1860 and second medium 1866 into their respective chambers on the syringe, e.g., by manual retraction bridge 1852. Adapter 1854 is disconnected from housing 1842 and then the dual-chamber syringe is connected to a mixer, such as one of those disclosed herein.

Referring to fig. 20, a two-component syringe 1840 is filled with a first medium 1860 and a second medium 1866. A connector 1805 may be provided at the distal end of the syringe 1840 for connection to an inflow port 1804 on a mixer, such as mixer 1800. As already described, the media extruded from the injector follows the flow path 1808, and the final fully mixed first and second media mixtures may be extruded into the mold via the needle 1868, as described elsewhere herein.

The following table relates to specific non-limiting embodiments and devices for delivering in situ formed hydrogels.

Peripheral Nerve Stimulation (PNS). As neurostimulators have evolved from the spine to the periphery, and the hardware and batteries have been miniaturized, specialized peripheral neurostimulators are being developed and developed for blocking pain, stimulating muscle contraction, stimulating or blocking nerves to modulate disease and/or symptoms (e.g., pain), and stimulating nerve regeneration. With the development of new applications and new neurostimulators, the need to be able to maintain the stimulation electrodes and catheters in direct or close proximity to the target nerve has increased because 1) programmatically, placing the electrodes in the vicinity of the nerve can be challenging, and even after it is ideally placed in the vicinity of the nerve, programmatically, the electrodes can move; and 2) after placement, the electrodes may drift through the patient's motion or manipulation as the muscles contract or the implant is better positioned within the tissue. This may result in loss of treatment to the target nerve and thus loss of efficacy.

Transdermal delivery. With the advent of higher resolution handheld ultrasound and interventional pain and better training for orthopedic surgeons, percutaneously delivered implantable neurostimulators are increasingly being used as an alternative method of treating chronic pain. In one embodiment, once the electrode is placed near the nerve using a percutaneous delivery system, the electrode can be held in place near the nerve by delivering approximately 0.1 to 3cc of a conductive hydrogel to form around the electrode and hold it in close proximity to the nerve. In this embodiment, the electrodes are placed at the desired location and the in situ formed hydrogel is then delivered to anchor its location. The hydrogel media may be delivered through the lumen of the catheter delivery system or the lumen of the electrode and will be formed in situ. In some embodiments, the surface of the electrodes may be designed such that the interface is rougher, allowing for stronger embedding between the hydrogel and the electrodes to prevent wire migration. In other embodiments, a coil or other screw-like design is placed at the tip of the electrode to provide better fixation between the electrode, the hydrogel, and the surrounding tissue. For transdermal administration, a more rapidly degradable PEG hydrogel is desirable for the treatment of nerve regeneration, maintaining mechanical strength for one or two weeks before being eroded and cleared from the site. While these in situ formed hydrogels have sufficient adhesion to hold the catheter or electrode at the delivery site, strong pull on the electrode of the catheter will allow percutaneous removal from the delivery site if the electrode or catheter needs to be removed. PEG hydrogels suitable for these applications are based on multi-arm PEGs that degrade faster, such as PEG-SS (PEG-succinimidyl succinate-NHS ester) or PEG-SG (PEG-succinimidyl glutarate-NHS ester). For therapeutic applications of chronic pain requiring indwelling peripheral electrodes, it is desirable to deliver growth-inhibiting hydrogels or hydrogels with moderate to long-term mechanical strength, such as multi-arm PEG based on more slowly degrading bonds. Again, mechanical strength needs to be maintained for long periods of time to maintain the position of the electrodes within the hydrogel until a chronic foreign body reaction is sufficient to hold the electrodes in place. For example, to maintain longer-term lead placement, it is desirable to select cross-linked PEG hydrogels containing more stable ester, carbamate, or amide linkages, such as PEG-SG (PEG-succinimidyl glutarate-NHS ester), PEG-SAP (PEG-succinimidyl adipate-NHS ester), PEG-SC (succinimidyl carbonate-NHS ester), or PEG-SGA (succinimidyl glutarate-NHS ester). These PEG-NHS esters are preferably mixed and subsequently cross-linked with PEG-amines to improve flexibility relative to small molecule cross-linking systems such as melamine.

In further embodiments, the nerve stimulating factor is an injectable wireless implant and is in the form of a pellet, rod, bead, wrap, sheet or envelope that is held in place by a hydrogel adjacent to a nerve, ganglion or plexus. In one embodiment, the hydrogel is first delivered to the target site and the neurostimulator is delivered into the hydrogel slurry. In another embodiment, the neurostimulator implant is first delivered, adjusted to the desired position, and then the hydrogel is delivered therearound to fix it in the desired position. Similarly, an external magnet may be used to adjust the position of the neurostimulator implant to orient the implant adjacent to or in contact with the nerve or nerve tissue. In this embodiment, the gelation time may be adjusted to provide sufficient time for proper alignment of the neurostimulator, for example, gelation for 15 seconds to 1 minute. In some embodiments, a plurality of injectable microstimulator implants are injected into the degradable or non-degradable in situ-forming hydrogel.

In yet another embodiment, a microstimulator in the form of a microrod or nanorod is implanted in the growth-permissive hydrogel between two nerve stumps to promote neurite extension and accelerate regeneration. These microstimulators may stimulate nerve regeneration by delivering a magnetic, chemical or electric field through the gel and possibly along the microstimulator implant. In one embodiment, the microstimulator is a nanofiber, and may be injected into a nerve through a low gauge needle or catheter.

In another embodiment, a short-or long-acting microstimulator may be delivered with an injectable biocompatible biomaterial (e.g., a hydrogel) to form a neurostimulator anisogel. The microstimulator is magnetic, allowing directional control of the microstimulator implant, and, for example, allowing parallel alignment of the microstimulator within the hydrogel prior to formation of the gel from the precursor solution. These hydrogels would be injected around or near the nerve bundle or tendrils, and then the microstimulator could physically provide areas where they can grow and orient, as well as provide chemical, electrical or magnetic field stimulation to support neurite outgrowth.

Referring to fig. 4A, a configuration for electrically stimulating nerve regeneration according to the present invention is schematically illustrated. The proximal nerve stump 100 and the distal nerve stump 102 are positioned within a temporary structure 104, such as a silicone wrap, in the manner previously disclosed herein. A growth-permitting gel 106 is introduced into the structure to span the gap between the proximal nerve stump 100 and the distal nerve stump 102. An electrode assembly 108 having a probe or support 110 is positioned within the temporary structure 104, the probe or support 110 having at least one conductive surface 112 and, in a bipolar system, a second conductive surface 114. A conductive hydrogel 116 is introduced into the structure 104 and cured to support the nerve stump and the growth-permitting gel, and to maintain the position of the electrodes 108 relative to the growth-permitting gel 106.

RF stimulation may be achieved using any of a variety of microneedle electrodes, such as a stainless steel needle electrode (0.35 mm outer diameter, 12mm length) connected to the stimulator negative core (cathode) (Trio 300; ito, tokyo, japan). The operating parameters may include low frequency stimulation, typically less than about 200Hz, preferably in the range of about 2Hz to about. The current may range from about 1ma to about 10ma or more. The voltage may be about 3V, a square wave with intermittent pulses of about 0.1 ms. The duration may be from about 1 hour to 2 weeks, depending on the desired clinical presentation.

Open surgery. For open surgical applications, the hydrogel may also be deposited in a similar manner around electrodes that directly contact and/or are adjacent to the nerve under direct visualization. Likewise, deposition of about 0.5 to 1cc, preferably 0.2 to 2cc, more preferably 0.5 to 1cc of hydrogel is sufficient to maintain the position of the electrode relative to the nerve. In one embodiment, prior to delivery of the hydrogel, electrodes may be inserted into recesses in the form of silicone adjacent to and connected to the nerve. A form having a second inlet region for the electrode is envisaged. In this way, for example, when a gel is applied, the electrodes may be aligned parallel to the nerve or directly conform to the nerve. For applications requiring only one day to several weeks for neurostimulation treatment, pulling the electrode will make it relatively easy to remove from the hydrogel. The combination of the above-described growth-inhibiting hydrogel and growth-permitting hydrogel may be selected for use according to the application. For example, where an electrode placed near a nerve need only be left in place for days or weeks, a short term degradable hydrogel may be used. This provides sufficient time for the hydrogel to remain in place while the therapy is delivered and then rapidly cleared from the tissue. An example of this is the selection of a cross-linked PEG hydrogel containing more reactive ester linkages, such as PEG-SS or PEG-SAZ. These hydrogels are electrically conductive and therefore suitable for applications involving neurostimulators. In other embodiments, non-conductive polymers may also be used to isolate the electrical signal from surrounding tissue.

Generally, the selection of a low-swelling formulation is critical to maintaining fit with the electrode; in one embodiment, the hydrogel swells less than 30%, more preferably less than 20%, to maintain conformance to nerves and electrodes.

Referring to fig. 4B, an in situ hydrogel anchor for securing an electrode in electrical communication with a nerve is illustrated. The stent 110 carries a conductor in electrical communication with at least a first conductive surface 112 and preferably at least a second conductive surface 114 for delivering RF energy from an external energy source. A second or third or more pairs of conductive surfaces may be provided. A quantity of electrically conductive hydrogel 116 may be introduced into the structure and cured in situ in the manner previously discussed. The conductive hydrogel 116 encapsulates the electrodes and stabilizes the electrodes relative to the adjacent nerve 120 such that the electrodes are in electrical communication with the nerve 120 through the conductive hydrogel 116. Alternatively, the electrodes may be pinned between the hydrogel anchor formed in situ and the nerve 120, or by forming a conductive hydrogel anchor around the nerve 120 and adjacent electrodes. The electrode may be configured to be removed from the conductive hydrogel. Alternatively, the electrode may be removed from the patient after absorption of the hydrogel. In one embodiment, the conductivity of PEG hydrogels can be enhanced by the incorporation of PSS in the PEG hydrogel matrix, resulting in the in situ formation of PEDOT: (Kim et al 2016.High purity connected and hydrated PEG-based hydrogels for the Functional application of a tissue engineering scaffold and Functional Polymers, DOI:10.1016/j. Reactive functionalized polymers.2016.09.003). In another embodiment, the metal nanoparticles and carbon-based material may be delivered in a hydrogel, including gold, silver, platinum, iron oxide, zinc oxide or polypyrrole (PPy), polyaniline (PANi), polythiophene (PT), PEDOT (above) or poly (p-phenylene vinylene) (PPV), such as Min et al 2018.Incorporation of Conductive Materials in Hydrogels for Tissue Engineering applications, polymers,10,1078; doi:10.3390/polym10101078, incorporated herein.

In yet another embodiment, the in situ formed hydrogel can be used to secure a convection enhanced delivery system to the site. Like an implantable neurostimulator, the drug delivery catheter may be fixed about 10mm near the site of the damaged nerve, with the tip about 5mm from the nerve damage. As with implantable neurostimulators, the silicone structure may be designed to include an access area or cut away from the top edge of the silicone structure to allow the catheter or stimulator lead to reside in the structure in preparation for the incorporation of the hydrogel. After delivery of the therapy (neurostimulation, convection enhanced drug delivery), the catheter or neurostimulator can be removed from the hydrogel without breaching the protective barrier around the hydrogel. For example, U.S. patent No.9,386,990, teaches the repair of nerves using DuraSeal for 2 to 4 weeks in vivo, and hydrogels do not provide the sustained mechanical strength necessary to prevent neuroma formation or relaxation (relaxation) of nerves during the regeneration process (e.g., within 3 months and 4 months after surgical repair). For example, cross-linked multi-arm PEGs containing rapidly degrading ester linkages (e.g., PEG-SS or PEG-SG) are suitable for applications that prevent acute and subacute adhesions from forming around nerves. As another example, low molecular weight linear PEG has been shown to act as a fusogenic agent when injected around damaged nerves and promote nerve repair and regeneration (but does not provide mechanical strength or durability to prevent neuroma formation). For example, PEG hydrogels (e.g., PEG tetraacrylate hydrogels) have been used to reattach nerves in preclinical models (Hubbell 2004/0195710).

Generally, PEG with readily hydrolyzable ester linkages does not contain degradable linkages necessary to support the desired mechanical strength or in vivo durability necessary for applications to prevent abnormal nerve growth and neuroma formation. Commercially available PEG hydrogels, particularly conventional PEG with hydrolyzed ester linkages, do not have suitable mechanical strength or in vivo durability to prevent neuroma formation for three to four months until nerve repair or neuroma prevention is achieved. These PEGs and PEG gels may initially have sufficient mechanical strength to temporarily help repair nerves at the anastomosis and/or prevent adhesion formation, but the hydrogel may not have sufficient mechanical strength to prevent abnormal neuroma formation two months, or more preferably three months, after administration and thus may not be suitable for the hydrogel cap. Figure 16 provides an example of the lack of durability of DuraSeal hydrogel in preventing neuroma formation in rat sciatic nerve transection constructs. Hydrogels containing ester linkages have either degraded sufficiently to no longer provide a barrier to nerve regeneration, or have been shed from nerves, or have been completely cleared. As a result, the initial mechanical barrier is not sufficient to act as a long-term barrier to prevent nerve growth, and neuroma formation occurs.

In another embodiment, mechanical unloading of the nerve is required. By designing the nerves to be covered with nerves of at least 8mm, preferably 10, 15 or 20mm length, there is sufficient adhesion on the circumference so that the tension is partially unloaded into the gel and better distributed. By providing mechanical unloading, the hydrogel can support the regenerating nerve (wrap). Given a nerve stump (S) of 10mm or greater length, the nerve can be embedded in the hydrogel through a bend or "S" bend, minimizing tension in the nerve anastomosis.

Other methods teach delivering the in situ formed hydrogel directly around the nerve without protecting the underlying muscle from adhesions, or provide methods of systematically circumferentially covering the proximal nerve endings with a hydrogel. The polymer systems formed in situ adhere to the surrounding tissue they contact during crosslinking or polymerization, although to varying degrees. If the non-target tissue (e.g., muscle or fascia) is not protected or shielded, the hydrogel also adheres to the tissue. Since it is preferred that nerves slide freely within the plane of the fascia (usually between muscles), it is undesirable to limit their movement and may cause pain and/or loss of therapeutic effectiveness. Some embodiments described herein provide a form that separates the in situ formed hydrogel from the surrounding environment to prevent binding between the nerve and surrounding tissue and to allow the nerve to slide within the fascial channel. Gliding can be achieved by two mechanisms, 1) lubricity and streamline structure of the hydrogel after forming a cap or wrap structure, or 2) hydrolysis of the hydrogel supporting 20 to 80% swelling, more preferably 40 to 70% swelling, with a more rapidly degrading PEG hydrogel by selecting a formulation with minimal equilibrium swelling in two to three weeks. The second stage of swelling, degradation swelling, allows the injured nerve to slide through the inner lumen of the hydrogel (lumen enlargement as swelling of the hydrogel transitions to outward swelling) after negligible equilibrium swelling to prevent both intraoperative and postoperative acute inflammatory reactions.

Nerve block. To block nerve regeneration, the biomaterials formed in situ need to have physical properties including negative or neutral charges, smaller pore sizes, hydrophilicity, and/or higher crosslink density that prevent migration of nerves into the biomaterials. While most research has focused on materials for nerve regeneration, there are also studies that record biomaterials in which nerves cannot grow (including poly (ethylene glycol) based hydrogels, agarose and alginate based hydrogels), especially at higher polymer concentrations. Higher concentrations generally have higher crosslink densities and therefore smaller pore sizes. These hydrogels, due to their charge, inert surface, hydrophilicity and pore size, are capable of preventing neurite outgrowth in vitro and in vivo. In one embodiment, agarose may be selected at a concentration of, e.g., 1.25% wt/vol to prevent nerve regeneration. In another example, the PEG hydrogel can prevent the formation of neuroma at a concentration of 4% w/v or greater. In other embodiments, even positively charged or naturally formed in situ biomaterials can provide a barrier to nerve regeneration if the solids content and crosslink density are such that the pores are too small for cell ingrowth.

To prevent neuroma formation, the biomaterial formed in situ needs to provide the necessary mechanical strength to act as a barrier to nerve regeneration over a period of two months, more preferably three months or more. Many in situ formed gels, including commercial in situ formed PEG hydrogels with biodegradable ester linkages, may initially have sufficient mechanical strength, but the rate of hydrolysis is such that their loss of crosslink density is large enough that their mechanical strength at 1-2 months is insufficient to prevent neuroma formation (see table 1). In vivo experiments in the rat sciatic nerve model demonstrated that bulbous neuroma formation 1-3 months after delivery of these hydrogels around transected nerve stumps, comparable to nerve transection alone. Preclinical testing has shown that a mechanical strength of at least 5kPa, preferably 10kPa, more preferably 20kPa or higher is necessary to prevent neuroma formation. At three months, in vivo studies have shown that these hydrogels have completely degraded and cleared from the site, or have lost sufficient mechanical integrity that nerves have grown to a soft, collapsed and/or fractured gel and formed neuroma. Thus, while the prior art teaches the use of PEG hydrogels for nerve repair purposes, not all hydrogels are suitable to support the long-term mechanical strength and durability requirements necessary to prevent neuroma formation and abnormal nerve growth. Preferably, the barrier has an in vivo persistence of at least about two months or at least about three months, preferably four months, more preferably 6 months, to reduce or prevent neuroma formation and reduce chronic neuropathic pain following surgery. The mechanical integrity of the hydrogel at various points in vitro and in vivo can be assessed by compression testing, as described further below.

And (4) durability. The in vivo durability of biodegradable hydrogels is related to the crosslink density and thus the mechanical integrity of the hydrogel. For applications to prevent neuroma formation, the hydrogel must degrade slowly enough that the hydrogel does not lose significant structural integrity during the weeks to months that nerves attempt to regenerate (which occurs within about 3 months and may be 6 months or more in humans). In this manner, the durability of the hydrogel and the durability of the mechanical integrity of the hydrogel are critical to provide sustained protection and prevent neuroma and abnormal nerve growth (preferably for 3 months or more, preferably 4 months or more). In embodiments where a degradable hydrogel is used, the mechanical strength must be maintained for more than 2 months, preferably 3 months, and thus the hydrogel must not degrade significantly during this period of time (preferably 3 months or more). Similarly, mechanical integrity and durability of the hydrogel are critical for the sustained load provided by the hydrogel around the nerve-nerve or nerve-graft interface, with a duration of preferably 2 months, more preferably 3 months, since even nerves that have been directly sutured to each other by direct engagement do not recover their original strength (nerves have approximately 60% original strength 3 months after transection).

It is challenging to develop in situ formed polymers, particularly in situ formed synthetic hydrogels, including PEG-based hydrogels with longer in vivo mechanical strength and longer durability of more than 2.5 to 3 months but less than 12 months. For example, there is a significant difference between the in vivo persistence of PEG hydrogels with biodegradable esters in and around the surgical environment of nerves (weeks less than 3 months) and PEG hydrogels containing biodegradable urethane or amide linkages (degradation characteristics at the subcutaneous extra-muscular site are approximately 9 months to 18 months or longer). Some embodiments concern in situ forming polymers (preferably multi-arm PEGs, including PEG-NHS-esters and PEG-amines in combination with biodegradable linkages) with the mechanical strength and durability required to prevent neuroma formation. In particular, the swelling, mechanical strength and in vivo durability of PEG hydrogels are described for long-term safety and efficacy in applications requiring long-term prevention of abnormal nerve growth and the ability to relax and unload nerves within a few months after surgical repair.

To achieve suitable mechanical strength and durability in vivo, conventional PEG hydrogels containing degradable ester linkages (which are widely commercially available as dural and lung sealants) are not suitable for peri-neurological applications because they lose mechanical strength and/or are eliminated within a few months. In short, degradation occurs at a rate fast enough that the mechanical integrity is not maintained for a long enough time, making these hydrogels suitable for adhesion prevention, but not for preventing nerve growth. In embodiments where a non-degradable PEG hydrogel is used, the mechanical strength of the hydrogel is based on the initial mechanical strength of the hydrogel, as the crosslinks do not degrade over time. In vitro and in vivo tests on a series of hydrogels with different molecular weights, degradable bonds, crosslink density showed that only hydrogels with sufficient mechanical strength (and in vivo durability) at 3 months could prevent neuroma formation. The following table provides examples of hydrogels, degradation times, and neuroma formation. Figure 16A shows the formation of neuroma following DuraSeal delivery.

Examples of Multi-arm PEG hydrogels with various hydrolytically unstable linkages

By in vivo persistence is meant that the biological material is not significantly absorbed, e.g., resorbed at a given point in time by less than 25%, preferably less than 15%. Depending on the biomaterial, this can be assessed by loss of mass, loss of crosslink density, or change in the form of the biomaterial. Active bonds with longer degradation times in vivo, such as PEG urea (e.g., PEG isocyanate, PEG-NCO), PEG urethane (PEG-succinimide carbonate) (PEG-SC), and PEG carbamate. Hydrogels comprising polyethylene glycol succinimide carbonates (PEG-SCs) with more than 2 arms, such as 4-, 6-or 8-arm PEG, with molecular weights ranging from 1K to 50K, preferably 10K to 20K, such as 10K, 15K or 20kDa, are preferably used for cap or nerve repair, wherein the hydrogel preferably has a longer in vivo persistence. In some embodiments, a 4 arm 10K PEG-SC, a 4 arm 20K PEG-SC, an 8 arm 10K PEG-SC, an 8 arm 15K PEG-SC, or an 8 arm 20K PEG-SC is selected, more preferably a 4 arm 10K PEG-SC or an 8 arm 20K PEG-SC is mixed with an 8 arm 20K amine or a 4 arm 10K amine. The following patent 20160331738A1 is incorporated herein by reference. In other embodiments, e.g., for encapsulation of nerves, a combination of 8-arm 20K PEG-SG or 8-arm 15K PEG-SAP with 8-arm 40K amine or a mixture of 8-arm 20K amine is preferred to provide structural support during nerve regeneration and/or to prevent nerve compression and scar tissue formation during acute and subchronic stages, which are then degraded and cleared from the site. For applications to prevent nerve compression or to support nerve regeneration after nerve injury, growth inhibitory PEG hydrogels with shorter in vivo degradation curves are preferred. For these applications, the hydrogel should provide sufficient mechanical strength to prevent abnormal nerve growth and to prevent immunoinfiltration into the healing nerve. PEG is suitable for these

And (3) compressive strength. The desired compressive strength (elastic modulus, young's modulus) of the growth-inhibiting hydrogel is greater than 10kPa, preferably greater than 20kPa, preferably greater than 30kPa. In a preferred embodiment, the compressive strength after 3 months in vivo is greater than 20kPa, more preferably greater than 40kPa at 3 months after administration.

After in vitro equilibration and after harvesting implant samples from the rat subcutaneous space, compressive strength was measured on a desktop computer, where hydrogel columns (d =6 mm) were cut to 100mm length, pre-equilibrated (12 hours at 37 ℃) and evaluated for compressive strength. The compressive properties of the hydrogel formulations were measured with an Instron at a rate of 1 mm/min. The modulus is calculated as the slope of the tangent to the linear region between 0.05 and 0.17 of the stress-strain curve.

Compressive strength of various formulations

Although the in vitro mechanical strength and durability of hydrogels (37 ℃, PBS) are generally less correlated with in vivo durability, the maintenance of mechanical strength of hydrogels for 3 months in vitro is a strong indicator of the ability of hydrogels to provide a sustained mechanical barrier to nerve regeneration in vivo.

In some embodiments, the cleavable carbamate, carbonate, or amide linker in the biodegradable hydrogel allows for a more stable slow degrading bond to maintain the necessary mechanical strength to prevent nerve growth for three months or more and, thus, maintain durability in vivo, thereby providing a durable mechanical barrier to nerve regeneration.

Typically, the structure of the multi-arm PEG is

C–[(PEG) _n -M-L-F] _m

Wherein

Core structure of C = multi-arm PEG

n = PEG repeat units on each arm (25 to 60 units)

M = modulator

L = cleavable or non-cleavable linkers (ester, carbamate, amide, urea, carbamate, carbonate, thiourea, thioester, disulfide, hydrazone, oxime, imine, amidine, triazole and thiol/maleimide).

F = reactive functional group for covalent crosslinking, such as maleimide, thiol or protected thiols, alcohols, acrylates, acrylamides, amines, protected amines, carboxylic acids or protected carboxylic acids, azides, alkynes, 1, 3-dienes, furans, a-halocarbonyls and N-hydroxysuccinimides, N-hydroxysulfosuccinimides or nitrophenyl esters or carbonates

m = number of PEG arms (e.g. 2, 3, 4, 6, 8, 10)

In some embodiments, the hydrolysis modulator (M) may be incorporated into the backbone of the hydrogel to slow the hydrolytic degradation of the ester linkages (L) in the hydrogel. This can be achieved by electron donating groups associated with the reaction or by increasing the length of the carbon chain adjacent to the ester bond to increase hydrophobicity and protect the bond from hydrolysis. For example, PEG-SAP, PEG-SAZ are examples of PEG-ester linkages having longer carbon chains than PEG-SG. In another embodiment, an aromatic group is placed next to the ester group to provide additional stability of the ester bond against hydrolysis, such as PEG-aromatic carboxyl esters, including benzoates or substituted benzoates.

In some embodiments, a more stable or slowly degrading bond, such as a urethane or amide bond, may be selected to provide the necessary mechanical strength and in vivo persistence to prevent neuroma formation.

In other embodiments, the hydrolysis modulator (M) may be designed in the backbone of the hydrogel to increase the hydrolytic degradation of the urethane in the hydrogel. This can be achieved by adding electron withdrawing groups that accelerate the reaction.

In one embodiment, the rate of hydrolysis of the urethane linkage can be modulated by adjacent groups, thereby modulating the in vivo persistence of the hydrogel. R1 and R3 can be any aliphatic hydrocarbon group (-CH 2-, -CHR-, -CRR' -), substituted aliphatic hydrocarbon groups, aromatic groups, and substituted aromatic groups in any permutation. The aromatic groups include, but are not limited to, phenyl, biphenyl, polycyclic aryl, and heterocyclic aryl. Substituents to the aliphatic and aromatic groups include, but are not limited to, halogen, alkyl, aryl, substituted alkyl, substituted aryl, substituted heteroaryl, alkenylalkyl, alkoxy, hydroxy, amine, phenolic ester, amide, carboalkoxy, carboxamide, aldehyde, carboxy, nitro, and cyanide. R2 may be H, and any of R1 and R3. Further, R1 may include isocyanate, aromatic isocyanate, diisocyanate (e.g., LDI). In one embodiment, R3 may be aniline, and in another embodiment, R1 may be phenyl.

In another embodiment, the rate of hydrolysis of the urethane linkage can be modulated by a modulator of the beta position. The modulator may be CF3PhSO2-, clPhSO2-, phSO2-, menPhSO2-, meOPhSO2-, meSO2-, O (CH 2CH 2) NSO2-, CN-, (Et) 2NSO2-. In other embodiments, these modulators may be useful for PEG hydrogels containing amide, carbonate, and urea linkages. Other modulators that affect the rate of hydrolysis of urethane linkages are described in 7,060,259, which is incorporated herein by reference. Other cleavable crosslinks are described by Henise et al (2019) In vitro-In vivo correlation for the differentiation of Tetra-PEG-hydrogel-microsheres with tunable b-electrophoretic crosslinking, international Journal of Polymer Science, which is incorporated In its entirety. These modifier groups M may be pendant to the backbone itself or nearby, with a β elimination linker as described in D.V. Santi et al (2012) predicable and tunable half-life extensions of therapeutic agents by controlled chemical formulations, PNAS,109 (6) 6211-6216 and US20170312368A1, which are incorporated herein by reference. In some embodiments, a soft chain extender, such as an amino acid peptide-based chain extender having an ester bond, is added. Such as poly (phosphoester urethanes) with chain extenders containing phosphate linkages. For example, poly (DL, lactide) is a chain extender or poly (caprolactone) to extend the PEG chain and add soft segments. Preferably, the molecular weight of the chain extender may be from 0.5kDa to 5kDa, preferably from 1 to 2kDa, more preferably 2kDa. The soft segment may provide additional properties to enhance the physical properties of the hydrogel, including thermal sensitivity, crystallinity, and the potential to cause physical and chemical crosslinking. These hydrogels may be composed of, for example, PEG with a molecular weight between 1,000da and 50kDa, including multi-arm PEG-succinimide carbonates (4 or 8 arms) with a molecular weight of 5 to 40kDa, with arm lengths of 1 to 3kDa, and PEG-amines (4 or 8 arms) with a molecular weight of 5 to 40kDa, preferably 10 or 20 kDa. In one embodiment, PEG-SC (4 arm 10K) is crosslinked with PEG-amine (8 arm 20K). Preferably, the solid content of PEG is between 6 and 10wt%, more preferably 8wt%. In one embodiment, PEG-SC (8 arm 15K) is crosslinked with trilysine amine. In one embodiment, PEG-SC (4 arm 20K) is crosslinked with a trilysine amine. Examples of other in situ formed PEG-SC formulations are described in 6,413,507, which is incorporated herein by reference. In another embodiment, 4-arm PEG succinimidyl glutaramide 4-arm 10K (PEG-SGA) may be used in combination with 8-arm PEG-amine 20K at a solids content of 8%.

Alternatively, functionalized PEG urethanes and esters can be covalently crosslinked with another reactive polymer or small molecule (e.g., a trilysine) containing an amine or protected amine, maleimide, thiol or protected thiol, acrylate, acrylamide, carboxylic acid or protected carboxylic acid, azide, alkyne (including cycloalkyne), 1-3 dienes and furans, alpha-hydroxycarbonyl and N-hydroxysuccinimide groups, N-hydroxysulfosuccinimide groups, or nitrophenyl or carbonate groups.

In other embodiments, blends of faster degrading PEG esters and slower degrading PEG-SGAs or PEG-SCs in a ratio of 10: 1 or 5: 1 may allow for a slowing of the in vivo degradation curve without significant loss of mechanical strength during the initial stages of nerve regeneration. Similarly, a blend of multi-armed PEG-SC and PEG-amine that crosslink to form urethane and PEG-carbonate linkages (delayed reaction of PEG-SC and hydroxyl functional groups) forms a mixed 60.

In other embodiments, multi-arm PEG may be combined with blocks of other hydrolytically degradable polymers that may be used to tailor the degradation time of the PEG hydrogel. For example, soft segments with polyester di-or tri-blocks can be synthesized with low molecular weight polyester regions to allow hydrogel formation in aqueous environment (polycaprolactone, polylactic acid, polyglycolic acid, polyurethane, polyhydroxyalkanoate (PHA), poly (ethylene adipate) (PEA), aliphatic diisocyanates such as isophorone diisocyanate (IPDI) or L-lysine ethyl ester diisocyanate (LDI)). These blocks can be composed of lactide, glycolide, or caprolactone regions, which can be used to provide additional mechanical strength to the hydrogel depending on the degree of crystallinity (D, L, or L, L), allowing adjustment of the degradation profile. Book-advances in biomaterial science and biomedical applications).

In some embodiments, heterobifunctional crosslinkers are used to enable the polyester to be conjugated with some arms and NHS esters or other functional groups with other arms.

In other embodiments, excipients may be incorporated into the hydrogel to alter the mechanical strength, density, surface tension, fluidity, and in vivo durability of the hydrogel. These modulators are encapsulated in the hydrogel as it is formed. The modulator may include an amphiphilic excipient (e.g., vitamin E TPGS), a low molecular weight polyester (e.g., caprolactone) or a solvent (e.g., ethanol). In one embodiment, ethanol is incorporated into the diluent or accelerator solution to produce 5% to 70% v/v ethanol-loaded hydrogel. The ethanol increases the elasticity of the hydrogel and decreases the density of the hydrogel precursor solution relative to the nerve density. In addition, low concentrations of ethanol may be incorporated into the hydrogel to increase the useful or functional life of the PEG/diluent solution after PEG powder suspension. In another embodiment, pluronic may be incorporated into the diluent or accelerator solution to 5-15% w/v, resulting in a PEG-SG hydrogel with improved elasticity and in vivo durability. In another embodiment, low molecular weight caprolactone is incorporated into the diluent solution to yield a 1-5% w/v PEG/caprolactone blended hydrogel. In another embodiment, the vitamin TPGS can be incorporated into a diluent solution resulting in a 5-20% w/v PEG/vitamin E TPGS blend.

Swelling. Another key element of these hydrogels is the swelling of the hydrogels applied around the nerve. When the hydrogel is delivered circumferentially around an object (e.g., a nerve), it undergoes a positive swelling in an outward radial direction. Initially, the hydrogel undergoes equilibrium-mediated swelling upon equilibration with fluids in the surrounding environment, and subsequently, when a critical number of hydrolytic bonds are broken, the hydrogel swells due to a loss of mechanical strength. The latter stage of degradation mediated swelling results in a gradual loss of mechanical strength and softening of the hydrogel, which collapses and is eventually cleared from the site. In vivo experiments in which transected rat sciatic nerves were surrounded by hydrogels swollen 5%, 10%, 20%, 30%, and 60% showed that hydrogels swollen more than 30% were more likely to be detached from the nerves due to the creation of a gap between the nerve and the hydrogel. Notably, PEG hydrogels with swelling degrees equal to or less than 0% shrink when equilibrated in vitro or in vivo, and the resulting compression may result in sustained hydrogel-mediated local neuropathic pain. For example, the DuraSeal hydrogel swells significantly when delivered in situ and has a tendency to slough off the proximal nerve stump.

Equilibrium swelling. For applications where the hydrogel is delivered to a nerve to prevent nerve regeneration, it is desirable to maintain a tight adhesion and fit between the nerve and the conformable hydrogel. Therefore, it is desirable to minimize equilibrium swelling after hydrogel delivery. When the hydrogel is equilibrated with a fluid in an in situ environment, equilibrium swelling occurs within minutes to days. In one embodiment, the hydrogel swells more than 0% but less than 40%, preferably more than 5% and less than 30%, more preferably more than 5% and less than 25%.

Furthermore, in some embodiments, it is desirable to avoid hydrogel shrinkage, as these hydrogels may stress nerves and cause abnormal nerve firing, so it is preferred to use hydrogels with a swelling greater than 0%. In addition, the nerve may swell after injury, and thus some swelling capacity is required to give the nerve some space for swelling.

The equilibrium swelling is preferably evaluated in vitro at body temperature (37 ℃ in PBS). Hydrogel samples were prepared in cylindrical silicone tubes (6 mm) and cut to dimensions of 6mm diameter by 12mm length. Samples were weighed and pooled into PBS at 37 ℃. After swelling in PBS at 37 ℃ for 12-24 hours, the sample was removed and weighed again. Swelling was calculated by the percentage of mass increase.

Degrading and swelling. A second feature of biodegradable or bioerodible hydrogels after initial equilibrium swelling is the recognition that a second, still ongoing swelling phase occurs as a result of hydrogel degradation. Swelling may occur through hydrolytically, enzymatically, or oxidatively sensitive bonds in the hydrogel. This is an equally important feature as the hydrogel needs to remain on the nerve for a period of one month or more, more preferably two months or more, more preferably three months. In animal models, this period is shorter, whereas in a clinical setting, this period is longer. In some cases, if the rate of degradation is too fast, the hydrogel may break and fall off the nerve or be cleared before the hydrogel can act to prevent nerve growth and/or prevent neuroma. In other cases, if the hydrogel appears intact on the nerves, but the mechanical integrity within the hydrogel may be significantly lost due to degradation, the nerves may extend into the softened or fractured hydrogel and form neuromas. Thus, it is preferred that the biodegradable system has no more than 50% of hydrolytically unstable bond breaks at 3 months, more preferably no more than 30% of bond breaks, even more preferably no more than 20% of bond breaks. After a period of time when the hydrogel provides a mechanical barrier for nerve regeneration, the crosslink density will decrease and degradation will continue until the hydrogel is completely cleared. Loss of bonds can be assessed in part by a decrease in the mechanical integrity of the hydrogel. Loss of bonds can be assessed in part by a decrease in the mechanical integrity of the hydrogel. Therefore, it is desirable that the hydrogel maintain a compressive modulus of 40kPa for 3 months after delivery, and that the hydrogel be sufficiently hard that nerves do not grow through it.

And (4) pressure. In addition to ensuring that the swelling is not so great as to cause the hydrogel to fall off the nerve, it is also necessary to confirm that swelling (or low swelling, shrinkage) does not result in nerve compression. In one experiment, a pressure sensor catheter was placed alongside a nerve and an in situ-forming hydrogel was delivered around the nerve/pressure sensor (Millar Mikro-Tip pressure catheter, 3.5F, straight, AD instrument). The hydrogel was then placed in PBS at 37 deg.C and the change in pressure over time was measured. Hydrogels with approximately 0% or negative swelling resulted in high and sustained increases (> 80 mmHg) in pressure applied to the embedded nerve, while hydrogels with 10% or more swelling did not result in significant increases in pressure (-20 mmHg). In a preferred embodiment, the pressure reading after equilibrium swelling is about 5mmHg. In preclinical and clinical models, the pressure at the site of nerve injury can be between 5 and 15mmHg (Khaing et al 2015-Injectable Hydrogels for Spinal Cord Repair). For example, although a number of materials that modulate nerve regeneration were evaluated in the spinal cord model, most materials do not have the linear compressive modulus (G) required to prevent neuroma formation (table 1, khaing et al, 2015).

Hardness. The hardness of the hydrogel can be measured/inferred by rheology (G' = storage modulus, G = shear modulus) or linear compression modulus (G). Preferably, the hardness of the hydrogel, measured by the linear compression modulus (G), is greater than 10kPa, preferably greater than 30kPa, more preferably greater than 50kPa. This stiffness prevents nerve growth into the surrounding hydrogel.

Compression and rebound. In addition to injectable gels with minimal swelling, compressible gels are also desirable. In this manner, the hydrogel implant does not break even if it is compressed. Compression and rebound tests were performed on cylindrical samples (6 mm diameter, 10mm long) which were incubated at 37 ℃ for at least 1 hour until equilibrium was reached. The sample was loaded into an Instron and a displacement perpendicular to the longitudinal axis of the cylinder was applied at a crosshead speed of 1 mm/min, so that the final displacement was 60% of the catheter diameter. It was verified that the hydrogel could withstand a compressive force of more than 0.25N and that no change in shape and diameter occurred after removal of the compressive force.

Flexibility. Another key parameter of these in situ formed polymers is the ability of the hydrogel to bend (bend) and flex (flex) at physiologically relevant angles in vivo. To evaluate the flexibility of the hydrogel, the hydrogel was formed in silicone tubing with an inner diameter of 0.1 to 0.25 "to form a 12 to 24" long cylindrical hydrogel rope. Preferably, the hydrogel has sufficient flexibility to bend more than 90 °, and more preferably, cylindrical hydrogel micelles can be easily knotted. Since flexibility and elasticity are determined in part by the distance between the core of a multi-arm PEG and the core of its neighboring multi-arm PEG, the PEG hydrogel has a core-to-core distance of 3kDa, more preferably 5kDa or greater. Flexible and strong hydrogels that do not break in highly mobile and stressed environments. Thus, a more flexible hydrogel is desired, e.g., a combination of 4-arm 10K or 20K PEG with 4-arm or 8-arm 20K PEG-amine may be desirable.

Viscosity. The low and medium viscosity precursor solutions can be selected to encapsulate hydrogels that generally have better adhesion in low viscosity solutions and improved nerve management capabilities in medium viscosity precursor solutions. In one embodiment of the invention, the flowable medium is a low viscosity hydrogel precursor solution having a viscosity of no more than about 100cP, in some embodiments no more than about 20cP or no more than about 5cP. In yet another embodiment, the flowable medium is a medium viscosity hydrogel precursor solution, preferably having a viscosity of 300 to 1000cP, more preferably 300 to 900cP. In one embodiment, a viscosity enhancing/modifying agent or thickening agent may be added to the gel precursor to modify the fluid properties and help locate the nerves in the cap prior to gelation. The viscosity modifier may be a natural hydrocolloid, a semi-synthetic hydrocolloid, a synthetic hydrocolloid, and a clay. Natural hydrocolloids include, but are not limited to, gum arabic, tragacanth gum, alginic acid, alginates, karaya gum, guar gum, locust bean gum, carrageenan, gelatin, collagen, hyaluronic acid, dextran, starch, xanthan gum, galactomannan, konjac mannan, tragacanth gum, chitosan, gellan gum, methoxy pectin, agar, gum arabic, and dammar gum. Semi-synthetic hydrocolloids include, but are not limited to, methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose (HPMC, 0.3%), modified starch, propylene glycol alginate. Synthetic hydrocolloids include, but are not limited to, PEG, polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, polyglycerol polyricinoleate. Clays include, but are not limited to, magnesium aluminum silicate (Veegum), bentonite, attapulgite.

In another embodiment, the viscosity can be adjusted by pre-crosslinking the PEG-amine and PEG-urethane. PEG-amine and PEG-urethane can be pre-crosslinked at a ratio of 10000: 1 to 1: 10000, wherein the total concentration of PEG is from 0.01% to 100%. The pre-crosslinking may be further by crosslinking itself, or with PEG-amine, or with PEG-urethane, or with both PEG-amine and PEG-urethane to form a higher viscosity precursor solution.

Density of the precursor solution. The nerve tissue formed by myelin and adipose tissue is hydrophobic and therefore tends to float in solutions with densities close to water (-1 g/cm 3). By adjusting the density of the flowable medium, the nerve location can be adjusted to reduce the tendency of smaller diameter nerves to float in the precursor solution without sacrificing adhesive strength with increasing precursor viscosity. In some embodiments, the density of the precursor solution or mediator solution is reduced such that the nerves are relatively denser than the solution, the density of the precursor solution or mediator solution being reduced to <1g/cm3, preferably <0.9g/cm3. In other embodiments, the density of the precursor solution is adjusted to be approximately equal to the density of the nerve. Less polar and less dense solvents including ethanol (10-70%), toluene (10%), ethyl acetate or chlorobenzene can be added to reduce the density of the precursor solution. In another embodiment, vitamin E TPGS (1-2kDa, 10-20%) may be added to reduce the tendency of the nerve to float upward. Some of these solvents also lower the surface tension of the precursor solution, leading to nerve sinking.

Open surgical neuroma surgery. After overtly exposing the surgical site and isolating the target nerve, a new transection of the nerve is required to clear the nerve end. In some embodiments, the clinician may choose to transect the nerve at an angle of 90 degrees or 45 degrees. In some embodiments, clinicians may choose to use other methods such as electrocautery of nerve endings, ligation of nerve stumps, application of low molecular weight end-capped PEGs (e.g., 1-5kda,50w/v% hypotonic solution), or other methods they develop for capping or ablating nerve endings.

In another embodiment, the clinician may choose to use the PEG fusion protocol described in US 10,398,438 or 10,136,894 on the nerve before applying the in situ formed hydrogel. These PEG fusion methods can be used to fuse nerves, such as for blocking nerve ends after transection to prevent neuroma. They may also be used to seal nerves together, e.g., the proximal and distal stumps are in close proximity, to improve the outcome of nerve regeneration. By applying such a sequence of solutions (Ca 2+, free, optional antioxidant, fusin, ca2+ solution) in advance, the nerve ends can be optimally blocked to improve neuron survival, or in the case of nerve regeneration, nerve growth. Kits comprising solutions of PEG fusion components described in the above patents and incorporated herein by reference in their entirety may be combined with in situ forming hydrogel kits.

And (4) shaft slurry. Since the axial slurry is a viscous substance that seeps out from the severed nerve end after severing, it may reduce the close fit between the nerve end and the hydrogel, and thus may need to be removed from the nerve end. This can be achieved by contact between the nerve and an absorbent material such as a swab. An absorbable swab may be provided to absorb any axoplasm following nerve transection. The tip of the swab is preferably less than 5mm, more preferably less than 2mm, so that it can fit properly within the structure and retain nerves while delivering the hydrogel. The swab may be provided as part of a kit. Alternatively, this can be achieved by contacting the nerve endings with the surrounding tissue, which results in a rapid formation of an adhesion between the nerve and the tissue, which must then be severed again. Alternatively, for neuroma prevention applications, the nerve tip may be washed with a Ca2+ -free solution to wash away excess axoplasm and growth factors prior to delivery of the in situ-formed hydrogel to the periphery of the nerve. Alternatively or in addition, the low molecular weight biofilm fusion agent can be delivered to the nerve in an in situ formed hydrogel to seal the membrane in line with delivery of the PEG hydrogel. As mentioned above, PEG concentrations between 40% and 70%, preferably 50% and molecular weights (< 5kDa,10-50 mM) have been widely demonstrated to block damaged plasma membranes. Several protocols for PEG fusion (for neuroma prevention applications by sealing severed nerves) or PEG occlusion (for nerve repair procedures) are widely disclosed in preclinical literature (PMID: 30586569, 22302626, 30737092, 30909624). While korton lei et al 2017 (PMID: 29053522) have described delivery of PEG fusion solutions and related solutions within a wrap structure, this can be better achieved by delivering the PEG fusion solutions and related solutions into a wrap or cap structure.

Covering the proximal nerve stump (apical). The hydrogel itself preferably extends at least 0.5mm, preferably 1mm to 20mm, preferably 2mm to 10mm beyond the proximal nerve stump.

The length is covered. Preferably, at least 10mm of the nerve is embedded/encapsulated in the hydrogel, although in some cases 5mm may be sufficient. Embedding longer nerves in hydrogels accomplishes several things: a) Increasing the surface area of fit between the nerve and the hydrogel, and b) reducing the likelihood of budding from near the transected Ranvier node due to the embedding of these proximal nodes in the hydrogel. Again, the longer the length of the encapsulated nerve, the higher the likelihood that even the damaged area, treated by forceps, previous trauma, etc., will still be embedded in the hydrogel, thereby preventing sprouting of nerve fibers.

Once approximately 10mm of the nerve portion is isolated, whether the nerve is attached to the side of a swab, forceps or rod, or gently grasped by a pair of forceps, the nerve is slightly elevated to allow placement of structures underneath the nerve. In one embodiment, the nerve is then gently lowered into the tunnel or entry zone to center the nerve on the structure. See, for example, fig. 1A and 1B. Once the desired position is reached, the structure remains there.

While one hand holds the nerve endings in the center of the structure, the clinician then delivers the in situ forming hydrogel with the other hand to fill the structure and hold the nerve in the center of the structure. The top of the silicone structure serves as a guide for when to stop filling the structure. When the hydrogel fills beyond the nerve head, the swab/forceps are removed, leaving the nerve within the hydrogel rather than within the tool. In this way, the nerve has no direct path for regeneration through the surrounding tissue, and the nerve is completely surrounded by hydrogel. In another embodiment, the posts are added in a cap structure to adhere the nerves to the cap structure prior to application of the hydrogel. The post is flexible and can be removed after the hydrogel is formed. In one embodiment, the post is comprised of a bioresorbable polymer that remains in place after delivery of the hydrogel and is detached from the cap structure after the hydrogel is formed inside the cap structure. Thus, the column remains embedded in the hydrogel cap, which remains in place. In one embodiment, the column is composed of the same material as the hydrogel cap. This allows similar or identical swelling behavior as the hydrogel cap formed around the post and nerve. In another embodiment, the column is composed of low molecular weight PEG, allowing the column to be cleared faster than the hydrogel cap. The column may be formed from a PEG solution that is freeze-dried inside the cap structure. Preferably, the post is retracted 2-3mm back from the tip of the transected nerve, so that the regenerated nerve now grows back and exits through the void created by the post.

Gel thickness. Preventing nerve regeneration requires providing a conformal barrier proximal to the transected nerve. The hydrogel also preferably radially surrounds the nerve at a thickness of 100 μm to 5 mm. In one embodiment, the hydrogel precursor solution is dropped onto the nerve to form a thin protective coating with a circumferential thickness of about 100 μm to 2mm around the proximal nerve stump and remains there to prevent neurite outgrowth. The thin coating is sufficient to provide a barrier for nerve regeneration, so in some embodiments, the nerve is dip coated in a flowable medium, and the hydrogel subsequently forms a thin layer around the nerve. In this embodiment, the hydrogel gel time is adjusted to 10 to 20 seconds to allow removal of the coated nerve before converting to a non-flowable form and forming a gel. It is desirable that the thin coating provide adhesion and coverage (on the order of 50 to 500 microns) to the circumference and tip of the nerve stump to cover the end of the nerve.

For applications where protection of long nerves is desired, wrap structures from 1cm to 100cm long, preferably from 1cm to 50cm long, more preferably from 1cm to 10cm long, can be designed. For longer nerve wraps, the gelation time of the in situ formed hydrogel can be extended, allowing coverage of the entire length of the nerve. Alternatively, the mixer/needle at the end of the hydrogel applicator may be interchangeable so that a longer wrap may be filled through a continuous gel-forming region. Alternatively, a plurality of wrappers of about 1.5cm to 2cm in length may be placed in sequence. The wrappers may each be filled with one applicator.

In yet another embodiment, it may be desirable to form the hydrogel around the nerve in the form of an implant or pellet to provide a firm adhesive layer of hydrogel around the nerve having a thickness of about 0.5-5mm, more preferably 1-2mm, around the circumference of the nerve from 1-5mm from the tip of the nerve stump.

And (4) the aperture. To prevent nerve regeneration into the biomaterial, the pore size of the hydrogel needs to be small enough to prevent infiltration of nerves and other supporting cells into the biomaterial. The nerve axons have a diameter of between about 0.5-30 μm. Preferably, the growth-inhibiting hydrogel is microporous or mesoporous, having a pore diameter of less than 1 μm, preferably less than 0.5 microns, more preferably less than 500nm.

And (4) charging. Neutral or negatively charged biomaterials are preferred as growth-inhibiting gels because neurites preferentially grow as positively charged biomaterials. Similarly, hydrophilic materials or amphiphiles are preferred over hydrophobic materials.

A non-degradable hydrogel. The same equilibrium swelling characteristics apply if a non-degradable hydrogel system is used, but since the hydrogel is non-degradable or biostable, degraded swelling is not relevant. For example, non-degradable, in situ-forming, thermo-responsive copolymers, pluronic (PEO-PPO-PEO), polyvinyl alcohol, or PEO may be used to form the hydrogel.

And (4) clearing. In a preferred embodiment, the hydrogel is clear, transparent, to confirm the location of the nerve after hydrogel formation. Of particular relevance to the case of nerve repair, transparency allows confirmation that nerve repair is optimal and that no split is constructed from the repair site or that the optimal distance between the two nerve stumps has been maintained. Visualization agents may be incorporated into the hydrogel to aid in contrast against background tissue. The colorant or colorant mixture may be contained in a polymer powder solution. Where multiple hydrogels are used (as described below), it may be desirable to use one or more different visualization agents to provide visual confirmation, e.g., that the growth-permissive hydrogel is properly delivered between the nerves, and that the growth-inhibiting hydrogel is delivered around the growth-permissive hydrogel.

The cap of the present invention may be formed from a hydrogel having sufficient optical clarity so that the nerves can be visually observed through the cap material after the cap is formed. Referring to fig. 1B and 1E, once released from the mold, the cap will have a first (lower) convex sidewall surface conforming to the concave shape of the mold, and a second (upper) surface aligned with the window 20, and having a relatively flat meniscus configuration. At least a portion of the first surface has a curvature that can substantially conform to a surface of a cylinder. This, together with the optically transparent nature of the hydrogel, acts as a lens, magnifying the appearance of the nerve when viewed through the first surface. Thus, the cap may function in the manner of a convex-concave lens (sometimes referred to as a negative meniscus), where the concave interface at the neural surface has a smaller radius of curvature than the radius of the outer first surface to produce magnification. The radius of the hydrogel lens is typically between about 1mm to about 12mm from the nerve center, and the thickness of the hydrogel lens passing through the first surface may be between about 0.5mm to about 5mm, preferably about 1mm to about 1.5mm thick. The hydrogel lenticulars have a refractive index similar to that of an object embedded in glass, about 1.45 to 2.00, and effectively bend light rays inward, making the nerves inside appear larger than they really are. Hydrogels are transparent, more than 90% water, and are therefore very suitable

And (4) elasticity. In some embodiments, the elasticity of the hydrogel can be modulated by incorporating hydrophobic domains into the hydrogel. The hydrophobic domains can be incorporated by cross-linking or mixing molecules, particles, fibers and micelles. The incorporated molecules may be amphiphilic molecules including pluronic, polysorbates, and tocopheryl polyethylene glycol succinates. The particles, fibers and micelles may be made of the above-mentioned amphiphilic molecules. In addition, many low molecular weight hydrophobic drugs (described below) incorporated into hydrogels can improve the elasticity of the hydrogel.

A kit. The in situ-forming hydrogel is delivered by a dual applicator system comprising a dual channel applicator, dual adapters, one or more mixers, and one or more blunt needles. Also included in the kit is a powder vial with associated vial adapter, diluent solution and accelerator solution for use in a dual applicator system. The kit may include one or more structures into which the hydrogel is delivered. The kit may also include one or more mixer-blunt syringes. The mixer injector may be a conventional single chamber mixer with static mixing elements, or the mixer may be one in which there is recirculation and turbulence of the contents to improve mixing of the precursor solution.

Chemical and physical. Preferably, the hydrogel network is predominantly hydrophilic, has a high water content, and is formed by physical or chemical crosslinking or synthetic or natural polymers, copolymers, block copolymers, or oligomers. Examples of non-growth-permissive synthetic hydrogels include agarose, PEG or alginate, PVA hydrogels having a solids content of 2% w/v or more, preferably 6% w/v, more preferably 8% w/v or more. PEGs. The multi-arm PEG is as described above, but may be selected from PEG-amine, pegaroxyl, PEG-SCM, PEG-SGA, PEG-nitrophenyl carbonate (carbonate linker), PEG-maleimide, PEG-acrylate, PEG-thiol, PEG-vinylsulfone, PEG-Succinimidyl Succinate (SS), PEG-Succinimidyl Glutarate (SG), PEG-isocyanurate (Isocianate), PEG-norbornene or PEG-azide depending on the desired properties. An alginate. Viscous injectable alginate sols (1-5%) can be delivered to the periphery of nerves. Also, agarose gels at concentrations of 1% weight/volume or higher can prevent nerve extension.

Hypotonic solutions. In one embodiment, a hypotonic solution (Ca 2+ -free, slightly hypotonic, salt solution containing 1-2mM EGTA) is delivered to the dissected nerve to help seal the compressed or transected axon ends prior to repair with the in situ forming biomaterial.

PEG fusion binds to the in situ nerve cap or wrap. As described in many publications summarizing the neural PEG fusion method (3.35-5kDa, 30-50% w/v solution), the PEG solution can be delivered to the nerve alone or in combination with methylene blue to fuse the nerve first. After sealing the membrane, a growth-permissive hydrogel is delivered between and around the compressed or severed nerves.

A cross-linked particle. In some embodiments, the hydrogel may be made of cross-linkable particles, fibers, or micelles. These particles, fibers or micelles are functionalized with reactive groups including, but not limited to, active esters, amines, carboxyls, aldehydes, isocyanates, isothiocyanates, thiols, azides and alkynes, which can crosslink with small molecules, polymers, particles, fibers or micelles having reactive groups to form bonds, including amides, carbamates, carbonates, ureas, thioureas, thioesters, disulfides, hydrazones, oximes, imines, amidines and triazoles. In some embodiments, the micelles, fibers and particles may be formed from amphiphilic macromolecules having hydrophilic and hydrophobic segments. The hydrophilic segment can be a natural or synthetic polymer including polyethylene glycol, polyacids, polyvinyl alcohol, polyamino acids, polyvinylpyrrolidone, polyglycerol, polyoxazolines, and polysaccharides. The hydrophobic segments can be fatty acids, lipids, PLA, PGA, PLGA, PCL and polymer ester copolymers in varying proportions. In another embodiment, the functionalized microparticles form physical crosslinks with each other upon a change in pH, and then, when placed in situ, the functionalized microparticles crosslink to form an interconnected network of microparticles.

And (3) a sealant. Some in situ-forming gels and sealants developed to prevent adhesions may also be suitable for this application to prevent neuroma and abnormal nerve growth into scar tissue, such as low molecular weight anhydrides of acids like sebacic acid, including poly (glycerol-co-sebacate) (PGSA) -based sealants (9,724,447, us20190071537, pelletnc et al (2019) clinical and clinical evaluation of a novel synthetic bioorganic, on demand activated sealant in vascular recovery, incorporated herein and suitable for use around nerves for reference). Another sealant that may be suitable for delivery around peripheral nerves is the adheus Dural sealant, which comprises an in situ formed PEG-Polyethyleneimine (PEI) copolymer, as it exhibits low swelling and degrades within about 90 days (9,878,066, incorporated herein). Other sealants include BioGlue surgical adhesive (Cryolife) consisting of bovine serum albumin and glutaraldehyde, omnex (Eticon), arterX (Baxter), coseal (Baxter), and TissuGlu (Cothera Medical) consisting of lysine-based urethane.

And (4) responding to light. In some embodiments, photoresponsive, photopolymerized or photocrosslinked biomaterials are contemplated that can be delivered in a liquid (low to medium viscosity) state into the structures surrounding the nerve (cap or wrap structures) and then, when the correct positioning of the nerve in the structure is obtained, the photopolymerization is initiated with ultraviolet, infrared, visible light. In one embodiment, the light source may be connected to the opening of the cap or wrap structure directly or through a fiber optic cable. By designing the light to illuminate the entire cap or wrap structure, uniformity of crosslink density can be achieved. The cap structure diffracts the light to ensure that the entire structure is sufficiently illuminated to achieve uniform cross-linking throughout the gel. In a preferred embodiment, the light source housing is directly coupled to the structure at the distal end of the cap facing the nerve stump face to ensure direct penetration of light. Optionally, the structure may be designed with embedded light emitting elements that allow light to pass circumferentially around the nerve. Hydrogels include PNIPAAM hydrogels modified with chromophores such as the trisodium salt of chlorophyll. Other biomaterials formed in situ include elastomers that can be crosslinked in situ, including US 10,035,871 (and PMID 31089086), the entire contents of which are incorporated by chemical or photo-crosslinking methods. For these biomaterials, the transparency and color of the cap and wrap structures may be adjusted to reflect ultraviolet light back to the structures, such as opaque white structures.

Other configurations. In addition to crosslinked networks, hydrogels can also be composed of dendrimers, self-assembled hydrogels, or low molecular weight synthetic polymer liquids.

In one embodiment, a low Molecular weight hydrogel (2 kDa, liquid) may be formed In Situ without water as a solvent, as described In Kelmansky et al (2017) In simple Dual Cross-Linking of New Biogel with Controlled Mechanical and Delivery Properties, molecular pharmaceuticals, 14 (10) 3609-3616, which is incorporated herein. In other embodiments, the hydrogel may be photocrosslinked to form a hydrogel, as is widely described in the literature. The crosslinking agent comprises eosin. In other embodiments, electrically conductive hydrogels are used, including poly (3, 4-ethylenedioxythiophene) (PEDOT), poly (pyrrole), polyaniline, polyacetylene, polythiophene, ester derivatives, 3, 4-propylenedioxythiophene (ProDOT), natural or synthetic melanin, derivatives, and combinations thereof.

Addition of sulfated proteoglycans. In some embodiments, it may be desirable to deliver inhibitory environmental cues to the nerve in addition to the mechanical barrier provided by the hydrogel. This can be achieved by adding inhibitory molecules and/or extracellular matrix to the hydrogel by physical mixing or chemical crosslinking. Sulfated proteoglycans, such as negatively charged side chains, such as glycosaminoglycans, are of interest. Of particular interest is Dermatan Sulfate (DS).

And (4) mixing. In some embodiments, it may be desirable to produce a mixture of two PEGs to increase the degradability of the system. In one embodiment, PEG-SC is combined with PEG-SG prior to cross-linking with the trilysine amine to produce a hydrogel with sufficient mechanical support to prevent nerve growth, but which subsequently degrades more rapidly than PEG-SC. The duration of the gel in vivo is fine-tuned by the ratio of PEG-SG and PEG-SC. As the PEG-SC content increased, the duration of the gel in vivo increased. In another embodiment, the PEG-carbamate is mixed with PEG-carbonate. Other hydrogels include PEG hydrogels composed of carbamate derivatives (7,060,259).

A growth-permissive gel. In some embodiments, the growth permissive solution comprises a low viscosity collagen solution of 1.5mg/ml or less, more preferably 0.6mg/ml or 0.8 mg/ml. In another embodiment. A laminin solution at a concentration of 0.4mg/ml is preferred. In another embodiment, HPMC or CMC formulations at 2% concentration provide low viscosity solutions through which nerves can seek without significant mechanical barriers.

And (4) incorporating the reagent. In some embodiments, the reagents may be dissolved or suspended in a diluent or accelerator solution, and a surfactant or ethanol may be added to stabilize the suspension. The drug may also be entrapped in microparticles, nanoparticles or micelles, which are then suspended in a diluent or enhancer. In some embodiments, the hydrogel may be made of cross-linkable particles and micelles. These particles or micelles have reactive groups such as active esters, amines, carboxyl groups, thiols, and those described in U.S. Pat. No. 7,347,850 B2, and can be crosslinked with small molecules, polymers, particles, or micelles having reactive groups that react with and form bonds with the aforementioned particles or micelles, including amides, carbamates, carbonates, ureas, thioureas, thioesters, disulfides, hydrazones, oximes, imines, amidines, and triazoles. In other embodiments, the gel may be formed by swelling of the particles. The voluminous swelling can increase particle contact and lock them in place where they form a gel.

The solids content. The solids content can be adjusted to fine tune the swelling and stretching properties of the hydrogel. For example, the solids content may be adjusted above the critical gelation concentration, such as a loading of 6-15%, more preferably a loading of 7-9%, more preferably a solids content of 8-8.5%.

And (4) crosslinking. Hydrogels can be formed in situ by electrophilic-nucleophilic, free radical, or photopolymerization.

Persistence in vivo. In some embodiments, longer in vivo persistence may be preferred, wherein the hydrogel remains in situ for 3 months to 3 years, more preferably 6 months to 18 months, more preferably 6 months to 12 months.

And (4) adhering. Adhesion strength is an important criterion for maintaining close contact of the hydrogel with the nerve. Adhesion can occur through a cross-linking reaction between the hydrogel and primary and secondary amines on the tissue surface, such as the extraneural membranes or amine groups found on the surface of nerves, glia and related cells. The adhesive strength should be greater than 10kPa, preferably greater than 50kPa, more preferably greater than 100kPa. The adhesion strength on the nerve can be estimated by embedding the sciatic nerve in a hydrogel. The ends of the nerves were embedded in super glue between sandpaper and placed in titanium clips in Bose Electronic Force 3200-ES. The nerves were pulsed at a rate of 0.08mm/s until failure. Care was taken to ensure that the nerves were used shortly after harvesting and that the hydrogel and nerves were equilibrated in PBS at 37 ℃ prior to testing.

Other hydrogels. The formation of polyanhydrides in situ is also advantageous for developing applications for nerves. In one embodiment, the polyanhydride-based polymers can be acrylated such that they can be formed in situ by free radical polymerization. Alternatively, they may be formed by photo-crosslinking. At lower concentrations, the polymer is water soluble, e.g., 10%. The prevention of nerve regeneration is due in part to its hydrophobic nature. US20180177913A1, US62/181,270 and US201562181270P are incorporated herein by reference.

An applicator. Dual channel Applicators for delivering in situ-forming hydrogels are commercially available (Nordson Medical fiber biomedical Applicators, medium Double System biomedical Delivery System, K-System). However, these mixers delivering 2.75ml to as much as 10ml of hydrogel include a single lumen with static mixing elements and are designed for adequate mixing and delivery of large volumes of hydrogel solution and are not ideal for delivering small volumes (< 1 ml) of hydrogel solution to one site. Thus, a mixer is needed to provide mixing of small volumes of two-component systems, since inevitably one of the two solutions will usually lead slightly to the other solution, which first results in a small volume fraction or insufficiently mixed gel at the needle. In one embodiment, the custom mixer is designed to be fitted to a Nordson Medical or K-System applicator via a luer fitting or snap fit, as necessary for dual lumen applicator systems, to recirculate some of the initial solution into the mixer to ensure better mixing of the hydrogel (including the initial components). Figure 18 shows the design of the central portion of the mixer (transparent) containing an inlet with at least one static mixer, a larger container through which the contents are delivered and recirculated, and a second port that captures the mixed recirculated fluid and delivers it to the mixer outlet. The second port may or may not contain an additional static mixer.

Example 1.

In some embodiments, the 4-arm PEG 10K-SC is crosslinked with an 8-arm PEG 20K amine. PEG-SC and PEG-amine were mixed at 1:1 in an acidic diluent. The suspension was mixed with the accelerator buffer and delivered through a static mixer to form a hydrogel. This formulation gelled in 4 seconds and provided a compressive strength of 50-70 kPa. Furthermore, the swelling is between 10 and 30 wt%.

Example 2.

In another embodiment, the 8-arm 15K PEG-SC is crosslinked with a trilysine. PEG-SC was suspended in a buffered trilysine solution and then mixed with the accelerator buffer by a static mixer. The formulation gelled within 2 seconds with a gel compressive strength of up to 200kPa.

Example 3.

In another example, an 8-arm 20K PEG-thioisocyanate is crosslinked with a trilysine in a ratio of 1: 1. The formulation gelled within 3 seconds, had a compressive strength of 120kPa and a swelling ratio of 5%.

A cap structure. The method may include the step of positioning a structure at the treatment site prior to the step of positioning the severed end. The structure is provided as part of a kit containing the delivery system and is composed of an inert, biocompatible, flexible, and non-tacky material to provide the desired shape to the material formed in situ. The structure is designed to be filled with a flowable medium such that it flows around the proximal nerve stump where it transforms into a non-flowable composition, conforming to the nerve stump and preventing the formation of neuroma. In a preferred embodiment, the structure produces a low profile nerve cap with a smooth transition between the nerve and the cap and an approximately cylindrical shape around the nerve.

And (4) shape. The structure is desirable not only because it reduces off-target diffusion of the in situ forming material, but also because it provides a low profile circumferentially smooth shape that cannot be achieved with the hydrogel alone. The contours and transitions of the structural design reduce friction and interference with surrounding tissue, allowing the hydrogel to slide and rotate relative to the surrounding tissue. The cap is designed to cover at least 5mm, preferably 10mm or longer, of the nerve.

According to another aspect of the present invention, there is provided a structure for generating an in situ formed nerve cap to inhibit neuroma formation or nerve wrap to prevent nerve compression or promote nerve regeneration. The structure includes a recessed wall defining a cavity, the wall having a top opening for accessing the cavity. The top opening lies in a first plane and has an area smaller than an area of a second plane that conforms to an inner dimension of the cavity and is spaced apart from the cavity and parallel to the first plane. A concave nerve guide is carried by the wall and provides lateral access to the cavity for receiving a nerve end. The wall is flexible so that it can be removed from the crosslinked nerve cap formed within the cavity, and it may comprise silicone, preferably having a hardness of 20 to 40, preferably 20 to 30, most preferably 20. The wall design of the structure has slight undercuts (undercuts) so that when the structure is filled to the top edge of the structure, the material forms a convex surface due in part to the surface tension of the medium, completing the cylindrical shape of the hydrogel. This hardness is much softer than FDA approved silicone neural tubes, which are too hard with a hardness of 50 or 60, resulting in shrinkage and chronic neuropathic pain.

A silicone resin. In one embodiment, the structure is composed of non-degradable materials that are not adherent. In a preferred embodiment, the material is medical grade silicone, which is sufficiently flexible to peel or pop off from the in situ formed material (e.g., hardness 20 or 30). After the media is converted to a substantially non-flowable state, the silicone form structure is removed and discarded. In one embodiment, the silicone structure is colored to provide contrast with surrounding tissue so that the non-degradable polymer does not accidentally remain in place. Darker colors are more beneficial to enhance the light that enters the cap and illuminates the nerves, such as dark blue, dark purple, or dark green.

While selected natural rubbers may be selected for neural structures, they are not ideal due to their lack of biocompatibility and poor properties in applications involving direct tissue contact. Thus, medical grade silicones such as those sold by NuSil/Avantor, elkem, dow and Momentive are preferred because most have been tested for USP grade biocompatibility. Most medical grade Liquid Silicone (LSR) is designed for high tensile strength applications at psi greater than 1200psi, rather than for handling relatively more fragile biomaterials. While these properties are desirable for some medical devices (e.g., implantable ICDs), their high tensile strength makes them a poor choice for applications that require flexibility to facilitate release of the biomaterial from the model. Thus, a series of LSRs were evaluated to determine which material was best suited for releasing the hydrogel from the structure. Soon, the higher tensile strength silicone was eliminated and a focus was placed on a smaller two-part silicone system for injection molding, cured by heat. For these applications where direct contact with tissue is brief, a material with a defined biocompatibility for implantation in humans for less than 29 days may be sufficient, although a material with a defined biocompatibility for longer implantation times may be required.

However, for this application, silicones with lower stiffness and lower tensile strength are preferred to facilitate removal of the hydrogel from the structure. Therefore, a silicone having a hardness of less than 45, preferably less than 30, and more preferably a hardness of about 20 is desirable. Although most commercially available Liquid Silicones (LSRs) have a tensile strength greater than 1000psi, for such applications LSRs with lower tensile strengths are preferred. For example, a tensile strength of less than 900psi, preferably less than 800psi, is desirable. Similarly, elongation is another factor that determines the ease with which the biodegradable biomaterial can be removed from the mold. Materials having an elongation of more than 400%, preferably between 400% and 2000%, more preferably between 400% and 1200%, more preferably between 400% and 800% are desirable. Examples of such materials include MED-4920 (NuSil, hardness A type 20, elongation 700%, tensile strength 750 psi), MED-4930 (hardness A type 30, elongation 450%, tensile strength 800 psi), LIM-6010 (hardness 15, elongation 440%, tensile strength 3 MPa), siloprene LSR 4020 (hardness 22, elongation 1000%, tensile strength 7 MPa) or MED50-5338 (hardness 30, elongation 350%, tensile strength 650 psi) and SIL-5940 (hardness 40, elongation 680%, tensile strength 1, 350psi), silicone LSR 4340 (hardness 40, elongation 605%, tensile strength 1250 psi), silibione 4325 (hardness 26, elongation 680%, tensile strength 1198 psi), MED-5840 (hardness 40, med elongation 680%, tensile strength 1350 psi) and MED-4840 (hardness 43, elongation 590%, tensile strength 0 psi), SILTIC Biochemical LSR 1430 (hardness 7-1430 LSR 1430) and SIL-700, preferably tensile strength of the raw materials are set up by hardness gradient VI, hardness 65, elongation 75%, tensile strength of Grail-75, tensile strength, preferably by tensile strength of Grail-75, grail hardness 33, 700-75 psi, tensile strength, and gradient III-75%.

Alternatively, but less preferably, a High Consistency Rubber (HCR) such as peroxide or platinum cured MED-4035 (hardness 35, elongation 1055%, tensile strength 1565 psi), MED-4025 (hardness 30, elongation 890%, tensile strength 1, 285psi), MED-4020 (hardness 25, elongation 1245%, tensile strength 1400), or preferably MED-4014 (hardness 15, elongation 1330%, tensile strength 700 psi), more preferably MED-4920 (hardness 20, elongation 700%, tensile strength 750 psi). Class VI high consistency rubbers may also be carefully selected, although these tend to have significantly higher tensile strengths at the higher end of the acceptable range above 1,000psi, for example 1300-1600 psi. Finally, room temperature vulcanizing silicones (e.g., RTV-2), such as P-44 (hardness 42, elongation 250%, tensile strength 600psi, silicones Inc. high Point, NC) are also suitable. In some embodiments, the LSR is white, rather than translucent (e.g., MED-4942), or colored with purple or blue healthcare color masterbatch of Nusil to contrast with tissue. In some embodiments, where longer or larger structures are desired, silicone having a higher hardness (e.g., shore a hardness 40) within this range may be desired to help maintain the shape of the wrap structure, e.g., 3cm in length. Examples of materials include MED-4940 (Nusil).

LSRs with stiffness above 50 and tensile strength above 1300psi are less desirable (e.g., silbaine LSR4370, silbaine LSR 4365). Similarly, some silicones are designed to be adhesive, for example for adhesive wound dressings such as sildione HC2 2031a &b. These silicones are less desirable because they are designed to adhere to tissue, rather than being designed to be smooth and easily peeled from tissue.

And (5) demolding. By reducing the adhesion of the nerve cap/wrap structure to the mold, easy demolding can be achieved. If the nerve cap/wrap precursor spreads well on the model, it will adhere to the surface after gelling. To achieve easy demolding, the wettability of the model by the nerve cap/wrap precursor may be reduced. The interfacial tension is largely determined by polarity, which is decisive for wettability. The worst wettability condition for adhesion is the condition where the liquid polarity is far from the surface polarity of the cap/wrap structure. In one embodiment, hydrophobic materials, including but not limited to biomedical grade silicone and mixtures thereof in varying proportions, can be used to make the model. The interfacial tension of a liquid with a particular solid can also be calculated from the contact angle of various solids with liquids.

In another embodiment, polar and non-polar interactions between the hydrogel nerve cap/wrap and the nerve cap/wrap structure will make the nerve cap/wrap difficult to demold. Polar interactions include, but are not limited to, dipole-dipole, dipole induced dipole, and hydrogen bonding. The model can be made using materials with less polar interactions with the nerve cap/wrap, especially hydrogen bonding.

In another embodiment, increasing the surface tension of the gel precursor will decrease the wettability of the gel precursor on the surface. Highly polar materials, including but not limited to salts, may be added to the gel formulation.

In another embodiment, a material that is chemically inert to the nerve cap/wrap may be used as the model material. Chemical reaction between the nerve cap/wrap and the model material increases adhesion.

In another embodiment, the surface smoothness of the mold affects the adhesion and release of the nerve cap/wrap. In general, the smoother the mold surface, the easier it is to demold. For example, the modeling material may be hydrophobic and rough on a microscopic scale. It can trap air on its surface, causing the unsolidified nerve cap/wrap to be supported by its own surface tension. Such surfaces are referred to as superhydrophobic. Superhydrophobic surfaces can be engineered by forming micro-scale roughness or patterns on hydrophobic materials.

In one embodiment, the hydrophobicity of the cap/wrap structure creates sufficient surface tension that when a hydrogel precursor solution is delivered to fill the cap/wrap structure, the hydrogel precursor solution forms a raised cap that is elevated above the structure itself, providing the wrap or cap structure with a cylindrical or elliptical cross-section.

Can be biologically degraded. The method may alternatively include the step of placing the biodegradable structure prior to the step of locating the severed end. The biodegradable structure may consist of a non-crosslinked lyophilized (or dried) synthetic biomaterial that remains in place for about 5 to 10 minutes during which the in situ-forming hydrogel is delivered, and then rapidly dissolves and clears from the site in less than, for example, one or two days. In one embodiment, the bioerodible structure consists of lyophilized, multi-arm, end-capped or non-crosslinked PEG, lyophilized, linear PEG (3.35 kDa), or crosslinked multi-arm PEG (e.g., 8-arm 15 kDa). The method may alternatively include the step of forming the biodegradable structure in situ prior to the step of locating the severed end. In alternative embodiments, the structure is composed of materials commonly used for nerve conduits and wraps, such as polyvinyl alcohol, chitosan, polylactic acid, polyglycolic acid, polycaprolactone.

In yet another embodiment, the ex vivo implantable structure is composed of the same material as the in situ forming material delivered into the structure. In this way, the properties of the equilibrated structure are similar and match well with the equilibrated in situ formed hydrogel. In these embodiments, the biodegradable structure remains in place after delivery of the hydrogel and is not removed, but instead clears from the implantation site at approximately the same rate as the material formed in situ. In yet another embodiment, the structure consists of lyophilized, non-crosslinked PEG into which the in situ-formed hydrogel media is delivered. The non-crosslinked PEG readily dissolves and clears from the site, making the structure a fast bioerodible structure. In another embodiment, the structure consists of a cross-linked PEG matrix that will rapidly clear from the site due to rapidly breaking hydrolytic bonds, such as may be obtained by ester bonds in PEG succinimidyl succinate (PEG-SS).

The structure may be synthesized by injection molding, cross-linking or polymerization in a cavity, solvent casting, or 3D printing. A range of synthetic and natural materials may be selected for the implantable structure, including collagen, PEG-PEI, alginate, chitosan or agarose.

The structure may be a fast dissolving structure that dissolves when wetted and clears within one hour after surgery, while leaving the biomaterial formed in situ in place. Alternatively, the structure may be a more slowly degrading structure that swells to a similar or greater extent than the in situ formed material delivered to its interior. The swelling prevents situations where the hydrogel swells during equilibrium swelling and compresses the nerve (if the structure to which it is delivered is contractible or has minimal conformability).

In some embodiments, the nerve is held in a desired position or orientation with forceps in one hand and the medium is delivered into the structure with the other hand. As the medium fills the structure, the nerves are released and the medium subsequently becomes non-flowable. Alternatively, an assisting physician or nurse may assist in the procedure. In another embodiment, the growth-inhibiting hydrogel is formed in two steps around the nerve. In a first step, a hydrogel is delivered to the nerve endings to encapsulate the nerve endings. In a second step, the hydrogel is applied to fill the entire structure, including the nerve endings. In another embodiment, the growth-inhibiting hydrogel is formed around the nerve in a first layer, and then a second layer of hydrogel is applied in a two-step process.

Conformability. Unlike the wrap (which still has a gap between the wrap and the nerve), the hydrogel conforms directly to the nerve itself, providing a barrier to the entry of inflammatory and pro-scar forming cells into the site, while allowing nutrients to pass through. Because the hydrogel is attached to the nerve, there is no need to suture the nerve to the hydrogel. The proximity of the hydrogel to the nerve also helps to prevent scarring and adhesions from forming around the nerve during the initial healing phase.

And (4) centering. Nerves, particularly smaller nerves, are likely to flow upward in low viscosity solutions due to their low density, high fat content and flexibility. The following embodiments are designed to ensure that the nerve does not float after delivery of the medium to the top of the surface.

Viscosity. As described above, the viscosity of the flowable solution can be increased to minimize neural uplift in the solution.

And (4) flowing. In another approach, the needle delivering the flowable medium is directed in such a way that the flow of the medium allows the solution to spread circumferentially around the nerve before the gel forms. The cap structure may also be designed to improve flow dynamics of the medium and improve neural alignment. In one embodiment of the invention, the steps of severing the target nerve and positioning the structure at the treatment site are performed by a single instrument. In another embodiment of the invention, the nerve cap structure is designed such that the delivery system and the structure are integral. In a preferred embodiment, the delivery system is connected to the structure by a catheter. The catheter inlet in the cap is located at the same inlet where the nerve enters the structure. The conduit allows material to flow circumferentially along the shaft and around the nerve such that the medium acts to self-center the nerve within the structure. Similarly, but with shorter gelation times, flow and neural movement are limited.

A stabilizer. In another embodiment, the stabilizing rod or sheet is arranged directly under or against the nerve such that it provides sufficient adhesive force such that the hydrogel can be delivered into the structure around the central nerve.

Strippable catheters in yet another embodiment, the nerve is positioned in the center of the biomaterial in situ by placing it in series within two strippable catheters. Briefly, a nerve is placed within a first peelable catheter, and an in situ forming material is delivered to the apex and around the distal end of the nerve. The peelable conduit may be an open-ended or closed-ended conduit. After the hydrogel is formed, the sheath is pulled along the line of weakness in the material and discarded. The resulting neuro-hydrogel is then placed in a second, larger, peelable catheter. By slightly rotating the nerve, the hydrogel surface can be placed at the bottom of the second sheath so that the nerve is approximately in the middle of the second sheath. The second application of the in situ forming hydrogel results in the formation of a ring-like hydrogel that accumulates around the nerve, protecting the nerve and centering it within the nerve cap. In one embodiment, the sheath is comprised of an extruded detachable PTFE tube with a vertical pull tab to assist in tearing the piece (piece) in the surgical environment, similar to a vascular introducer.

In another embodiment, the nerve is placed such that the proximal stump rests at a 90 degree angle down in a cup-shaped structure, and the hydrogel is delivered into the cup-shaped structure to shape around the nerve. The cup-shaped structure is then removed and discarded, and the proximal stump is adjusted back to its resting position in the tissue.

In yet another embodiment, the nerves can be delivered in an amphiphilic or hydrophobic solution to prevent the nerves from floating to the surface of the medium. In yet another embodiment, the in situ-forming material may be more viscous to prevent migration of nerves within the structure.

And (4) inclining. Alternatively, the structure may have a slope in form to bias the nerve filling from the distal end to the proximal end. In this manner, the nerve may be positioned in such a way that the hydrogel is first formed circumferentially around the proximal nerve ending and then fills the remainder of the structure by a second application or continuation of the first application.

The inlet is centered. In one embodiment, the structure is designed in such a way that the nerve enters at a lower level relative to the top of the structure to allow circumferential delivery of the material around the nerve. In another embodiment, the entry region of the structure is sloped such that nerves enter the structure at a downward angle, biasing the proximal nerve ending position downward.

Ribs (rib). A pull tab or rib is provided on the outer surface of the non-degradable temporary structure (e.g., silicone structure) to assist in removing the structure after gel formation. These tabs are placed in such positions to provide additional stability to the cap structure on irregular surfaces, or to provide a surface for grasping with forceps or other surgical instruments. In yet another embodiment, the structure is designed to be self-centering (self centering). In other words, the structure may naturally sit such that the top surface of the structure is horizontal, thereby providing for delivery of the in situ formed hydrogel.

And (4) a hole. In some embodiments, an aperture or an introducer sheath is provided to guide the needle to deliver media into the structure in a particular direction. The direction of the media flow can be designed to better position the nerves in the channel. In one embodiment, an aperture is provided near or at the top of the site where the nerve enters the structure to direct the solution from the proximal end to the distal end in the catheter and promote laminar flow within the structure.

And a cover. In some embodiments, the structure comprises a partially or fully hinged lid to allow centering of the nerve depending on the direction of flow within the structure.

The volume delivered. As with the cap structure, the volume of media delivered can range from 0.1cc to 10cc, typically 0.2cc to 5cc, more typically 0.3cc to 1cc.

Needle size. The kit contains 21 gauge (gauge) or 23 gauge needles for delivering smaller volumes to smaller wrap (or cap) structures, and 18 gauge needles for filling larger structures. These provide additional control over the delivery rate of the in situ forming material, allowing deposition of hydrogel beads to rapidly fill larger conduits.

Gel time. Similarly, the gel time may be adjusted depending on the fill volume of the wrap or cap structure, providing a longer gel time of 10 to 20 seconds for larger wraps, and a shorter gel time of no more than about 10 seconds or no more than about 5 seconds, but typically at least about 2 or 3 seconds for smaller wraps or caps.

Structural dimensions and hydrogel thickness. The range of structural sizes is designed to have an inlet zone to accommodate nerve diameters ± 1 to 3mm, or more preferably ± 1mm. The diameter of the structure determines the thickness of the gel formed around the nerve. The thickness of the hydrogel formed around the nerve may be 0.05mm to 10mm, more preferably 1mm to 5mm, more preferably 1mm to 3mm, depending on the size of the nerve.

And (5) designing a kit. Unlike the case where each kit contains one structure for only one size of nerve, such as an implantable nerve conduit and wrap, the kit will contain one to ten, more typically one to one cap structure (or wrap structure, or a combination thereof), allowing the physician to select the appropriate size for the procedure, and the ability to switch structures without the need for an additional kit. The kit may be labeled according to the structure selected, for example, a structure suitable for a range of nerve sizes, a structure suitable for a certain type of surgery (neuroprotection for inguinal repair), or a structure suitable for a specific location of the surgery (nerve cap for hand surgery, neuroprotection for upper limbs, nerve structure for brachial plexus).

A sheet. In some embodiments, the in situ forming Material may be delivered to nerves that are placed on temporary, non-adherent biocompatible sheets, such as esparch bandages or other biocompatible sheets or backgrounds (Mercian Surgical visual Background materials) that are commonly used to isolate nerves from surrounding tissue. The gelation time can be shortened to limit the diffusion of the hydrogel around the nerve, for example, to 10 seconds or less, preferably 5 seconds or less. Any excess hydrogel may then be removed from the surgical site and discarded.

A liquid cap structure. In another embodiment, the structure is not a physical structure, but is produced by injection of a soluble hydrophobic solution, preferably a viscous solution, such as glycerol. For example, while ensuring that the nerve is undisturbed, a viscous oil can be delivered into the surrounding tissue to cover it and prevent the hydrogel from adhering to the surrounding tissue. If the solution is sufficiently viscous, a rapidly bioerodible structure can be formed for delivery of the in situ formed hydrogel. In a preferred embodiment, solution a is delivered first to block amine and tissue binding sites and create a space or region into which solution B can be delivered. In the next step, solution B is delivered into the space created by solution a, or it is delivered to the center of solution B, and solution B is removed from the site.

And has no structure. In some embodiments, the space or access does not allow for the use of structures. In some cases, such as in brachial plexus injury, the surgical window is so small or there is little concern about damaging adjacent tissue that it is not possible to place structures at the site of delivery of the hydrogel. In these cases, the hydrogel may be delivered directly into a surgical pocket or region within or around the nerve. If the area around the nerve is used as a natural structure, the cap has an irregular shape defined by the tissue boundaries of the nerve base and sides. In one embodiment, since the hydrogel adheres to both the nerve and tissue, the material formed in situ should be carefully peeled away from the muscle and fascia so that it forms a free floating mass in contact with the nerve. This will allow the nerve to continue to move within the area without being bound by surrounding tissue.

In other embodiments, it is desirable that the hydrogel take the shape of the surrounding tissue surrounding the nerve. For example, in embodiments where the nerve is to be ablated and the hydrogel needs to fill the potential space and surrounding area where the nerve is/was located to prevent regeneration. Alternatively, when the hydrogel is delivered around the splanchnic nerve, there is usually a loose and fine network of tiny nerve fibers, the space around these nerve fibers needs to be filled. In another embodiment, the hydrogel fills the surrounding of irregularly shaped nerve or nerve fiber bundles/clusters and/or cell bodies. In this way, the hydrogel may most effectively deliver the therapeutic agent to the area.

A hydrogel placed in situ in a controlled manner around a nerve. However, in another embodiment, it is not desirable for the nerve to be bound to the surrounding tissue through the hydrogel, particularly in a dynamic environment where the nerve slides during movement between muscles, joints, bones, or tendons (e.g., between surrounding muscles). Instead, it is desirable to develop solutions where the nerve can slide freely within the channel. In these embodiments, the hydrogel may be physically separated from the surrounding tissue during in situ crosslinking or in situ polymerization. This can be achieved by a method as simple as a non-stick sterile sheet that can be placed in the site and then removed after gel formation. This can also be achieved by placing structures in/around the nerve. The structure may take a variety of forms depending on the size and location of the nerve, the presence or absence of a sheath, the therapeutic target delivered to the site (nerve stimulation, nerve block, nerve ablation, or nerve regeneration disorder). In one embodiment, the structure is a cap that can be gently placed around the end of the nerve and the in situ forming gel injected into the structure so that it takes the shape of the structure. The material in the cap can then be pushed out and the cap structure removed and discarded. In other embodiments, the cap is biocompatible and therefore not removed. In another embodiment, half cylinders (longitudinal halves) may be placed under the nerve and the in situ forming material delivered into the half cylinders. In the same way, the gel can be delivered circumferentially around the nerve without the gel adhering to the surrounding tissue.

An optional cap structure. To reduce the chance of adhesion formation, a 3 to 10mm nerve segment can be placed into the syringe barrel (the luer lock tip portion removed) and the plunger pulled back to the appropriate gel distance required on the nerve tip. The hydrogel is delivered into and around the nerve within the plunger where the gel is located. After the gel was formed, the plunger was gently pressed to squeeze out the nerves encapsulated in the hydrogel. Again, this method requires minimal or no suturing to avoid additional damage or over-treatment of the nerve. Preferably, the syringe barrel has a lubricious coating.

Laparoscopic or endoscopic procedures. During laparoscopic or endoscopic surgery, the structure may be advanced along a channel and placed under the nerve, similar to the approach in open surgery. The structure may be folded to allow passage through a smaller catheter and then released at the surgical site. Alternatively, the device may be designed to build neural structures (caps or wraps) to the tip of one device, delivering the in situ forming biomaterial through the lumen of the other device. If a catheter is used and gel mixing occurs at a distance from the gel formation, the gel time of the in situ formed biomaterial needs to be adjusted to 20 to 30 seconds or more to allow time for the hydrogel to travel along the lumen of the device. The treatment of hernias is particularly suitable for blocking nerve regeneration in transected nerves during inguinal hernia repair, for example by open, robotic or laparoscopic methods

In the needlescopic method of the procedure, a first material can be injected which covers the external tissue to prevent direct adhesion between the gel and the surrounding tissue. Thereafter, the hydrogel can be delivered to the same site, forming a depot around the nerve, and transferring the first material to the periphery of the injection site. This can be achieved by hydrophobic substances such as oils or viscous substances such as glycerol. This can also be achieved with a low molecular weight PEG solution which has the added benefit of helping to seal the nerve membrane before the hydrogel forms the nerve block/cap around the nerve.

In another embodiment, the nerve is immersed in a solution of flowable material prior to crosslinking of the nerve to form a thin protective surface on the hydrogel. In some embodiments, only a thin coating of biodegradable polymer is required around the nerve. The coating may be as thick as 100 microns to 500 microns. In other embodiments, the coating is insufficient to prevent inflammatory infiltration and/or prevent early degradation-in these cases, it is desirable to use a coating 0.5mm to 10mm thick.

To date, attempts to develop neural caps have focused on solid physical caps that are sutured around the ends of transected nerves. These caps necessarily have a gap around and circumferentially around the nerve end of the proximal stump. As a result, neuroma formation occurs at the end of the cap. Examples include resorbable poly (D, L-lactide-co-caprolactone) implants, oriented Silk Fibroin (SF) (SF/P (LLA-CL)) nanofiber scaffolds blended with poly (L-lactic acid-co-e-caprolactone), poly (lactic acid) -co- (glycolic acid)/arginyl glycine granulic acid) modified poly (lactic acid-co-glycolic acid-alt-L-lysine) (PRGD-PDLLA) implants with pores of about 10 microns in diameter (Yi et al 2018, adv Sci). PRGD/PDLLA catheters were 10mm long, 2mm inner diameter, 200 micron wall thickness, SF/P (LLA-CL) catheters were 1.5cm long, 1.5mm inner diameter. These caps require suture placement. The other is called as

Is a synthetic nerve cap device comprising a solid tube with a closed end, which is placed over the nerve bundle and then must be sutured both to the nerve to retain it within the cap and to the surrounding tissue to retain the cap in place, as disclosed in WO2016144166 A1. Another approach also uses solid implants, as disclosed in US20140094932 A1. In contrast, the injectable gel method can flow around nerves of any size, from tiny fibers to large fascicles, does not require cutting or suturing, and reduces pain and neuropathic pain. In short, the injectable flowable system is not limited to nerve stumps, but also prevents abnormal nerve outgrowth where the fibers are too thin to pick up.

Neuroprotective/wrap. According to another aspect of the invention, methods and devices for protecting intact or stressed nerves are provided. In some cases, it may be desirable to protect nerves that are surgically exposed as a result of the procedure or adjacent nerves or tissue, for example, in cases where these nerves would otherwise dry out. In some cases, it may be desirable to protect and mark nerves exposed as part of another procedure, so that additional handling, stretching, bruising, and/or compression may be reduced or avoided. In one embodiment, the in situ forming material is delivered around the nerve to provide a protective layer and prevent damage to the nerve in the area by forceps and other surgical devices. In addition, the dye in the hydrogel may provide sufficient contrast with surrounding tissue so that the physician may also be visually remote from the nerve during the procedure. This can significantly reduce the incidence of iatrogenic nerve damage during surgery.

According to another aspect of the present invention, a structure for creating an in situ formed capsule around a nerve-to-nerve connection is provided. The structure includes a recessed wall defining a cavity, the wall having a top opening for accessing the cavity. The top opening lies in a first plane and has an area smaller than a second plane that conforms to the interior dimensions of the cavity and is spaced apart from the cavity and parallel to the first plane. A first concave nerve guide carried by the wall and providing a first lateral access for positioning a first nerve end in the cavity; and a second concave nerve guide carried by the wall and providing a second lateral access for positioning a second nerve end in the cavity.

An example of this is the prophylactic treatment of the iliac inguinal and infrailiac nerves during hernia repair, in particular inguinal hernia repair. During hernia repair, these nerves may be partially or completely exposed, resulting in compression, contusion, and partial or complete transection. After surgery, the damaged nerve may send abnormal nerve buds out into the scar tissue after surgery, which may lead to neuroma formation and nerve entrapment, resulting in a high percentage of chronic pain after surgery. In addition, these nerves can be surgically severed accidentally or purposefully in an attempt to prevent the nerves from being surgically trapped or tangled in the mesh used to repair the hernia. In one embodiment, a kit comprising an in situ forming growth-inhibiting hydrogel and a suitable structure allows a surgeon to select a structure to provide a structural cavity in the shape of a "cap" or "wrap". Depending on the surgical situation, the surgeon may select the wrap if the nerve is not severed, the surgeon may wish to protect the nerve from further damage if the nerve is severed, or the surgeon may select the cap if the nerve is severed, the surgeon wishes to prevent a distal neuroma from forming at the end of the severed nerve.

Another example is exposure of the sciatic nerve in hip surgery. Although nerves are not the target of these procedures, nerves are often exposed and in traction, and therefore risk being damaged and/or drying out during the procedure. In one embodiment, a wrap-shaped structure is provided to deliver the hydrogel around the area of risk of the sciatic nerve. For larger nerves, these regions may be 5 to 50cm or more. A wrap-shaped structure may be provided to span the entire length, or multiple cap structures may be placed in series along the nerve to provide protection. In another embodiment, an anti-inflammatory agent is delivered into the hydrogel to reduce inflammation around the nerve that may result from locating or moving the nerve during surgery. In another embodiment, the local anesthetic is delivered into a hydrogel placed around the nerve.

In another embodiment, the hydrogel is delivered around nerves to reduce inflammatory neuropathy that may result after surgery, particularly peripheral neuropathy, which may result in slowly progressing severe pain and/or weakness in the affected limb. The in situ formed hydrogel may be delivered after open surgery or by percutaneous image guided procedures. For percutaneous ultrasound guided or fluoroscopic delivery, echogenic needles need to confirm not only the depth, but also the position of the needle relative to the relevant structure.

In another embodiment, the in situ-forming hydrogel is delivered around the nerve in a cavity of a "wrap" structure to form a protective conformable wrap around the nerve. The cavity of the wrap structure is left in place, which provides additional support and protection during the procedure, and then after the procedure is complete, the wrap structure is removed and discarded before closing the site. The hydrogel remains in place to protect the nerve, prevent abnormal nerve growth, and any scar tissue infiltration into the nerve.

A bonding aid. In some embodiments, a nerve that has been directly engaged with a suture may be placed in a cavity in the form of a wrap. The direct anastomosis site may be filled with an injected growth-permitting hydrogel or temporary spacer material (e.g. fibrin glue) which may diffuse into the interstices of the site, and the growth-inhibiting hydrogel is delivered directly around the anastomosis site using a cavity in the form of a wrap.

Wrap or protector structures. The cavity of the wrap structure comprises a structure having two nerve entry regions with variable cavity lengths around the nerve region to be protected. In a shorter wrap structure, the cavity is designed such that the nerves rest on the entry area and the nerves "float" between the areas and do not contact the walls of the structure. Delivery of in situ hydrogel-forming needles deliver a flowable hydrogel solution into the structure surrounding the nerve, where it forms a protective hydrogel around the nerve. The structure prevents unintended diffusion of the hydrogel to adjacent tissue and maintains a consistent hydrogel thickness around the nerve.

A longer length. In the case of longer nerve lengths that need to be protected, a longer cavity of the wrap structure can be used and a small strut designed at the bottom of the structure to support and provide stability to the nerve over a longer distance. When the structure is removed, these struts are removed, leaving only a small exposure between the nerve and the surrounding environment in non-critical locations (if present) away from the proximal nerve stump tip. In another embodiment, where longer nerves are desired to be protected, the in situ forming hydrogel solution may be delivered in multiple layers or regions. In one embodiment, the structure is filled in multiple portions so as to keep nerves in the center of the structure. In another embodiment, a first layer of in situ-formed hydrogel is delivered to the bottom of the structure, with or without nerve embedding, in whole or in part, and then a second layer of hydrogel is delivered to the top of the first layer to completely cover and protect the nerve.

In one embodiment, a kit is provided that contains an appropriate volume of in situ-forming hydrogel to fill the wrap structure, and a plurality of mixer and needle assemblies to allow the physician to switch the mixer-needle tip as needed and continue to deliver more media in a second or third application as needed.

Protecting the anastomotic site. With the increased use of allografts and autografts in the repair of larger nerve spaces, it is increasingly recognized that abnormal nerve growth from the peripheral nerve stumps into the peripheral tissue at the nerve-allograft, nerve-autograft or nerve-conduit junction can cause local pain and reduce the effectiveness of nerve repair. Furthermore, the mismatch in conformability between the solid implantable catheter used to secure the two nerve stumps and the nerve itself may cause friction at the interface between the nerve and the catheter, resulting in additional abnormal nerve tethering to the surrounding tissue. In one embodiment, it is contemplated to deliver the hydrogel formed in situ at the interface between the proximal nerve and the allograft or autograft anastomosis, or between the allograft or autograft and the distal nerve stump, or similarly at the junction between where the nerve stump enters and emerges from the catheter. The smaller volume of hydrogel delivered directly or in shorter encapsulated segments provides protection against neurite outgrowth and reduces scar formation and infiltration of immune cells into grafts and catheters. Alternatively, the wrap structure may span the entire length of the anastomosis to cover the allograft/autograft/conduit except for the nerve.

The nerve slides. Some peripheral nerves undergo considerable movement in the plane of the fascia in which they are located, and thus scarring and binding of these nerves is particularly painful. For example, the median nerve in the carpal tunnel or the ulnar nerve position relative to the elbow are important locations for providing sliding motion. For implantable catheters, wraps and protections, the form of the implant is such that the sliding of the nerve is not enhanced by the biomaterial and may be further inhibited. In one embodiment, the in situ-forming hydrogel is delivered as a protector around these nerves to allow the nerves to continue to slide within their fascial plane. One way to achieve this is by delivering a higher swelling in situ forming material that swells significantly after delivery around the nerve, e.g., greater than 30%, preferably greater than 60% radially outward, so that the nerve can slide within the channel created after the hydrogel reaches equilibrium. In this way, even if the hydrogel eventually becomes embedded in a thin capsular layer, the nerves within the hydrogel are themselves free to slide within the tunnel, and there is no significant scar tissue, nerve growth into the surrounding tissue, and there is no compression at these critical locations.

Thus, the hydrogel forms a hollow cylindrical sleeve with a central lumen in which the nerve or tendon can slide. Sliding can occur in one of two ways, sliding can occur on the outer surface of the formed hydrogel, the hydrogel moving with the nerve or tendon. Alternatively, preferably, the nerve or tendon slides within the lumen of the hydrogel structure while the hydrogel is anchored in place with a minimal capsule. In this way, the nerve or tendon is free to move through the hydrogel in a soft, conformable, scar-free manner.

Is suitable for the indications. In situ forming hydrogels can also be introduced intraoperatively to help maintain a successful microsurgical anastomosis of donor and recipient nerves using the wrap structure. The hydrogel can be injected in the form of a wrap to the anastomotic junction to protect against abnormal inflammatory responses, scar tissue formation, and to aid in the adaptation of the donor and recipient nerves. The transfer of non-critical nerves to reactivate more important sensory or motor nerves is called neurogenesis (neuralization). In one example, a patient undergoing a breast reconstruction after a mastectomy may choose an autologous skin flap reconstruction to connect the nerve and chest wall. A wrap structure may be placed over and sutured to the junction between the proximal nerve stump and the distal nerve tissue, and a hydrogel delivered to protect the anastomosis site. Such repair may lead to a restoration of sensory function and improve the physical and quality of life of the woman.

And (4) repairing tendons. In another embodiment, the hydrogel can be used to aid in tendon repair and to prevent the formation of adhesion/scar tissue around the tendon. In some embodiments, the hydrogel is placed around the tendon using a similar structure that allows for delivery of the solution to form a gel around the tendon. In this way, the gel allows the tendon to slide in the same way as the gel around the nerve.

And (5) pressing and repairing. In another embodiment, the in situ-formed hydrogel can be delivered in a wrap around the nerve to act as a barrier to the attachment of surrounding tissue during nerve repair. This approach allows the hydrogel to penetrate and conform to the nerve and serves as an alternative to venous encapsulation, where autologous veins are wrapped around the nerve in a spiral wrapping technique to provide a barrier to attachment of surrounding tissue. This also provides an alternative to the AxoGuard neuroprotection which must be wrapped around the nerve, which can stretch and further damage the nerve. Solid neuroprotective articles require extensive handling of the nerve with forceps, pulling the solid wrap apart to hold it open, and then suturing the nerve to the wrap. Using a soft, conformable, hydrogel-based approach, a liquid or viscous liquid can be delivered directly to the nerve periphery in a form that minimizes nerve processing. Soft tissue attachment is minimized, swelling is minimized, and the mechanical support provided by the gel reduces the tension and stress at the junction. The nutrients may diffuse through the hydrogel network. In addition, hydrogels can reduce the surgeon's surgical time.

In one embodiment, the solution is based on hyaluronic acid. In another embodiment, the solution is based on a hydrogel slurry (TraceIT, boston science).

The distance of application. The hydrogel may be delivered circumferentially around the nerve with a syringe or applicator tip in such a way that the nerve has protection over the length of the lesion. Ideally, the hydrogel would be applied at every site of the damaged or transected nerve, for example 5-15mm. The volume administered may be between 100 microliters to 10ml, more preferably 0.2 to 3ml. The syringe containing the hydrogel (or two precursor components of the hydrogel) can be designed to have a precise volume to be delivered to allow for controlled automated delivery of the in situ formed hydrogel. Alternatively, an extra amount may be provided to allow the individual to use his/her judgment to decide how much to deliver around the site. The hydrogel should form a cellular barrier about 200 microns thick around at least the lateral side of the nerve, although the hydrogel may also be delivered to fill the site, forming a circumferential mass with a radius of 2cm around the hydrogel site.

End-to-side (end-to-side) repair. A window is formed in the outer nerve sheath and connects the nerve transition to one side of the nerve. After suturing, the in situ-forming hydrogel is delivered around to keep them in close proximity to each other.

Internal nerve lysis. Internal scarring and swelling can occur after the nerve is stretched or compressed for a long period of time. The outer sheaths of these nerves can open to relieve pressure and assist blood flow.

External nerve lysis. If the nerve has scarred or developed neuroma, stretching or movement may result in additional nerve damage, pain and additional nerve scarring. Neurolysis can be used to remove scar tissue around nerves without accessing the nerves themselves. The in situ formed hydrogel can be delivered around the nerve after the outer nerve is released to prevent additional nerve scarring and reduce pain.

And (4) carrying out nerve transformation. In one embodiment, the transcutaneous neuroprotection is delivered around a damaged or stressed nerve. In such embodiments, the local inflammatory response may be reduced if administered within one to several days after the injury. In situ forming a hydrogel that 1) is biocompatible, 2) is biodegradable or bioerodible, 3) allows diffusion of nutrients and oxygen into and out of tissue while preventing inflammation, 4) is flexible and conformable so axons are protected from compression, 5) does not swell or minimally swells, and 6) prevents ingrowth of fibers to the site of injury.

Location. Injectable conformable hydrogels also allow the same product to be delivered to multiple nerve diameters and multiple locations (between bone, fascia, ligaments, muscles) because the material will flow in the peri-nerve area.

A delivery location. In some embodiments, the location of the needle affects the delivery of the hydrogel. In some embodiments, the needle delivers the hydrogel directly to the top of the target nerve or region. In another embodiment, the needle is advanced from distal to proximal to fill the tip and distal nerve stump of the structure first, and then the rest of the catheter.

And (4) TMR. In another embodiment, the hydrogel may be delivered to the periphery of nerves reconnected as part of a targeted muscle innervation (TMR) program. Since the size of the severed donor nerve and the generally smaller denervated recipient nerve often do not match, it may be necessary to apply a hydrogel at the junction to help guide the regenerated fibers into the targeted recipient motor nerve. For these indications, the hydrogel may be administered directly or in a structure. Typically, this is done between mixed motor and sensory nerves.

Inhibiting the drug into the cap and wrap. Depending on the desired clinical presentation, the mechanical barrier may be aided or enhanced by any of a variety of chemical agents that inhibit or prevent nerve regeneration (sometimes referred to as "anti-regenerative agents"). These agents include inorganic and organic chemical agents, including small molecule organic chemical agents, biochemical agents, which may be derived from a patient and/or an external source, such as an animal source and/or a synthetic biochemical source, and cell-based therapies. The anti-regenerative agent may be applied directly to the target tissue before or after the nerve ends are formed. Alternatively, the anti-regenerative agents may be carried in a medium where they are captured in the medium and then released over time near the nerve ends.

Some specific examples of anti-regeneration agents that may be used in conjunction with some embodiments of the present invention include, inter alia: (a) Capsaicin, resiniferatoxins and other capsaicinoids (see, e.g., J.Szolcsanyi et al, "Resiniferatoxin: an ultrasonic selective modulator of capsaicin-sensitive primary enzymes", J Pharmacol exp.1990 November;255 (2): 923-8); (b) Taxols (taxol), including paclitaxel (paclitaxel) and docetaxel (i.e., because lower concentrations of paclitaxel promote nerve regeneration, their concentrations rise sufficiently to slow or stop nerve regeneration; see, e.g., W.B. Derrice, et al, "society combining of taxol requirements microtubule dynamics," Biochemistry 1995february 21 (7): 2203-11), botulinum, purine analogs (see, e.g., L A Greene et al, "human biology association in nerve growth factor-modified nerve outgrowth by systemic and sensory nerves," The Journal of Neuroscience,1May 1990,10 (5): 1479-1485); (c) Organic solvents (e.g., acetone, aniline, cyclohexane, ethylene glycol, ethanol, etc.); (d) Vinca alkaloids, including vincristine, vindesine, and vinorelbine, and other antimicrotubule agents, such as nocodazole and colchicine; (e) Platinum antineoplastic agents (platins) such as cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, and triplatin; znso.sub.4 (i.e., neurodegenerative factor); (g) latarcins (short linear antibacterial and cytolytic peptides, possibly from venom of the spider Lachasana tarabaevi); (h) Chondroitin Sulfate Proteoglycans (CSPGs), such as aggrecan (CSPG 1), pluripotent proteoglycan (CSPG 2), neuroproteoglycan (CSPG 3), melanoma-associated chondroitin sulfate proteoglycan or NG2 (CSPG 4), CSPGS, SMC3 (CSPG 6), short proteoglycan (CSPG 7), CD44 (CSPG 8), and phosphoproteoglycan (see, e.g., shen Y et al, "PTPsigma a receiver for chloride protein, an inhibitor of neural regeneration", science,2009October 23 (326 (5952): 592-6); (i) Myelin Associated Glycoprotein (MAG); (j) oligodendrocytes; (k) oligodendrocyte-myelin glycoprotein; and (I) reticulin-4, also known as neurite growth inhibitory factor or Nogo, a protein encoded by the RTN4 gene in humans (see, e.g., lynda J. -S. Yang et al., "Axon regeneration inhibitors, neurological Research,1Dec.2008, volume 30, issue 10, pp.1047-1052); (m) ethanol or glycerol.

Further examples of anti-regenerative agents include agents that induce inhibitory scar tissue formation, which may be selected from the following, among others: (a) laminin, fibronectin, tenascin-C and proteoglycans, which have been shown to inhibit axonal Regeneration (see, e.g., stephen J.A. Davies et al, "Regeneration of adult axons in white matter tracks of The central nervous vous system," Nature 390,680-683 (18Dec.1997); (b) reactive astrocytes, which are The major cellular component of glial scars, form dense plasma membrane extended networks and modify The extracellular matrix by secreting a number of molecules including laminin, fibronectin, tenascin-C and proteoglycans, (C) molecular mediators known to induce glial scar formation, including transforming growth factor beta (TGF β), such as TGF β -1 and TGF β -2, interleukins, cytokines, such as interferon gamma (IFN γ), fibroblast growth factor 2 (FGF 2) and ciliary neurotrophic factors, (d) glycoproteins and proteoglycan basement membranes that promote growth (see, e.g., CC Stichel et al, "" The CNS clearance scan: new vistas on rolled Regeneration, 294 (1): 1-9) and (e) substances that inactivate Schwann cells-resistant agents, including Semaphorin-3A (a protein) which can be incorporated into The hydrogel to induce The release of calcium in The nerve growth cone by local paralysis, which is induced by The intracellular calcium release, which is increased by The release of local calcium ion (1, 2) and which can be induced by The release of local nerve growth cone collapse of The growth factor), f) An inhibitory dye such as methylene blue, and g) a radioactive particle. Other inhibitory drugs include ciguatoxin (ciguatoxin), cannabinoid, HA-1004, phenamil, mnTBAB, AM580, PGD2, topoisomerase I inhibitor (10-HCT), anti-NGF, and anti-BDNF.

Pain and inflammation. The medium may additionally include one or more agents for relieving pain within a short time frame post-surgery where an increase in pain beyond baseline may be experienced due to local tissue reactions depending on the ablation procedure. Examples of suitable anesthetics that can be incorporated into the hydrogel for this purpose include, for example, bupivacaine, ropivacaine, lidocaine, and the like, which can be released to provide short-term local pain relief around the treatment area after surgery. By incorporating methylprednisolone in the hydrogel, inflammation and scar tissue in the surrounding tissue can also be minimized.

The permissive structure is grown. With respect to the examples of fig. 5A-5E, in some instances it may be desirable to provide a growth-permitting substance between the proximal and distal stumps of the nerve to promote nerve regeneration rather than inhibit growth. In some embodiments, the growth-permitting substance simply provides a temporary barrier to leakage of the growth-inhibiting gel into the anastomosis site or damaged nerve tissue and inhibits regeneration. In other embodiments, the growth-permitting substance provides a medium through which nerves can regenerate without the need for autograft/allograft or catheter/wrap.

According to another aspect of the present invention, methods and devices are provided that facilitate guided nerve growth, such as spanning a gap between two opposing nerve stumps and restoring nerve function or filling a small gap between nerves that have been directly joined with a suture. The method may comprise the steps of: placing a first nerve end and a second nerve end in the first structural cavity; introducing an in situ forming growth-permitting medium into the cavity and into contact with the first nerve end and the second nerve end to form a junction; the medium transitions from a flowable state to a non-flowable state. The nerves coupled together by the in situ formed medium are then removed from the first structural cavity and placed into a second, larger structural cavity; and introducing a growth-suppressing medium into the second structural cavity to encapsulate the connection. The growth-inhibiting medium transitioning from a flowable state to a non-flowable state, overlying the nerve and the growth-permitting medium; the second structure is then removed and discarded. In another embodiment, the first and/or second structures remain in place.

Also provided is an in situ formed nerve regeneration construct comprising a growth-permissive hydrogel bridge having first and second ends and configured to span the space between the two nerve ends and promote nerve transaxle regeneration; and a growth-inhibiting hydrogel sheath encapsulating the growth-permitting hydrogel bridge and configured to extend beyond the first and second ends of the growth-permitting region to directly contact proximal and distal nerves, respectively. In yet another embodiment, the growth-permitting medium is delivered into the inhibitory structural cavity where it undergoes a change from a substantially flowable state to a non-flowable state. The structure remains in situ and acts as a growth-inhibiting matrix through which nerves cannot regenerate.

Preferably, the growth-permitting medium consists of an in situ forming gel, such as a hydrogel, and the growth-inhibiting medium consists of an in situ forming gel, such as a hydrogel. However, the growth-permissive medium may consist of an in situ-forming gel, and the structure to which it is delivered consists of an ex vivo cross-linked gel.

In some embodiments, it is desirable that the growth-permitting hydrogel adhere to neural tissue, providing a method for anastomosing tissue without suturing. In this way, the nerve growth-permitting gel-nerve units can be picked up and handled as one continuous nerve unit, allowing them to later place the unit in the growth-inhibiting hydrogel. In other embodiments, the growth-permissive hydrogel may provide temporary adhesion for about half an hour. The glue is strong enough to easily stick to both nerves, but the mechanical strength can be comparable to, for example, fibrin glue.

And (4) repairing without tension. Another advantage of forming hydrogels in situ is that they can be designed to provide tensionless repair of nerves. In one embodiment, the wrap structure of the catheter is deep enough that the directly repaired/anastomosed nerve end is placed in the structure while the repair area is relaxed inside the structure. When the hydrogel is formed around a relaxed nerve, the nerve-nerve repair is not under tension; any tension is carried by the hydrogel around it. In this way, the nerve is not under tension and the hydrogel carries the load in a more evenly distributed manner than suture repair.

In another embodiment, the growth-inhibiting hydrogel additionally provides a tensionless repair. In the described embodiment, the proximal and distal nerves are placed in a catheter, and a growth-inhibiting hydrogel is delivered at the nerve-catheter interface to prevent the nerves from escaping the catheter and binding together with the surrounding scar tissue. In another embodiment, the nerves are purposefully relaxed within the growth-permissive hydrogel by creating relaxation in the nerves within the structure. In the case of direct nerve re-anastomosis, care is taken to ensure that tension, if any, is located at the interface between the nerve and the structure entrances on either side of the wrap structure and that the nerve is relaxed or not tensioned inside the wrap before the growth-permissive hydrogel precursor solution is applied to the wrap. In this way, the nerve anastomosis, nerve-gel-nerve or nerve-nerve interface is tension free. In a preferred embodiment, the nerve growth permitting hydrogel-nerve unit is located entirely within the cavity of the second neural structure. Delivery of the inhibitory hydrogel provides additional protection and stress relief, providing circumferential coverage of approximately 3 to 10mm around the bilateral nerve of the injury.

And (6) covering. In one embodiment, the growth-permissive medium is located substantially between two nerve ends and does not significantly cover the outer surface of the nerve. Thus, the diameter of the growth-permissive medium is very close to the diameter of the nerve. Due to the location of the growth-permitting medium, the growth-inhibiting medium is delivered to the outer surface of the proximal and distal nerves or to the extraneural surface and around the growth-permitting medium, preferably covering 10mm or more than 5mm of healthy nerves on both sides. This allows the nerve to be guided from the proximal nerve stump directly to the distal nerve stump. The additional covering provides adhesive strength and protection against abnormal nerve growth at the proximal nerve-gel junction.

And (4) color. In one embodiment, the growth-permitting hydrogel is one color, such as blue, while the growth-inhibiting hydrogel is not colored. In another embodiment, the growth-permitting hydrogel is blue in color and the growth-inhibiting hydrogel is green or blue-green in color.

Preferably, the growth-permitting substance is an in situ-forming hydrogel. Preferably, the growth-permitting substance comprises growth-inhibitory and growth-permitting microdomains. The nerve will naturally seek ways along the growth-inhibitory region and within the growth-permissive region. Growth-permissive hydrogels that utilize in situ formation of a PEG platform are desirable. These hydrogels may be chemically crosslinked or crosslinked using photocrosslinking methods, such as the non-growth-permissive hydrogels described above. The in situ growth-permissive hydrogel preferably degrades faster than growth-inhibitory hydrogels, promotes cell ingrowth, and replaces synthetic matrices with natural extracellular matrices. Thus, preferred PEG hydrogels for these applications are formed by the crosslinking of PEG-NHS esters with hydrolytically labile ester bonds (PEG-SS, PEG-SG, PEG-SAZ, PEG-SAP, preferably PEG-SS). For example, these PEGs may be cross-linked with PEG-amine or trilysine.

Other hydrogels may be selected to provide the non-growth-permissive regions of the growth-permissive hydrogel, including PEG-PPO-PEG, PEG-polyester (tri-block, di-block), alginate, agar, and agarose. Other synthetic hydrogels include PEG-poly (amidoamine) hydrogels, PEO, PVA, PPF, PNIPAAm, PEG-PPO-PEO, PLGA-PEG-PLGA, poly (aldehydo guluronate), or polyanhydrides. A series of suitable hydrogel matrices for in situ formation are listed in Hoffman (2012) Hydrogels for biological applications, advanced Drug Delivery Reviews,64, which is incorporated herein by reference. Another potentially useful soft water gel includes InnoCore liquid polymer (LQP) (PCLA-PEG-PCLA), which is a liquid polymer that forms a soft macroscopic reservoir after in vivo delivery and slowly degrades over a period of two to three months. Ext> Anotherext> potentiallyext> suitableext> hydrogelext> includesext> sixext> -ext> armedext> starext> -ext> shapedext> polyext> (ext> ethyleneext> oxideext> -ext> statext> -ext> propyleneext> oxideext>)ext> withext> acrylateext> endext> groupsext> (ext> starext> -ext> PEGext> -ext> Aext>)ext>,ext> whichext> canext> beext> photoext> -ext> curedext>.ext> Other star-shaped PEGs include 6-or 8-arm NHS ester PEGs, including mPEG-SCM (PEG-NHS: succinimidylcarboxylate methyl ester) and mPEG-SG (PEG-NHS: succinimidylutamate), PEG-co-poly (lactic acid)/poly (trimethylene carbonate), PEG-NHS and trilysine, PEG-NHS and PEG-thiol, PEG-NHS and PEG-amine, PEG-NHS and albumin, dextran aldehyde and PEG-amine functionalized with tris (2-aminoethyl) amine. The concentration of PEG. If PEG is used in the growth-permissive matrix, the concentration of PEG in these hydrogels is preferably 3-8wt%, more preferably 3-5wt%, for applications that support nerve extension.

In some embodiments, the growth-permissive region is directly conjugated or chemically linked to a non-growth-permissive hydrogel region. For example, chitosan may be coupled to the inhibition zone. The chitosan may have a molecular weight of 100 to 350kDa, more preferably 130 to 160kDa, and a degree of deacetylation of 0.85. In another embodiment, an interpenetrating network of gelatin methacrylamide is polymerized with the PEG framework.

An optional growth-permissive substrate. In addition to containing positively charged matrix components that promote glial cell invasion, cell division, and three-dimensional cellular organization, the growth-permitting component may also support nerve ingrowth with or without supporting cells. These growth promoting substances are applied at concentrations sufficient to support growth, but not so high as to affect the mechanical properties of the hydrogel. The growth-permissive hydrogel comprises a combination of natural growth-promoting biomaterials, for example the natural polymer type I collagen (0.01-5 wt%, preferably 0.3-0.5wt%,1.28 mg/ml), laminin (4 mg/ml), hyaluronic acid, fibrin (9-50 mg/ml, strength 2.1 kPa) or synthetic/semisynthetic polymers such as poly-L-arginine or poly-L-lysine (0.001-10 wt%). These mixtures support 1) the establishment of pathways through which regenerating nerves can seek, 2) the provision of a matrix to which neurites can adhere and to which schwann cells can migrate. In one embodiment, the hydrogel is an 8-arm 15K PEG-succinimide succinate (PEG-SS) cross-linked with trilysine, which contains 5wt% collagen. In another embodiment, the hydrogel comprises 4-percent PEG (4-arm 10K PEG-SG crosslinked with 4-arm 20K PEG-amine) containing 0.01% poly-L-lysine. By reducing the concentration of the cross-linked PEG solution relative to the growth inhibitory PEG for neuroma blocking applications and increasing the concentration of the positively charged growth permissive biomaterial, an in situ forming hydrogel with inhibitory and permissive regions can be created to promote nerve growth.

In another example, a non-growth-permissive Hydrogel (e.g., a cross-linked PEG Hydrogel, alginate, methacryl-substituted Tropoelastin MeTro Hydrogel) may be mixed with a growth-permissive Hydrogel (e.g., fibrin Gelatin-methacryloyl GelM, gelM/PEG or GelMA/MeTro composite), soucy et al (2018) Photosorosslinkable gel-Troopectin Hydrogel for Peripheral Nerve Repair, tissue Engineering, PMID:29580168. And adding polylysine. Polylysine, whether in the D, L or L form, may be incorporated into the growth-permitting hydrogel regions. For example Epsiliseen (Siveele, ε -poly-L-lysine). The growth-permissive hydrogel may be an in situ-forming hydrogel comprising chitosan and polylysine (https:// pubs. Acs. Org/doi/10.1021/acs. Biomac.5b01550). The growth-permissive hydrogel may be an in situ-forming hydrogel comprising PEG and polylysine (https:// pubs. Acs. Org/doi/abs/10.1021/bm201763 n).

The growth-permitting component of the invention may alternatively comprise a decellularized peripheral nerve-specific scaffold formulated into an injectable hydrogel structure. It is based, at least in part, on the use of decellularized tissue that is substantially devoid of immunogenic cellular components, but retains a sufficient amount of nerve-specific components to effectively support nerve regeneration and reduce or prevent muscle atrophy. In certain non-limiting embodiments, the decellularized tissue scaffold can be formulated into a hydrogel by using enzymatic degradation. These hydrogels may be non-cytotoxic to neurons and also support neuronal growth of cultured cells. Thus, one aspect of the method of the invention comprises applying or injecting a growth-permissive medium comprising a hydrogel comprising an decellularized peripheral nerve scaffold that has been enzymatically degraded such that it bridges a gap in a nerve. The growth-permissive medium may be wrapped in the growth-inhibitory medium in any manner discussed elsewhere herein. The peripheral nerve scaffold may be derived from an organism that is not autologous to the intended recipient of the hydrogel, derived from an organism that is the same species as the intended recipient, or derived from an organism that is heterologous to the intended recipient. The harvested nerve may include, but is not limited to, the sciatic nerve, femoral nerve, median nerve, ulnar nerve, peroneal nerve, or other motor/sensory nerves. Additional details of acellular scaffolds can be found in U.S. patent No.9,737,635 to Brown et al, entitled "injectable peripheral nerve-specific hydrogel," issued on 22.8.2017, the disclosure of which is incorporated herein by reference in its entirety.

In addition to the neuro-derived or tissue-specific materials, other growth-permissive components produced from human (autologous) or animal (non-autologous bovine, porcine) sources may be used in whole or in part in the growth-permissive solution delivered between the proximal and distal stumps. These include inactive human umbilical cord allografts, amniotic membrane and/or chorion grafts (submucosa of human placenta), autologous skin grafts, untreated allogenic cadaver/porcine skin grafts, autologous connective tissue, autologous or allogeneic tendon tissue, bovine collagen, fibrous collagen, fibronectin, laminin, proteoglycans. These components may take the form of micronized or dehydrated or lyophilized powders, gels, tablets or rolled sheets, or dehydrated powders that may be rehydrated prior to use. The dehydrated product can be rehydrated before use in an aqueous solution (e.g., saline) or a solution containing 6-9% low molecular weight polyethylene glycol (e.g., PEG 3350 or PEG 5000). In yet another embodiment, the growth-permissive environment may comprise cells.

Growth permissive solutions can be used to support nerve regeneration in direct anastomosis repair, small slope repair (< 7 mm), and larger gap repair (> 7 mm). The growth-permissive and/or growth-inhibitory solutions may be delivered acutely after injury, subacute, or several days after injury, where the injury has a chance to manifest itself, or for chronic nerve repair.

PEG + collagen in the backbone. Alternatively, natural polymers, such as type I Collagen, can be crosslinked to PEG hydrogels (e.g., 8-arm 15K SG) at Collagen concentrations ranging from 30 to 60mg/ml and at PEG concentrations of 50 or 100mg/ml (Sargent et al 2012.an in situ forming Collagen-PEG hydrogel for Tissue regeneration. Acta biomaterials 8,124-132 and Chan et al (2012) Robust and semi-interactive hydrogels from PEG and Collagen for Elastomeric Tissue scales.12 (11) 1490-1501).

Other gels. In yet another embodiment, the first growth-permitting material may comprise a viscous solution, a nanoparticle or microparticle-based gel, a slurry, or a macrogel. In one embodiment, the fibrin glue may be delivered around the nerve. In another embodiment, the solution is a slurry of biocompatible nanoparticles or microparticles through which nerves can be regenerated. In another embodiment, a microgel or a modular gel (model) is delivered to the site. Microgels are stable dispersions of uniform size and large surface area produced by precipitation polymerization. Modular gels are scaffolds formed from microgels whose properties can be altered by the degree of crosslinking and scaffold stiffness (preparation of said gels, including PEG-based hydrogels, can be referred to Scott et al (2011) Modular Poly (ethylene glycol) coatings from the properties and protein concentrations on PC12 cells acta biomaterer 7 (11) 3841-3849, which is incorporated herein by reference). Furthermore, the use of electrically conductive hydrogels, such as piezoelectric polymers that generate transient surface charges under mechanical strain, such as polyvinylidene fluoride (PVDF), may be beneficial in supporting nerve growth through the hydrogel. For example, dendrimers composed of metabolites such as succinic acid, glycerol and beta-alanine may be incorporated into Hydrogels to promote extracellular matrix infiltration (degericcija et al (2008) Hydrogels for Osteochondral Repair base on Photonanosystemable carbohydrate polymers, biomacromolecules,9 (10) 2863).

Common natural hydrogels. In another embodiment, a fully growth-permissive hydrogel is provided without growth-inhibiting microdomains. In one embodiment, fibrin hydrogels (e.g., those crosslinked with thrombin) are selected that have a lower linear compressive modulus. Many other biomaterials have also been demonstrated to support nerve regeneration in 2D and 3D scaffolds, including chitosan, chitosan-coupled alginate hydrogels, viscous fibronectin, type I collagen (-1.2 mg/ml), assisted regeneration, fibrin (9 to 50 mg/ml), fibronectin, laminin (https:// w.ncbi.nlm.nih.gov/pubmed/15978668), puramatrix, heparan sulfate proteoglycans, hyaluronic acid (1% sodium hyaluronate viscous solution), polylysine (poly (D, or L, or D, L) lysine), xyloglucan, polyornithine, agarose (0.5% to 1 w/v) and mixtures of these materials. Other growth-permissive hydrogels include thermosensitive hydrogels, such as chitosan- β -glycerophosphate hydrogel (C/GP) mixtures. Other thermosensitive hydrogels include poly (N-isopropylacrylamide) (PNIPAAM). In one embodiment, polypropylene fumarate (PPF) can be injected as a liquid and crosslinked chemically, thermally or photo-in situ to form a gel, thereby providing a hydrogel that supports growth. In another embodiment, an interpenetrating network of HA and photocrosslinkable Glycidyl Methacrylate Hyaluronic Acid (GMHA) provides a growth permissive matrix. Other growth-permissive hydrogels include: cross-linked hyaluronic acid gel (Hyaloglide gel) or ADCON-T/N gel (Gliatch). These materials may be physically or covalently crosslinked. Other scaffold materials are contemplated for growth-permissive areas (https:// www. Ncbi. Nlm. Nih. Gov/PMC/articles/PMC5899851 /).

And (4) charging. It is well known in the art that nerves prefer to grow on or through positively charged surfaces. In some embodiments, a positive charge is incorporated into the polymer backbone. In other embodiments, these charges are incorporated into other components, such as extracellular matrix proteins that are trapped in the hydrogel as it is formed in situ.

Incorporation of adjuvants. In some embodiments, an anti-inhibitory molecule may be incorporated into the hydrogel to improve the growth permissive environment, such as chondroitinase, which breaks down Chondroitin Sulfate Proteoglycans (CSPGs). https:// www.ncbi.nlm.nih.gov/pubmed/20620201. These may be incorporated with polymer powders, diluents or accelerators depending on the stability requirements of the adjuvant.

Incorporation of lipid domains. Lipid domains may be added to the backbone or side chains or to these polymers to promote nerve growth. Hydrophobic domains may also be incorporated into the backbone of the hydrogel to support nerve ingrowth through the soft and hard regions of the hydrogel. In one embodiment, lipids are added to diffuse between the polymer chains and act as plasticizers for the polymer material, promoting chain movement and increasing elasticity.

Adhesive strength. The growth-permitting medium and the growth-inhibiting medium can be converted into a hydrogel and have sufficient adhesion that once formed, the nerve ends can be easily picked up and handled. However, the adhesive strength of the subsequently formed non-flowable growth-permissive medium allows for the pick-up of the nerve-gel-nerve units with forceps. The cells may be gently placed in the second configuration to allow circumferential delivery of the inhibitory hydrogel. The adhesive strength also allows good coupling between the nerve ends and the hydrogel.

Rigidity. Since the matrix stiffness and compressive strength of hydrogels play an important role in promoting or inhibiting nerve regeneration, the mechanical properties of growth-inhibiting hydrogels and growth-permissive hydrogels differ greatly. The growth permits the hydrogel to be significantly softer and less rigid to support and promote regeneration of the nerve growth cone in the medium. The growth-permitting hydrogel preferably has a softer and more elastic gel stiffness (G.about.dynes/sq cm), characterized in that G.about.is less than 800dynes/sq cm, more preferably less than 200dynes/sq cm. In some embodiments, soft substrate regions (100-500 Pa) are placed near harder substrate regions (1000-10000 Pa). The elasticity of the growth-permitting substrate should preferably be less than 0.1-0.2MPa, preferably less than 1.5KPa. On the other hand, growth-inhibiting hydrogels provide the necessary mechanical strength to maintain the coupling and relationship between the proximal and distal nerve stumps, reduce or eliminate the need for sutures, and potentially allow for tension-free repair. The elongation in the growth-permissive matrix strongly depends on the matrix stiffness and pore interconnectivity of the pores, the charge. As the gel degrades, nerve extension may also be affected by matrix hydrolysis, oxidation, or enzymatic degradation. For growth-permitting hydrogels, the rigidity of the gel should be closer to the elastic modulus of the neural tissue, equal to or lower than 1kPa, preferably 200-300Pa. Swelling. Based on the placement of the growth-inhibiting hydrogel around the growth-permitting hydrogel, the swelling of the growth-permitting hydrogel must be less than or equal to the swelling of the growth-inhibiting hydrogel. Alternatively, the growth-permitting hydrogel must be sufficiently flexible that it does not have the strength to push the growth-inhibiting hydrogel. And (4) porosity. In some embodiments, the growth-permissive medium comprises a growth-inhibiting hydrogel filled with highly interconnected macroscopic growth-permissive pores that provide a channel through which the regenerated nerve can seek. Pores can be created in the hydrogel by porogen leaching (solid, liquid), gas foaming, emulsion templating to create macroporosity. The pores may be created by growth-permitting pore-forming agents and/or contain therapeutic agents or simply be filled with saline. The pores may be created by phase separation during hydrogel formation. The average pore size, pore size distribution and pore interconnections are difficult to quantify and are therefore included in a term called tortuosity (tortuosity). Preferably, the hydrogel is a macroporous hydrogel with pores larger than 1 μm, more preferably larger than 10 μm, preferably between >100 μm, more preferably >150 μm, and an average pore radius of 0.5 to 5%. The density of the pores should be greater than 60%, or preferably greater than 90%, of the pore volume, which is sufficient to interconnect the pores. In this manner, the remaining PEG hydrogel provides a non-growth-permissive scaffold through which neurons can grow. In one embodiment, the porosity is created by shaking to create a bubble of air or nitrogen in the hydrogel, pushing a plunger back and forth in the applicator, or introducing air through another port in the applicator. In another embodiment, a surfactant, such as Sodium Dodecyl Sulfate (SDS), is used as an air retention agent to create porosity in the hydrogel. In situ gas foaming has a porosity of up to 60% and pores of 50 to 500 microns and a compressive modulus of 20-40MPa as described in https:// www.ncbi.nlm.nih.gov/PMC/articles/PMC 3842433. In another embodiment, a foaming agent is created that creates macroscopic-sized pores to allow cell migration and proliferation. In some embodiments, the porogen is a degradation enhancer. Preferably, the concentration of pores is sufficient to interconnect the pores. Preferably >70% of the pores are interconnected, more preferably 80% or more. These holes form and define growth-permissive zones and preferably the interconnectivity is sufficiently high that the tortuosity is low and the nerves will extend into them. Furthermore, if the walls of the pores are formed of PEG, nerves can seek along the walls of the hydrogel. Pores can be created by low molecular weight end-capped PEGs (e.g., PEG 3350) that can be delivered in solution up to 50 wt%. The growth-permissive zone or pore may comprise a natural biomaterial such as collagen/gelatin, chitosan, hyaluronic acid, laminin (matrix gel), fibrin, which provide a growth-permissive matrix for nerve growth.

A channel. In another embodiment, channels are created in situ in the hydrogel to allow for nerve guidance. In one embodiment, the channels are about 150 μm, 300 μm in diameter, and more preferably 500 μm to 1mm in diameter. Preferably, the channel is filled with saline in situ.

Fibers and other structural elements. It may be desirable to add fibers or structural elements (e.g., beads, macrospheres, gel particle slurries, microspheres, rods, nanoparticles, liposomes, rods, filaments, sponges) to enhance the structural integrity of the hydrogel, improve the durability of the hydrogel in the body, and/or provide a matrix along which neurites may extend and grow for guidance. The nanofibers can be flexible or rigid, can range in size from nanometers to micrometers, and can be linear or irregularly shaped. In a preferred embodiment, the deposition of the fibers into the structure through the needles containing the media allows for a generally longitudinal parallel alignment of the fibers within the conduit. The fiber-laden media is placed in the catheter by first filling the distal end and advancing the needle in a smooth continuous motion to the proximal end of the structure (while depositing the hydrogel). Rapid gelation (less than 20 seconds, preferably less than 10 seconds, more preferably less than 7 seconds) allows the fibers to be captured in the desired direction when the media becomes non-flowable. In another embodiment, the medium solution is more viscous, between 10 and 20cP, allowing the fibers to be suspended in the growth permitting medium. In another embodiment, the fibers are provided in a kit and placed in the lumen with forceps.

Fibers, rods, filaments, sponges. In another embodiment, fibers are added to the delivery wrap structure immediately before or after the growth-permitting hydrogel solution in the structure to provide a surface along which nerves can grow to the distal stump. The fibers may be added using forceps, another syringe, or sprayed within the catheter. The gel time of the hydrogel medium is sufficiently delayed so that the fibers can be embedded in the medium before the medium becomes substantially non-flowable.

Injection of nanorods similarly, shorter nanorods can be incorporated into a polymer solution, polymer powder, diluent, or accelerator, and then injected in situ. The alignment of these fibers can be improved by smoothly injecting in one direction and using a rapidly gelling hydrogel. The fibers may be made of non-degradable or biodegradable materials. In some embodiments, the fibers are made of chitosan, polycaprolactone, polylactic acid, or glycolic acid, or a combination thereof. The fibers may be inert, functionalized with a positive charge or coated (e.g., laminin). https:// www.ncbi.nlm.nih.gov/pubmed/24083073. In another embodiment, the fibers undergo molecular self-assembly to form fibers or cables.

In one embodiment, fibers will be added randomly or in an aligned manner to provide support for nerve regeneration across the gel. The filaments and sponges may be formed of collagen. The rod may be formed from collagen-glycosaminoglycans, fibrin, hyaluronic acid, polyamides, polyacrylonitrile-co-methacrylate, PAN-MA, PGA, PHBV/PLGA blends, PLLA, PLGA, or PP. The filaments may have a diameter of between 0.5 and 500 μm, more preferably a diameter of between 15 and 250 μm. In one embodiment, the rods, fibers and filaments may be coated with laminin.

Nanofibers can be incorporated into hydrogels to provide structural support. The fibers may be composed of PEG, PGA, PLA, PCL mixed with gelatin, PCL with laminin coating, chitosan, hyaluronic acid, gelatin, hyaluronic acid, fibrin, or fibrinogen (10 mg/ml). In one embodiment, fibrous fibrin hydrogel (AFG), or P (D, L, LA) fibers made by electrospinning are incorporated into the in situ-formed gel. (McMurtrey (2014) Patterned and functionalized nanofiber scaffold in a three-dimensional hydrogel construction process for treating a fibrous outer and functional control. J. The neural Eng 11,1-15 describes an electrospinning process, which is incorporated herein). In another embodiment, polyethylene glycol is incorporated as a pore former and Nanofibers (e.g., cellulose Nanofibers) are used to provide structural integrity to the soft Porous hydrogel (Naseri et al (2016) 3-Dimensional Porous Nanocomposite coatings Based on Cellulose Nanofibers fibers for Tissue Engineering: tailling of Porosity and Mechanical performance. RSC Advances,6,5999-6007, which is incorporated herein by reference).

And (3) microparticles. In yet another embodiment, microparticles, nanoparticles or micelles may be introduced into the growth-permissive medium to deliver the drug to the neural tissue. In one embodiment, the microparticles consist of PEG hydrogel (e.g., 8 arm 15K sg, 10%), poly (D, lysine) microparticles. For example, ex vivo formed cross-linked PEG particles can be formulated as a slurry lubricated with low molecular weight PEG (1-6%, 12 kDa). Alternatively, the particles may be suspended in a collagen or hyaluronic acid solution to provide a growth-permissive matrix through which nerves can be regenerated. Similarly, hydrophobic particles and oils may be incorporated to create voids in the hydrogel that allow growth to promote nerve growth.

Growth promotes the compressive modulus of the hydrogel. It may also be advantageous to match The compressive modulus of nerve tissue with a growth-permissive hydrogel-from about 2.6 to as high as 9.2kPa (Seidlis et al (2010) The effects of hydrophilic acids with a long lasting functional properties on neural promoter cell differentiation and promotion (Biomaterials 31, 3930-3940). Similarly, the linear compressive modulus is less than 20kPa, preferably less than 10kPa, more preferably less than 1kPa, to promote nerve and Schwann cell ingrowth into The gel.

It is desirable to form in situ a growth-permissive hydrogel that can be delivered in a wrap structure or lamina around a partially transected, compressed, or fully transected nerve end. The use of in situ forming gel eliminates the need to transect an otherwise substantially intact nerve and provides a mechanism to support nerve regeneration through the matrix and into the distal tissue. Coupling the growth-permissive hydrogel with the growth-inhibiting hydrogel helps to guide and direct these neurites in the growth-permissive region. In one embodiment, the in situ-forming hydrogel has sufficient adhesive strength and rigidity such that it can be delivered between nerve stumps, into a suitable structure, then picked up and removed from the first wrap structure and placed in a larger second wrap structure into which the growth-inhibitory in situ-forming hydrogel is delivered.

Hydrogel thickness. A growth-permissive gel. The thickness or diameter of the growth-permissive gel should be approximately close to the diameter of the nerve to which it is delivered. In the case of only small defects in the nerve, the growth-permitting gel may be dropped directly onto the injured tissue to form a thin layer. A growth inhibiting gel. In view of the generally rigorous environment in which the nerve is located (typically between muscles or along the fascial plane of a muscle), in some embodiments it is desirable to maintain a minimum thickness of growth-inhibiting hydrogel around the nerve, preferably 1mm, more preferably 2-3mm in the circumferential direction. For example, for structures placed around the nerve of a common finger (about 2-2.5mm in diameter), a catheter of about 3-4mm in diameter is used to provide a 0.5-2mm hydrogel layer around the nerve. For a finger nerve of about 1 to 1.5mm, a catheter of about 2 to 2.5mm in diameter was selected. For larger nerves (between 2 to 10 mm) embedded in the arm or thigh, the gel thickness is preferably 2 to 6mm, preferably 2 to 3mm, circumferentially around the nerve.

Gel time. The hydrogel is formed around the nerve after 30 seconds or less, preferably 20 seconds or less, more preferably 10 seconds or less, more preferably 3 to 7 seconds. The hydrogel is transparent so that the location of the nerve can be visually confirmed in the hydrogel. The clinician visually or mechanically confirms the formation of the hydrogel and slides the silicone structure off the hydrogel cap and discards it. See fig. 2. The surrounding tissue (muscle, skin) is then sutured again according to standard surgical techniques.

Growth permits in vivo persistence of the gel or slurry. The in vivo persistence of the growth-permissive hydrogel may be much lower than that of the growth-inhibitory gel, allowing progressive invasion of schwann cells and regeneration of nerve fibers. For growth-permissive hydrogels, more rapidly degrading hydrogel networks are needed to allow cellular infiltration and subsequent nerve regeneration. Preferably, the hydrogel should degrade between 2 months and 6 months, more preferably 3 months. Inhibiting degradation of the zone. The inhibitory leads are preferably maintained in situ for 1 month or more, more preferably 3 months or more, to provide support to the regenerating nerve. In some embodiments, the growth-permissive hydrogel is degraded for a period of days to months, preferably days to weeks, thereby allowing material to be removed as cellular tissue is replaced and regenerated.

An electric charge. Preferably, the growth-permitting hydrogel is positively charged or comprises positively charged regions. Adding PEG fusogenic agent. In some embodiments, it may be desirable to add a non-reactive fusogenic agent to the hydrogel formulation. Thus, in addition to the mechanical blocking properties of the hydrogel, damaged proximal surviving nerves can be protected from excitotoxic damage and their membranes resealed. In addition, hyperexcitability of cell bodies such as dorsal root ganglia is reduced, reducing neurogenic paresthesia and discomfort associated with nerve damage. In one embodiment, a low molecular weight end-capped or non-reactive PEG (methoxy-PEG) is added to the formulation. For example, the trilysine buffer may comprise a non-reactive low molecular weight linear PEG (0.2 kDa, 2kDa, 3.35kDa, 4kDa or 5 kDa). When mixed with 8-arm 15K star PEG, the resulting hydrogel will have low molecular weight PEG (2kDa, 10-50% w/v), which may help seal damaged nerve endings, thereby further reducing ion influx and efflux. In this way, lysosome formation, demyelination and other membrane debris can be prevented from accumulating at the site. In another embodiment, cyclosporin a may be applied with the solution to improve survival of ablated axons.

In another embodiment, a six-arm star-capped PEG (polyethylene oxide-stat-propylene oxide) or star PEG-OH can be added as a fusogenic agent. The linear PEG incorporated in the polymer mixture can diffuse out of the cross-linked network, creating pores up to 1 micron in diameter, facilitating diffusion of nutrients, but not neurite extension. The linear PEG-based hydrogel is stiffer than the star PEG-based fusogenic additive. The addition of linear PEG is based on the discovery that 2kDa PEG has been shown to be beneficial in rapidly restoring axonal integrity, termed "PEG fusion" between cut and compressed axons (Britt et al 2010, j. Neurophysiol, 104. It is theorized that this is due in part to the occlusion of the plasma and axial membranes at the site of the lesion.

Reapplied or relocated. If the clinician is not satisfied with the location of the nerve, the hydrogel "cap" can be removed with forceps and the procedure repeated. The nerves are ready for use. In yet another embodiment, it may be desirable to deliver the in situ-forming hydrogel around the nerve to reduce handling of the nerve during surgery. By delivering it into and around the nerve bundle, the hydrogel can be fixed during surgery and prevent forceps or any other micromanipulator from squeezing it. In addition to protecting nerves from mechanical damage, hydrogels can also prevent thermal damage, for example by cauterization or RF ablation, cryoablation.

There are several embodiments where existing nerve wraps (e.g., a slit at the top of the catheter that can push the nerve into a semi-rigid wrap) and/or catheters remain desirable, but the physician wishes to provide additional support for regeneration with the application of a structure of a growth-permissive hydrogel, a growth-inhibitory hydrogel, or a combination thereof.

The structures of the growth-permissive hydrogels are designed to be substantially the same size as the nerve in which they are placed. In one embodiment, a silicon tube structure is selected, which is a half-tube with an inner diameter approximately equal to the outer diameter of the nerve. The nerves are placed in the structure either in direct apposition, in close proximity, or with a gap to prevent tension so that they can be placed in the structure without tension. Nerves are located directly on the surface of the structure itself for delivery of the growth-permissive hydrogel.

A medicine for promoting nerve regeneration. The drug may be delivered directly to the nerve prior to placement of the structure. For example, local anesthetics, anti-inflammatory agents, growth factor agents can be delivered directly to the nerve prior to encapsulation with hydrogel. Alternatively, the drug may be incorporated directly into the hydrogel, or by encapsulation into drug-loaded microspheres, micelles, liposomes or free base to achieve an improved sustained release profile.

Analgesic drugs. In some embodiments, drugs for the treatment of chronic neuropathic pain may be delivered in hydrogels, including tricyclic antidepressants, selective serotonin and norepinephrine reuptake inhibitors, antiepileptics, and opioids. For example, pregabalin and gabapentin may be selected for their analgesic properties. Similarly, duloxetine, venlafaxine, SNRI inhibitors, and combinations thereof may provide more complete pain relief. Anti-inflammatory agents such as diclofenac may also be expected. Other potential targets include ligands of the FK506 binding protein family, neuroimmunophilin ligands, which are neurotrophic, neuroprotective, and neuroregenerative agents.

Topical administration of paclitaxel and cetuximab also shows promise for improved neuronal survival and regeneration, and may be suitable for stimulating nerve regeneration when delivered locally in an in situ-formed hydrogel. In another embodiment, cyclic adenosine monophosphate (cAMP) or the cAMP analog dibutyryl cAMP promotes nerve regeneration and may be incorporated into an in situ-forming hydrogel to promote nerve regeneration after injury. In another embodiment, drugs of Kindlin-1 and Kindlin-2 (Fermitin family) and the integrin superfamily that bind to cell surface receptors allow nerve extension through inhibitory matrices and can be incorporated into hydrogels to enhance regeneration across inhibitory extracellular matrices.

In another embodiment, the immunosuppressant, tacrolimus (FK 506), may be incorporated into a hydrogel to enhance neurite outgrowth and speed. The final concentration of FK506 in the resulting hydrogel was between 100ug/ml and 10mg/ml, more preferably 0.1mg/ml. The drug is released for several weeks to several months, preferably at least one month, more preferably at least 3 months, to help immunosuppression and enhance nerve growth. The drug includes FK506, a drug selected for selective inhibition of FKBP12 or FKBP 51.

Drugs that are P2X receptor antagonists (P2 XR), P2X3 receptor antagonists (e.g., AF-219Gefapivant, AF-130), P2X4 and P2X7 receptor antagonists associated with visceral and neuropathic pain (as well as migraine and cancer pain) are of interest. A P2X7 receptor antagonist. The purinergic receptor antagonist Brilliant Blue G (BBG) and the structurally similar analogue brilliant Blue FCF (BB FCF) are of particular interest due to their ability to modulate the neuronal environment following injury (Wang et al.2013.The food dye FD & C Blue No.1is a selective inhibitor of the ATP release channel panx1.J. Gen. Physiol.141 (5) 649-656)). Other dyes of interest include FD & C Green No.3 dye, which like BBG and BB FCF, inhibits ATP release from the Pannexin1 channel with an IC50 between 0.2 and 3 uM. A structurally similar analog, brilliant Blue FCF (BB FCF), also known as FD & C #1 (https:// pubchem. Ncbi. Nlm. Nih. Gov/compound/Acid _ Blue-9), has also been shown to improve post-injury nerve survival and regeneration when used in conjunction with low molecular weight end-capped PEG 3350Da (https:// www. Ncbi. Nlm. Nih. Gov/pubmed/23731685). Similar efficacy was demonstrated using BBG in the rat sciatic nerve crush injury model (Ribeiro et al, 2017) and the intertuscular plexus ischemia model (Palombit et al, 2019). Furthermore, by reducing extracellular high ATP concentrations and high calcium influx after nerve injury, BBG is thought to have anti-inflammatory and antinociceptive effects. In one embodiment, the brilliant blue FCF is incorporated into an in situ-forming hydrogel. The dye may be incorporated into the polymer vial, diluent or accelerator solution to produce a final concentration in the gel of 0.0001 to 5%, preferably 0.001 to 0.25%, more preferably 0.01 to 0.02% wt% or about 1 to 1000ppm, preferably 10 to 100 ppm. On an anatomical basis per site, a local dose of dye of 5 μ g up to 25mg can be delivered locally in the hydrogel. For example, FD & C #1 dye can be delivered at a concentration of 0.01% in a hydrogel to reduce neuronal damage following stroke (Arbeloa et al, 2012 — reference Palmobit et al, 2019). By incorporating a dye into the hydrogel, the dye may help increase the survival rate of transected axons, reducing local inflammation, while the hydrogel provides a regenerative barrier.

In another embodiment, a TRPV1 agonist, such as capsaicin, is delivered to a nerve to precondition the nerve for injury, resulting in a downstream nerve regeneration response to enhance nerve regeneration (PMID: 29854941). In one embodiment, the capsaicin-loaded hydrogel (1 to 8wt% drug load) is delivered transdermally to intact nerves to reduce diabetic neuropathic pain. In another embodiment, the fisetin-u (pifithrin-u) or acetyl-L-carnitine is delivered in a hydrogel to reduce and treat chemotherapy-induced peripheral neuropathy (CIPN) by reducing neuronal mitochondrial damage.

In another embodiment, drugs that block deregulated long non-coding RNAs can also be incorporated into the hydrogel, such as targets of endogenous Kcna2 antisense RNAs. In one embodiment, mitomycin C is incorporated into the in situ forming hydrogel to inhibit Schwann cell proliferation and stimulate fibroblast apoptosis. In one embodiment, 0.1 to 5mg of mitomycin C is loaded into a polymer powder and used to form an in situ formed gel, wherein 0.01 to 0.5wt% of mitomycin C is released at a daily rate of between 0.1 and 0.5mg/ml, preferably for 7 days or more. In yet another embodiment, a Rho kinase (ROCK) inhibitor or ROK antagonist or Rac1 antagonist, such as lissudil hydrochloride, may be incorporated.

Other drugs include anti-inflammatory curcumins, rapamycin, paclitaxel, cyclosporin A, pyrimidine derivatives that stimulate remyelination (RG 2 and RG 5), axon homing molecule Slit 3, triptolide, KMUP-1. Calcium modulators including calcitonin, the calcium antagonists nifedipine, nimodipine, nerve growth factor (NGF, 500 ng), insulin-like growth factor (IGF-1), thymoquinone, duloxetine (10-30 mg), melatonin, c-Jun or mTORC1 agonists may help support schwann cell differentiation and nerve remyelination, nicotine and adrenomedullin-for use as neuroprotective and neurotrophic agents.

Example 1. A growth inhibiting hydrogel. To a vial containing 80mg of PEG with NHS ester reactive groups, 80 μ g of BB FCF was added to produce a dye concentration of 0.1% in the PEG hydrogel.

Example 2. Growth inhibitor hydrogels and fusions. To a vial containing 80PEG with NHS ester reactive groups was added 80 μ g BB FCF and 500mg PEG 3350.

Example 3. Phospholipids (such as cephalins) are incorporated into PEG hydrogels to improve fusion. Phospholipids are surface-active amphiphilic molecules that can be incorporated as emulsifiers, wetting agents, solubilizers and membrane fusion agents. These may include phosphatidylcholine, phosphatidylethanolamine or phosphatidylglycerol (https:// www.ncbi.nlm.nih.gov/PMC/articles/PMC4207189/, which is incorporated herein by reference).

Example 4. In some embodiments, the hydrogel is loaded with amiodarone, with or without the addition of ethanol. For example, loadings of 0.1 to 5wt% amiodarone or more may be achieved. This can also be achieved and improved by incorporating ethanol in the solution. For example, 50 to 75% ethanol may be incorporated with 0.25wt% amiodarone to achieve a burst release of amiodarone between 3 to 5 days. Likewise, 1% amiodarone may be delivered from the hydrogel for 30-60 days.

The following examples are in situ formed growth inhibitory wrap formulations suitable for preventing abnormal nerve growth, scar tissue formation and supporting nerve slippage within a hydrogel.

Example 5. In some embodiments, 8-arm 20K PEG-SAP is cross-linked with 4-arm 10K PEG amine, the PEG-SAP being in excess of the PEG-amine. For example, PEG-SAP and PEG-amine are dissolved in an acidic diluent in a ratio of 1.2: 1. The suspension is mixed with an accelerator buffer and delivered through a static mixer to form a hydrogel. The formulation gels within 3 seconds providing a compressive strength of 70-100kPa and a degree of swelling of between 10-30 wt%.

Example 6. In some embodiments, the 8-arm 15K PEG-SAP is crosslinked with an 8-arm 40K PEG amine. PEG-SAP and PEG-amine were dissolved in an acidic diluent in a ratio of 1.6: 1. The suspension is mixed with an accelerator buffer and delivered through a static mixer to form a hydrogel. The formulation gelled within 4 seconds, providing a compressive strength of 30-80kPa and an equilibrium swelling of 20-60% by weight.

Example 7. In some embodiments, 8 arm 20K PEG-SG is crosslinked with 4 arm 20K PEG-amine. PEG-SG and PEG-amine were dissolved in an acidic diluent at a ratio of 1.0: 1. The suspension is mixed with an accelerator buffer and delivered through a static mixer to form a hydrogel. The formulation gels within 5 seconds, provides a compressive strength of 20-70kPa, and undergoes an equilibrium swelling of 40-80 wt%.

The following examples support the formation of in situ growth inhibitory wrap formulations suitable for preventing abnormal nerve growth, scar tissue formation, and supporting nerve glide within the hydrogel as it degrades and gradually softens, while preventing cellular infiltration.

Example 8. In some embodiments, 8 arm 40K PEG-SG is crosslinked with 8 arm 40K PEG-amine. PEG-SG and PEG-amine were dissolved in an acidic diluent at a ratio of 1.2: 1. The suspension is mixed with an accelerator buffer and delivered through a static mixer to form a hydrogel. The formulation gelled within 4 seconds, provided compressive strength of 30-60kPa, and underwent equilibrium swelling of 40-80 wt%.

Example 9. In some embodiments, an 8-arm 20K PEG-SAZ (PEG-succinimidyl azelate) is crosslinked with a 4-arm 40K PEG amine. PEG-SAZ and PEG-amine were dissolved in an acidic diluent at a ratio of 1.2: 1. The suspension is mixed with an accelerator buffer and delivered through a static mixer to form a hydrogel. The formulation gelled within 4 seconds and provided a compressive strength of 20-50 kPa. Furthermore, the equilibrium swelling is between 50 and 100 wt%.

Example 10. In some embodiments, the 4-arm 15K PEG-SAZ is crosslinked with a 4-arm 40K PEG amine. PEG-SAZ and PEG-amine were dissolved in an acidic diluent at a ratio of 1.2: 1. The suspension is mixed with an accelerator buffer and delivered through a static mixer to form a hydrogel. The formulation gelled within 8 seconds and provided a compressive strength of 10 to 40 kPa. Furthermore, the equilibrium swelling is between 70 and 130 wt%.

Example 11. In another example, a 3 arm 15K PEG-SS (succinimidyl succinate) is crosslinked with a 4 arm 40K PEG amine.

The following are examples of materials that may be used in biodegradable structures or sheets that may be delivered around nerve ends in need of repair.

Example 12. In some embodiments, the material of the biodegradable structure is crosslinked or uncrosslinked chitosan. The degree of deacetylation of chitosan is in the range of 70% to 100% and the thickness of the structure is between 10 μm to 100 μm, preferably about 30 μm.

Example 13. In some embodiments, the material of the biodegradable structure consists of cross-linked or non-cross-linked chitosan mixed with HPMC (hydroxypropylmethylcellulose), CMC (carboxymethylcellulose) or HA (hyaluronic acid). The degree of deacetylation of chitosan is between 70% and 100%. The chitosan layer has a thickness in the range of 10 to 100 μm. The layers of the other components (HPMC, CMC or HA) were 5 μm to 50 μm.

Example 14. In some embodiments, the material of the biodegradable structure is crosslinked or uncrosslinked gelatin. The thickness of the conduit ranges from 10 μm to 100 μm.

Example 15. In some embodiments, the material of the conduit is a crosslinked or non-crosslinked CMC/HA mixture. The thickness of the conduit ranges from 10 μm to 100 μm, preferably about 40 μm.

Example 16. In some embodiments, the growth-permissive solution comprises 3 to 5wt% PEG-diacrylate, preferably 3wt% PEG-DA, having a compressive modulus of about 70 Pa.

Example 17. In one embodiment, the growth-permissive solution consists of a collagen solution at 800 μ g/ml.

Example 18. In one embodiment, the growth-permitting solution consists of a 2% solution of Hydroxypropylmethylcellulose (HPMC) in water, having a viscosity of between 7500 and 14000 mPa-s.

Example 19. In one embodiment, the growth-permitting solution consists of a fibrin solution.

Example 20. In another embodiment, the growth-permissive solution consists of a collagen-chondroitin-6-sulfatoprotein solution.

Many other modifications, adaptations and alternative designs are of course possible in light of the above teaching. It is, therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein. It is contemplated that various combinations or subcombinations of the specific features and aspects of the embodiments disclosed above may be made and still fall within one or more of the inventions. Moreover, the disclosure herein of any particular feature, aspect, method, characteristic, feature, quality, attribute, element, etc., associated with an embodiment can be used in all other embodiments set forth herein. Thus, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes of the disclosed inventions. Therefore, it is intended that the scope of the invention herein disclosed should not be limited by the particular disclosed embodiments described above. In addition, while the invention is susceptible to various modifications and alternative forms, specific examples thereof have been shown in the drawings and are herein described in detail. It should be understood, however, that the invention is not to be limited to the particular forms or methods disclosed, but to the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the various embodiments and the appended claims. Any of the methods disclosed herein need not be performed in the order recited. The methods disclosed herein include certain actions taken by the practitioner; however, they may also include any third party indication of such actions, either explicitly or implicitly. For example, actions such as "enter the spleen-kidney ligament" include "directing entry into the spleen-kidney ligament. The scope disclosed herein also includes any and all overlaps, sub-ranges, and combinations thereof. Language such as "at most," "at least," "greater than," "less than," "between," and the like includes the listed numbers. As used herein, numbers modified with terms such as "about," "about," and "substantially" include the listed numbers (e.g., about 10% = 10%) and also represent quantities close to the recited quantity that still perform the desired function or achieve the desired result. For example, the terms "about," "about," and "substantially" may refer to an amount within less than 10%, within less than 5%, within less than 1%, within less than 0.1%, within less than 0.01% of the recited amount. Further, a variety of theories and possible mechanisms of action are discussed herein, but are not intended to be limiting.

Claims

1. A kit for forming an implant in situ for directing nerve regeneration between two nerve ends, comprising:

a first component for producing a first growth-permissive hydrogel;

a second component for producing a second growth-inhibiting hydrogel;

at least one structure having a concave shape;

a first applicator for delivering a growth-permissive hydrogel into the cavity; and

a second applicator for delivering the growth-inhibiting hydrogel into the cavity.

2. The kit of claim 1, wherein the first component comprises a powdered growth-permitting hydrogel precursor, a reconstituting solution, and an accelerator solution.

3. The kit of claim 1, wherein the powdered growth-permissive hydrogel precursor comprises one or more agents that stimulate nerve regeneration.

4. The kit of claim 1, wherein the second component comprises a powdered growth-inhibiting hydrogel precursor, a reconstituting solution, and an accelerator solution.

5. The kit of claim 1, wherein the first component comprises a powdered growth-permitting gel precursor and a reconstituting solution.

6. The kit of claim 1, wherein the second component comprises a pre-filled syringe containing a growth-permissive gel.

7. The kit of claim 1, comprising a first structure having a first configuration for receiving the growth-inhibiting hydrogel and a second structure having a second, different configuration for receiving the growth-permissive hydrogel.

8. The kit of claim 1, wherein the concave shape has a surface of hydrophobic character.

9. The kit of claim 7, wherein at least the growth-permitting hydrogel has hydrophilic properties.

10. The kit of claim 7, wherein the second structure comprises biocompatible biodegradable half cylinders or sheets.

11. The kit of claim 10, wherein the sheet has a thickness of less than about 60 microns.

12. The kit of claim 10, wherein the thickness is less than about 40 microns.

13. The kit of claim 7, wherein the second structure degrades within one week.

14. A kit for forming a hydrogel neuropil in situ, comprising:

a dual applicator system;

a vial containing a powdered hydrogel precursor;

reconstituting the solution;

an accelerator solution; and

at least one nerve cap structure.

15. The kit of claim 14, wherein the powdered hydrogel precursor comprises an anti-inflammatory or anti-infective agent.

16. An in situ formed nerve regeneration construct comprising:

a growth-permissive hydrogel bridge having a first end and a second end and configured to span the space between the two nerve ends and promote nerve transaxle regeneration; and

a growth-inhibiting hydrogel sheath encapsulating the growth-permissive hydrogel bridge and configured to extend beyond the first and second ends to directly contact the two nerve ends.

17. A structure for creating an in situ nerve cap to inhibit neuroma formation, comprising:

a recessed wall defining a cavity, the wall having a top opening for accessing the cavity, the top opening lying in a first plane and having an area smaller than an area of a second plane conforming to an interior dimension of the cavity and spaced apart from the cavity and parallel to the first plane; and

a concave nerve guide carried by the wall and providing lateral access to the cavity;

wherein the recessed walls have hydrophobic characteristics.

18. A structure for manufacturing an in situ nerve conduit for promoting regeneration of nerve-to-nerve junctions and scar tissue prevention, the structure comprising:

A recessed wall defining a cavity, the wall having a top opening for accessing the cavity, the top opening lying in a first plane and having an area smaller than an area of a second plane conforming to an interior dimension of the cavity and spaced apart from the cavity and parallel to the first plane;

a first concave nerve guide carried by the wall and providing a first lateral access for positioning a first nerve end in the cavity; and

a second concave nerve guide carried by the wall and providing a second lateral access for positioning a second nerve end in the cavity.

19. The structure of claim 18, wherein the structure is configured to receive a second biodegradable structure containing a nerve end to be repaired.

20. The structure of claim 18, wherein the structure promotes nerve regeneration across a nerve-to-nerve junction.

21. The structure of claim 18, wherein the structure promotes hydrogel formation to prevent nerve compression.

22. A composition for forming a growth-inhibiting hydrogel in situ, having:

compressive strength of greater than 10kDa for more than 3 months

In vivo persistence for at least 3 months, including less than 15% mass loss,

And less than 30% swelling for more than 3 months.

23. The composition of claim 22, wherein degradation of the hydrogel causes the hydrogel to swell radially outward.

24. The composition of claim 22, comprising a poly (ethylene glycol) having a biodegradable amide or urethane linkage.

25. The composition of claim 22, comprising poly (ethylene glycol) succinimide carbonate.

26. The composition of claim 22, wherein the hydrogel comprises a P2XR receptor antagonist.

27. The composition of claim 22, wherein the hydrogel comprises a P2X7 receptor antagonist.

28. The composition of claim 27, wherein the P2X7 receptor antagonist is brilliant blue FCF (BB FCF) or Brilliant Blue G (BBG).

29. The composition of claim 22, comprising a poly (ethylene glycol) having biodegradable ester linkages.

30. The composition of claim 22, comprising poly (ethylene glycol) succinimide adipate.

31. The composition of claim 22, comprising a multi-arm PEG with arm lengths between 1 and 10 kDa.

32. The composition of claim 22, comprising at least one multi-arm PEG with an arm length of 5 kDa.

33. A composition for forming a growth-inhibiting hydrogel in situ, comprising a hydrogel having:

a compressive strength greater than about 10 kPa;

in vivo persistence for at least about 2 weeks; and

initial swelling greater than about 20% but less than about 100%

34. The composition of claim 33, wherein degradation of the hydrogel results in outward radial swelling of the hydrogel with a volume swell of less than about 160%.

35. The kit of claim 33, wherein degradation of the hydrogel occurs in less than about 16 weeks.

36. The composition of claim 33, wherein the growth-inhibiting hydrogel is configured to encapsulate a growth-permissive gel with:

young's modulus of less than about 10kPa

The viscosity is greater than about 5,000cp.

37. The composition of claim 33, comprising a poly (ethylene glycol) having biodegradable ester linkages.

38. The composition of claim 33, comprising poly (ethylene glycol) succinimide adipate.

39. The composition of claim 33, comprising a multi-arm PEG having arms between 1 and 10kDa in length.

40. The composition of claim 33, comprising at least one multi-arm PEG with an arm length of 5 kDa.

41. The composition of claim 33, wherein the growth-permissive gel allows for a beam-like arrangement.

42. The composition of claim 33, wherein the growth-permitting gel comprises HPMC.

43. The composition of claim 33, wherein the growth-permissive gel comprises collagen.

44. The composition of claim 33, wherein the growth-permitting gel comprises a biodegradable rod or fiber.

45. An absorbable, in situ-formed electrode anchor comprising a quantity of hydrogel polymerized in situ around an electrode and configured to maintain the electrode in electrical communication with a nerve.

46. The electrode anchor of claim 45, wherein the hydrogel is electrically conductive.

47. An in situ forming implant comprising an amount of hydrogel that transitions from a relatively flowable state to a relatively non-flowable state upon contact with a structure and is removed from the structure by a pulling force of no more than about 5N, wherein removal is aided by the hydrophilic properties of the hydrogel and the hydrophobic properties of the structure.

48. The in situ forming implant of claim 47, comprising a nerve cap.

49. The in situ forming implant of claim 47, comprising a nerve conduit for guiding nerve regeneration.

50. The in situ forming implant of claim 47, comprising a nerve wrap for preventing scar formation and nerve tethering.